1,997 results on '"Xylosidases"'
Search Results
202. Biochemical properties of a novel thermostable and highly xylose-tolerant β-xylosidase/ a-arabinosidase from Thermotoga thermarum.
- Author
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Hao Shi, Xun Li, Huaxiang Gu, Yu Zhang, Yingjuan Huang, Liangliang Wang, and Fei Wang
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XYLOSIDASES , *HEMICELLULOSE , *THERMOTOGA maritima , *ENERGY industries , *BIOCONVERSION , *ESCHERICHIA coli , *HEAT stability in proteins - Abstract
Background: β-Xylosidase is an important constituent of the hemicellulase system and it plays an important role in hydrolyzing xylooligosaccharides to xylose. Xylose, a useful monose, has been utilized in a wide range of applications such as food, light, chemical as well as energy industry. Therefore, the xylose-tolerant β-xylosidase with high specific activity for bioconversion of xylooligosaccharides has a great potential in the fields as above. Results: A β-xylosidase gene (Tth xynB3) of 2,322 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 774 amino acid residues, and was expressed in Escherichia coli BL21 (DE3). The phylogenetic trees of β-xylosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. The phylogeny and amino acid analysis indicated that the Tth xynB3 β-xylosidase was a novel β-xylosidase of GH3. The optimal activity of the Tth xynB3 β-xylosidase was obtained at pH 6.0 and 95°C and was stable over a pH range of 5.0-7.5 and exhibited 2 h half-life at 85°C. The kinetic parameters Km and Vmax values for p-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside were 0.27 mM and 223.3 U/mg, 0.21 mM and 75 U/mg, respectively. The kcat/Km values for p-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside were 1,173.4 mM-1 s-1 and 505.9 mM-1 s-1, respectively. It displayed high tolerance to xylose, with Ki value approximately 1000 mM. It was stimulated by xylose at higher concentration up to 500 mM, above which the enzyme activity of Tth xynB3 β-xylosidase was gradually decreased. However, it still remained approximately 50% of its original activity even if the concentration of xylose was as high as 1000 mM. It was also discovered that the Tth xynB3 β-xylosidase exhibited a high hydrolytic activity on xylooligosaccharides. When 5% substrate was incubated with 0.3 U Tth xynB3 β-xylosidase in 200 μL reaction system for 3 h, almost all the substrate was biodegraded into xylose. Conclusions: The article provides a useful and novel β-xylosidase displaying extraordinary and desirable properties: high xylose tolerance and catalytic activity at temperatures above 75°C, thermally stable and excellent hydrolytic activity on xylooligosaccharides [ABSTRACT FROM AUTHOR]
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- 2013
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203. Expression profile of transcripts encoding cell wall remodelling proteins in tomato fruit cv. Micro-Tom subjected to 15°C storage.
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Müller, Gabriela L., Budde, Claudio O., Lauxmann, Martin A., Triassi, Agustina, Andreo, Carlos S., Drincovich, María F., and Lara, María V.
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TOMATOES , *ETHYLENE , *GENETIC transcription , *XYLOSIDASES , *PLANT cell walls - Abstract
To extend fruit market life, tomatoes are harvested before red ripe and kept at temperatures below optimum (20°C). In this work, Micro-Tom tomatoes stored at 20°C (normal ripening) were compared with those stored at 15°C or 4°C (chilling injury inducer) for 7 days. In contrast to 4°C, storage at 15°C delayed ripening with the benefit of not enhancing oxidative metabolism and of enabling ripening upon being transferred to 20°C. The transcriptional expression profile of enzymes related to cell wall metabolism was compared at the three temperatures. Although endo-β-1 ,4-glucanase (Cel1), which is associated with fruit decay, was largely increased after removal from 4°C storage, its expression was not modified in fruits stored at 15°C. Enhanced transcriptional expression of xyloglucan endotransgylcosylase/hydrolases (XTHs) XTH1, -2, -10 and -11, and of two β-xylosidases (Xyl1-2) was detected in fruits stored at 15°C with respect to those at 20°C. Following 2 days at 20°C, these transcripts remained higher in fruits stored at 15 °C and XHT3 and -9 also increased. Ethylene evolution was similar in fruits kept at 15°C and 20°C; thus, the changes in the transcript profile and fruit properties between these treatments may be under the control of factors other than ethylene. [ABSTRACT FROM AUTHOR]
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- 2013
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204. Activation of a GH43 β-xylosidase by divalent metal cations: Slow binding of divalent metal and high substrate specificity.
- Author
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Jordan, Douglas B., Lee, Charles C., Wagschal, Kurt, and Braker, Jay D.
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XYLOSIDASES , *GLYCOSIDASES , *CATIONS , *NITROPHENYL compounds , *ISOMERIZATION , *ENZYME activation - Abstract
RS223-BX of glycoside hydrolase family 43 is a β-d-xylosidase that is strongly activated (k cat/K m as much as 116-fold) by the addition of divalent metal cations, Ca2+, Co2+, Fe2+, Mg2+, Mn2+ and Ni2+. Slow activation by Mg2+ was demonstrated (k on 0.013s−1 mM−1, k off 0.008s−1) at pH 7.0 and 25°C. k off and k on values are independent of Mg2+ concentration, but k off and k on are slower in the presence of increasing levels of substrate 4-nitrophenyl-β-d-xylopyranoside. The kinetics strongly suggest that M2+ binds to the enzyme rapidly, forming E M2+, followed by slow isomerization to the activated enzyme, E∗ M2+. Moderately high values of k cat (7–30s−1) were found for M2+-activated RS223-BX acting on xylobiose (natural substrate) at pH 7.0 and 25°C. Certain M2+-activated RS223-BX exhibit the highest reported values of k cat/K m of any β-xylosidase acting on natural substrates: for example, at pH 7.0 and 25°C, xylobiose (Mn2+, 190s−1 mM−1), xylotriose (Ca2+, 150s−1 mM−1) and xylotetraose (Ca2+, 260s−1 mM−1). There is potential for the enzyme to add value to industrial saccharification operations at low substrate and high d-glucose and high d-xylose concentrations. [ABSTRACT FROM AUTHOR]
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- 2013
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205. High-level production of thermotolerant β-xylosidase of Aspergillus sp. BCC125 in Pichia pastoris: Characterization and its application in ethanol production
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Wongwisansri, Sriwan, Promdonkoy, Peerada, Matetaviparee, Panida, Roongsawang, Niran, Eurwilaichitr, Lily, and Tanapongpipat, Sutipa
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XYLOSIDASES , *BIOMASS production , *ASPERGILLUS , *PICHIA pastoris , *ETHANOL , *TEMPERATURE effect , *HYDROGEN-ion concentration , *FERMENTATION - Abstract
Abstract: A gene coding for thermotolerant β-xylosidase from Aspergillus sp. BCC125 was characterized. The recombinant enzyme was expressed in methylotrophic yeast Pichia pastoris KM71 and especially high yield of secreted enzyme was obtained. β-xylosidase possessed high enzyme efficiency (K cat/K m =198.8mM−1 s−1) toward pNP-β-D-xylopyranoside (pNPβX) with optimal temperature and pH for activity of 60°C and pH 4.0–5.0, respectively. The identified β-xylosidase showed clear synergism with previously identified xylanase for hydrolysis of xylan in vitro as well as simultaneous saccharification and fermentation process (SSF) in vivo with Pichia stipitis. [Copyright &y& Elsevier]
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- 2013
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206. Purification and characterization of β-d-xylosidase from Lactobacillus brevis grown on xylo-oligosaccharides
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Lasrado, Lyned D. and Gudipati, Muralikrishna
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XYLOSIDASES , *LACTOBACILLUS brevis , *OLIGOSACCHARIDES , *GEL permeation chromatography , *DIETHYLAMINOETHANOL , *CELLULOSE - Abstract
Abstract: In the recent years there has been a growing interest in the use of oligosaccharides as prebiotics to modulate gut microbiota with an aim to improve the gut health. Though xylo-oligosaccharides (XOS) have been increasingly used as prebiotics, information pertaining to the enzymes used by lactobacilli to degrade these substrates is scanty. Present investigation reports the purification and characterization of β-d-xylosidase from Lactobacillus brevis NCDC01 grown on XOS. Three sequential steps consisting of ultra-filtration, DEAE cellulose ion-exchange and Sephacryl S-100 gel filtration chromatographies were employed to purify the enzyme to apparent homogeneity and it was found to be monomeric on SDS-PAGE with an apparent molecular mass of ∼58.0kDa. The pH and temperature optima were 6.0 and 40°C respectively. The enzyme remained stable over a pH range of 5.5–7.5 and up to 50°C for 30min. Under optimum pH and temperature with p-nitrophenyl β-d-xylopyranoside as a substrate, the enzyme exhibited a K m of 0.87mM. The enzyme does not require any metal ion for activity or stability but is completely inhibited by Hg2+, Pb2+, p-chloromercuribenzoate (PCMB), oxalic acid and citric acid. This is perhaps the first report on the purification and characterization of β-d-xylosidase from Lactobacillus brevis NCDC01. [Copyright &y& Elsevier]
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- 2013
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207. Divalent metal activation of a GH43 β-xylosidase
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Lee, Charles C., Braker, Jay D., Grigorescu, Arabela A., Wagschal, Kurt, and Jordan, Douglas B.
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METAL activation , *XYLOSIDASES , *XYLANS , *POLYSACCHARIDES , *LIGNOCELLULOSE , *XYLOSE , *AMINO acids , *GLUCOSE , *ARABINOFURANOSIDASES - Abstract
Abstract: Depolymerization of xylan, a major fraction of lignocellulosic biomass, releases xylose which can be converted into transportation fuels and chemical feedstocks. A requisite enzyme for the breakdown of xylan is β-xylosidase. A gene encoding the 324-amino acid β-xylosidase, RS223-BX, was cloned from an anaerobic mixed microbial culture. This glycoside hydrolase belongs to family 43. Unlike other GH43 enzymes, RS223-BX can be strongly activated by exogenously supplied Ca2+, Co2+, Fe2+, Mg2+, Mn2+ and Ni2+ (e.g., 28-fold by Mg2+) and it is inhibited by Cu2+ or Zn2+. Sedimentation equilibrium centrifugation experiments indicated that the divalent metal cations mediate multimerization of the enzyme from a dimeric to a tetrameric state, which have equal catalytic activity on an active-site basis. Compared to the determined active sites of other GH43 β-xylosidases, the predicted active site of RS223-BX contains two additional amino acids with carboxylated side chains that provide potential sites for divalent metal cations to reside. Thus, the divalent metal cations likely occupy the active site and participate in the catalytic mechanism. RS223-BX accepts as substrate xylobiose, arabinobiose, 4-nitrophenyl-β-d-xylopyranoside, and 4-nitrophenyl-α-l-arabinofuranoside. Additionally, the enzyme has good pH and temperature stabilities and a large K i for d-glucose (1.3M), favorable properties for performance in saccharification reactors. [Copyright &y& Elsevier]
- Published
- 2013
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208. Xylanase and β-Xylosidase from Penicillium janczewskii: Production, Physico-chemical Properties, and Application of the Crude Extract to Pulp Biobleaching.
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Terrasan, César R. F., Temer, Beatriz, Sarto, Camila, Júnior, Francides G. Silva, and Carmona, Eleonora C.
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XYLANASES , *XYLOSIDASES , *PENICILLIUM , *WOOD pulp bleaching , *BLEACHING (Chemistry) , *HYDROGEN-ion concentration - Abstract
Extracellular xylanase and β-xylosidase production by a Penicillium janczewskii strain were investigated in liquid cultures with xylan from oat spelts under different physical and chemical conditions. The selected conditions for optimized production of xylanase and β-xylosidase were 7 days, pH 6.5, at 30 °C and 8 days, pH 5.0, at 25 °C, respectively. The xylanase exhibited optimal activity in pH 5.0 at 50 °C and the β- xylosidase in pH 4.0 at 75 °C. The xylanase was more stable at pH 6.0 to 9.5, while the β-xylosidase remained stable at pH ranging from 1.6 to 5.5. The xylanase half-life (T50) at 40, 50, and 60 °C was 183, 15, and 3 min, respectively. β-xylosidase half-life was 144, 8, and 4 min at 50, 65, and 75 °C, respectively. When applied to the biobleaching of Eucalyptus kraft pulp, xylanase dosages of 2 and 4 U/g dried pulp reduced, respectively, kappa number by 3.0 and 3.3 units after 1 h treatment, demonstrating that the use of P. janczewskii xylanases in this process is quite promising. The pulp viscosity was not altered, confirming the absence of cellulolytic enzymes in the fungal extract. [ABSTRACT FROM AUTHOR]
- Published
- 2013
209. A New Approach in the Active Site Investigation of an Inverting β- d-Xylosidase from Thermobifida fusca TM51.
- Author
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Fekete, Csaba and Kiss, László
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BINDING sites , *XYLOSIDASES , *AMINO acids , *HYDROGEN-ion concentration , *THERMOPHILIC bacteria , *SOIL microbiology - Abstract
The catalytic amino acid residues of the β- d-xylosidase (EC 3.2.1.37; GH43), from Thermobifida fusca TM51 (TfBXyl43), were investigated by direct chemical modifications. The pH dependence curves of the kinetic parameters (k and k/K) gave pK values for the free enzyme (5.55 ± 0.19; 6.44 ± 0.19), and pK values of for the enzyme-substrate complex (4.85 ± 0.23; 7.60 ± 0.28) respectively, by using an artificial substrate p-nitrophenyl-β- d-xylopyranoside (pNP-Xyl). The detailed inhibition studies demonstrated well the hydrophobic character of the glycon binding site. Carbodiimide-mediated chemical modifications of the enzyme with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) in the presence of glycine methyl ester supports the conclusion that a carboxylate residue can be fundamental in the catalytic process. We have also synthesized and tested N-bromoacetyl-β- d-xylopyranosylamine (NBAXA) for TfBXyl43 as an affinity label, which also inactivated the enzyme irreversible. The pH dependence studies in both cases of inactivation revealed that the modified group is the catalytic proton donor (NBAXA pK = 6.68 ± 0,1; EDAC pK = 7.42 ± 0.22) which displays its essential role in the hydrolytic process. The β- d-xylopyranosylazide as competitive inhibitor protected the enzyme in all cases against the inactivation, suggesting that the chemical modification which has an impact on the activity took place in the active center. Changing of the enzyme conformation was followed by CD spectroscopy, as a result of the NBAXA inactivation. Our study is important because to our knowledge no similar investigations were made in the case of an inverting β- d-xylosidase. [ABSTRACT FROM AUTHOR]
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- 2013
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210. Optimization of ß-Glucosidase, ß-Xylosidase and Xylanase Production by Colletotrichum graminicola under Solid-State Fermentation and Application in Raw Sugarcane Trash Saccharification.
- Author
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Zimbardi, Ana L. R. L., Sehn, Cesar, Meleiro, Luana P., Souza, Flavio H. M., Masui, Douglas C., Nozawa, Monica S. F., Guimarães, Luis H. S., Jorge, João A., and Furriel, Rosa P. M.
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GLUCOSIDASES , *XYLOSIDASES , *XYLANASES , *COLLETOTRICHUM graminicola , *SOLID-state fermentation , *SUGARCANE , *HYDROGEN-ion concentration - Abstract
Efficient, low-cost enzymatic hydrolysis of lignocellulosic residues is essential for cost-effective production of bioethanol. The production of ß-glucosidase, ß-xylosidase and xylanase by Colletotrichum graminicola was optimized using Response Surface Methodology (RSM). Maximal production occurred in wheat bran. Sugarcane trash, peanut hulls and corncob enhanced ß-glucosidase, ß-xylosidase and xylanase production, respectively. Maximal levels after optimization reached 159.3 ± 12.7 U g-1, 128.1 ± 6.4 U g-1 and 378.1 ± 23.3 U g-1, respectively, but the enzymes were produced simultaneously at good levels under culture conditions optimized for each one of them. Optima of pH and temperature were 5.0 and 65 °C for the three enzymes, which maintained full activity for 72 h at 50 °C and for 120 min at 60 °C (ß-glucosidase) or 65 °C (ß-xylosidase and xylanase). Mixed with Trichoderma reesei cellulases, C. graminicola crude extract hydrolyzed raw sugarcane trash with glucose yield of 33.1% after 48 h, demonstrating good potential to compose efficient cocktails for lignocellulosic materials hydrolysis. [ABSTRACT FROM AUTHOR]
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- 2013
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211. Heterologous expression of β-xylosidase gene from Paecilomyces thermophila in Pichia pastoris.
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Juturu, Veeresh and Wu, Jin
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XYLOSIDASES , *GENE expression , *PAECILOMYCES , *PICHIA pastoris , *ENZYME activation , *ALCOHOL oxidase , *C-terminal binding proteins , *PH effect - Abstract
β-xylosidase from thermophilic fungi Paecilomyces thermophila was functionally expressed in Pichia pastoris with a his tag in the C-terminal under the alcohol oxidase 1 (AOX1) promoter and secreted into the medium at 0.22 mg l. Its molecular mass was estimated to be 52.3 kDa based on the SDS-PAGE analysis, which is 1.3 times higher than the predicted 39.31 kDa from its amino acid compositions, although no potential N- or O- glycosylation sites were predicted from its amino acid sequence. This is presumed to be caused by some unpredictable posttranslational modifications based on mass spectrum analysis of the recombinant protein. The enzyme was most active at 60 °C and pH 7. It showed not only a β-xylosidase activity with a K of 8 mM and a V of 54 μmol min mg for hydrolysis of p-nitrophenyl β- d-xylopyranoside but also an arabinofuranosidase activity (6.2 U mg) on p-nitrophenyl arabinofuranoside. [ABSTRACT FROM AUTHOR]
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- 2013
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212. Cellulolytic and Xylanolytic Enzymes from Yeasts: Properties and Industrial Applications.
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Sohail M, Barzkar N, Michaud P, Tamadoni Jahromi S, Babich O, Sukhikh S, Das R, and Nahavandi R
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- Cellulose, Endo-1,4-beta Xylanases chemistry, Yeasts, Cellulase chemistry, Cellulases, Xylosidases
- Abstract
Lignocellulose, the main component of plant cell walls, comprises polyaromatic lignin and fermentable materials, cellulose and hemicellulose. It is a plentiful and renewable feedstock for chemicals and energy. It can serve as a raw material for the production of various value-added products, including cellulase and xylanase. Cellulase is essentially required in lignocellulose-based biorefineries and is applied in many commercial processes. Likewise, xylanases are industrially important enzymes applied in papermaking and in the manufacture of prebiotics and pharmaceuticals. Owing to the widespread application of these enzymes, many prokaryotes and eukaryotes have been exploited to produce cellulase and xylanases in good yields, yet yeasts have rarely been explored for their plant-cell-wall-degrading activities. This review is focused on summarizing reports about cellulolytic and xylanolytic yeasts, their properties, and their biotechnological applications.
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- 2022
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213. The substrate/product-binding modes of a novel GH120 ß-xylosidase (XylC) from Thermoanaerobacterium saccharolyticum JW/SL-YS485.
- Author
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HUANG, Chun-Hsiang, Yu SUN, KO, Tzu-Ping, CHEN, Chun-Chi, Yingying ZHENG, CHAN, Hsiu-Chien, Xuefei PANG, WIEGE, Juergen, Weilan SHAO, and GUO, Rey-Ting
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XYLANS , *BINDING sites , *XYLOSIDASES , *ENTEROBACTER , *GLYCOSIDASES , *CRYSTAL structure - Abstract
Xylan-1,4-ß-xylosidase (ß-xylosidase) hydrolyses xylooligomers at their non-reducing ends into individual xylose units. Recently, XylC, a ß-xylosidase from Thermoanaerobacterium saccharolyticum JW/SL-YS485, was found to be structurally different from corresponding glycosyl hydrolases in the CAZy database (http://www.cazy.org/), and was subsequently classified as the first member of a novel family of glycoside hydrolases (GH120). In the present paper, we report three crystal structures of XylC in complex with Tris, xylobiose and xylose at 1.48-2.05 Å (1 Å=0.1 nm) resolution. XylC assembles into a tetramer, and each monomer comprises two distinct domains. The core domain is a right-handed parallel ß-helix (residues 1-75 and 201-638) and the flanking region (residues 76-200) folds into a ß-sandwich domain. The enzyme contains an open carbohydrate-binding cleft, allowing accommodation of longer xylo-oligosaccharides. On the basis of the crystal structures and in agreement with previous kinetic data, we propose that XylC cleaves the glycosidic bond by the retaining mechanism using two acidic residues Asp382 (nucleophile) and Glu405 (general acid/base). In addition to the active site, nine other xylose-binding sites were consistently observed in each of the four monomers, providing a possible reason for the high tolerance of product inhibition. [ABSTRACT FROM AUTHOR]
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- 2012
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214. Purification and Characterization of a Recombinant β- d-xylosidase from Thermobifida fusca TM51.
- Author
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Fekete, Csaba and Kiss, László
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XYLOSIDASES , *RECOMBINANT proteins , *PH effect , *NITROPHENYL compounds , *GLYCOSIDASES , *BIOSENSORS , *SODIUM dodecyl sulfate , *POLYACRYLAMIDE gel electrophoresis - Abstract
The subject of our investigations was a recombinant β- d-xylosidase (TfBXyl43) from Thermobifida fusca TM51 which was expressed in E. coli BL21DE3 and was purified to apparent homogeneity. The SDS-PAGE investigations demonstrated that the molecular weight of the monomer unit is 62.5 kDa but the native-PAGE studies indicated that the mass of the enzyme is 240-250 kDa which proves the presence of a characteristic homo oligomer quaternary structure in solution phase. Optimal parameters of the enzyme activity were at pH 6.0 and 50 °C. The enzyme showed little stability under pH 4.5 and above 60 °C. The substrate specificity investigations indicated that the TfBXyl43 is an exo-glycosidase, hydrolyzing only xylobiose and -triose from the nonreducing end. Besides the enzyme shows very high specificity on the glycon part of the substrate, since it can only hydrolyze β- d-xylopyranoside derivatives. The importance of hydrophobic interactions in the binding of the substrates are supported that the enzyme can hydrolize about four times more efficiently the artificial p-nitrophenyl-β- d-xylopyranoside substrate compared to the natural one, xylobiose. Furthermore we could detect transxylosidase activity both in the case of xylobiose and p-nitrophenyl-β- d-xylopyranoside donors which is the first example among the inverting β- d-xylosidases from T. fusca. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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215. SCREENING OF ENZYMATIC ACTIVITIES IN NON- SACCHAROMYCES CIDER YEASTS.
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PANDO BEDRIÑANA, R., LASTRA QUEIPO, A., and SUÁREZ VALLES, B.
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ENZYME kinetics , *SACCHAROMYCES , *POLYGALACTURONASE , *PECTIN lyase , *RESTRICTION fragment length polymorphisms , *XYLOSIDASES - Abstract
ABSTRACT The activities of polygalacturonase, pectin lyase, β-glucosidase, β-xylosidase and protease were determined using solid media in 420 wild non- Saccharomyces cider strains identified by internal transcribed spacer-restriction fragment length polymorphism. The identified species corresponded to Hanseniaspora valbyensis, Hanseniaspora uvarum, Hanseniaspora osmophila, Metschnikowia pulcherrima, Candida parapsilosis and Pichia guilliermondii. The most common activity exhibited was that of β-glucosidase (33%), with all the analyzed species having some strains able to develop this activity. Strains of M. pulcherrima showed the greatest capacity to produce β-glucosidase and protease. β-xylosidase was detected in 17 yeast strains belonging to the genera Hanseniaspora, Pichia and Metschnikowia. All of the tested species have some strains with the capacity to develop β-xylosidase activity, except for C. parapsilosis. No strains were able to secrete pectin lyase, while polygalacturonase activity was observed in eight Hanseniaspora strains. Only two strains, belonging to the species H. uvarum and M. pulcherrima, developed three enzymatic activities, namely β-glucosidase, β-xylosidase and protease. PRACTICAL APPLICATIONS The non- Saccharomyces yeasts are very interesting in cider making because of the low level of ethanol in cider. In this paper, it is demonstrated that the yeast isolated from Asturian cider represents a source of several enzymes, which have capacity to release flavor compounds. In this sense non- Saccharomyces strains are being used as inoculated cultures to ferment apple pomace and apple must, with the aim of enhancing the aroma and flavor of the products. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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216. Analysis of the saccharification capability of high-functional cellulase JN11 for various pretreated biomasses through a comparison with commercially available counterparts.
- Author
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Kawai, Tetsushi, Nakazawa, Hikaru, Ida, Noriko, Okada, Hirofumi, Tani, Shuji, Sumitani, Jun-ichi, Kawaguchi, Takashi, Ogasawara, Wataru, Morikawa, Yasushi, and Kobayashi, Yoshinori
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BIOMASS , *CELLULASE , *ENZYMES , *EUCALYPTUS , *XYLOSIDASES - Abstract
Although the capabilities of Trichoderma reesei cellulases have been greatly improved, these enzymes are still too costly for commercial use. The aim of this research was to assess the biomass saccharification capability of JN11, a recombinant cellulase, compared with that of the commercially available cellulases Accellerase 1500 and Cellic CTec. The activities of JN11, Accellerase 1500, and Cellic CTec were compared by using various types of cellulosic biomass, including rice straw, Erianthus, eucalyptus, and Japanese cedar. JN11 had higher saccharification capability for rice straw, Erianthus, eucalyptus, and Japanese cedar compared with the commercial cellulases. The JN11 saccharification of cellulosic biomasses, including hemicellulose (NaOH-pretreated biomasses), resulted in high glucose and xylose yields because of the high xylanase/xylosidase activity of JN11. Moreover, even JN11 saccharification of hemicellulose-free biomasses (sulfuric acid-, hydrothermally, and steam exploded-pretreated biomasses) resulted in high glucose yields. The cellulase activity of JN11, however, was comparable to that of its commercial counterparts. These findings indicate that the saccharification ability of cellulase is unrelated to its cellulase activity when measured against Avicel, CMC, pNP-lactoside, and other substrates. JN11 showed high activity for all types of pretreated cellulosic biomasses, indicating its usefulness for saccharification of various cellulosic biomasses. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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217. Characterization of an ethanol-tolerant 1,4-β-xylosidase produced by Pichia membranifaciens.
- Author
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Romero, A. M., Mateo, J. J., and Maicas, S.
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ETHANOL , *XYLOSIDASES , *PICHIA , *WINES , *YEAST , *MOLECULAR weights - Abstract
Aims: The purification and biochemical properties of the 1,4-β-xylosidase of an oenological yeast were investigated. Methods and Results: An ethanol-tolerant 1,4-β-xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G-100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS-PAGE. The activity of 1,4-β-xylosidase was optimum at pH 6·0 and at 35°C. The activity had a Km of 0·48 ± 0·06 mmol l-1 and a Vmax of 7·4 ± 0·1 μmol min-1 mg-1 protein for p-nitrophenyl-β-d-xylopyranoside. Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4-β-xylosidase from P. membranifaciens to be used in the bioethanol production process. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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218. Secretory expression of a β-xylosidase gene from Thermomyces lanuginosus in Escherichia coli and characterization of its recombinant enzyme.
- Author
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Chen, Z., Jia, H., Yang, Y., Yan, Q., Jiang, Z., and Teng, C.
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XYLOSIDASES , *GENE expression , *RECOMBINANT proteins , *THERMOPHILIC fungi , *HEMICELLULOSE , *XYLANS , *ESCHERICHIA coli - Abstract
Aims: To characterize a β-xylosidase from the thermophilic fungus Thermomyces lanuginosus and to investigate its potential in saccharification of hemicellulosic xylans. Methods and Results: A gene (designated TlXyl43) encoding β-xylosidase was cloned from T. lanuginosus CAU44 and expressed in Escherichia coli. The gene consists of a 1017-bp open reading frame without introns. It encodes a mature protein of 338 residues with no predicted signal peptide, belonging to glycoside hydrolase (GH) family 43. Over 60% of the recombinant β-xylosidase (TlXyl43) was secreted into the culture medium. TlXyl43 was purified 2·6-fold to homogeneity with an estimated mass of 51·6 kDa by SDS-PAGE. The purified enzyme exhibited optimal activity at pH 6·5 and 55°C and was stable at 50°C. It was competitively inhibited by xylose with a Ki value of 63 mmol l-1. Conclusions: In this study, a GH family 43 β-xylosidase gene (TlXyl43) from T. lanuginosus CAU44 was cloned and functionally expressed in E. coli, and over 60% of recombinant protein was secreted into the culture. Significance and Impact of the Study: This is the first report of the cloning and functional expression of a β-xylosidase gene from Thermomyces species. TlXyl43 holds great potential for variety of industries. [ABSTRACT FROM AUTHOR]
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- 2012
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219. A novel xylan degrading β- d-xylosidase: purification and biochemical characterization.
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Michelin, Michele, Peixoto-Nogueira, Simone, Silva, Tony, Jorge, João, Terenzi, Héctor, Teixeira, José, and Lourdes T. M. Polizeli, Maria
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ASPERGILLUS , *XYLOSIDASES , *XYLANS , *BIOCHEMISTRY , *CELLULOSE , *ION exchange chromatography , *GLYCOPROTEINS - Abstract
Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular β-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. β-xylosidase is a glycoprotein (39 % carbohydrate content) and has a molecular mass of 137 kDa by SDS-PAGE, with optimal temperature and pH at 70 °C and 3.0-5.5, respectively. β-xylosidase was stable in acidic pH (3.0-6.0) and 70 °C for 1 h. The enzyme was activated by 5 mM MnCl (28 %) and MgCl (20 %) salts. The β-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-β- d-xylopyranoside, exhibiting apparent K and V values of 0.66 mM and 39 U (mg protein) respectively, and to a lesser extent p-nitrophenyl-β- d-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel β- d-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by β-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose. [ABSTRACT FROM AUTHOR]
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- 2012
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220. Effects of the fungicide tebuconazole on microbial capacities for litter breakdown in streams
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Artigas, Joan, Majerholc, Joy, Foulquier, Arnaud, Margoum, Christelle, Volat, Bernadette, Neyra, Marc, and Pesce, Stéphane
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PHYSIOLOGICAL effects of fungicides , *TEBUCONAZOLE , *AQUATIC microbiology , *MICROBIAL ecology , *BLACK poplar , *ALNUS glutinosa , *XYLOSIDASES , *BETA-glucosidase , *BIODEGRADATION - Abstract
Abstract: Streams draining agricultural basins are subjected to the input of fungicides which can affect aquatic microbial communities. We analyzed the effect of the fungicide tebuconazole (TBZ) on Alnus glutinosa and Populus nigra litter breakdown by aquatic microorganisms. For six weeks, fungal and bacterial responses were analyzed in indoor stream channels subjected to TBZ-contaminated (33.1±12.4μgL−1) and uncontaminated conditions. Litter breakdown rates decreased in presence of TBZ. The decrease was explained by reductions in microbial biomass development and shifts in community structure. At the same time, TBZ modified the kinetics of β-glucosidase, β-xylosidase and cellobiohydrolase enzymes resulting in lower affinities for cellulose and hemicellulose decomposition in leaves. These alterations were modulated by the litter quality; the greatest structural impairment was observed in Populus whereas Alnus were more affected in terms of leaf breakdown rate. Our results suggest that chronic exposure to TBZ can affect aquatic microbial communities and their capacity to break down leaf litter in streams. [Copyright &y& Elsevier]
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- 2012
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221. The accessory domain changes the accessibility and molecular topography of the catalytic interface in monomeric GH39 β-xylosidases.
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Ramos Santos, Camila, Polo, Carla Cristina, Corrêa, Juliana Moço, Simão, Rita de Cássia Garcia, Seixas, Flavio Augusto Vicente, and Murakami, Mario Tyago
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MOLECULAR topography , *XYLOSIDASES , *CATALYSIS , *MONOMERS , *OLIGOSACCHARIDES , *OLIGOMERIZATION , *HYDROLASES , *CRYSTAL structure - Abstract
β-Xylosidases (EC 3.2.1.37) are among the principal glycosyl hydrolases involved in the breakdown of hemicelluloses, catalyzing the reduction of xylooligosaccharides to free xylose. All GH39 β-xylosidases structurally characterized to date display a modular multi-domain organization that assembles a tetrameric quaternary structure. In this work, the crystal structure and the SAXS molecular envelope of a new GH39 β-xylosidase from Caulobacter crescentus (CcXynB2) have been determined. Interestingly, CcXynB2 is a monomer in solution and comparative structural analyses suggest that the shortened C-terminus prevents the formation of a stable tetramer. Moreover, CcXynB2 has a longer loop from the auxiliary domain (the long α-helix-containing loop) which makes a number of polar and hydrophobic contacts with the parental (α/β)8-barrel domain, modifying the accessibility and the molecular topography of the catalytic interface. These interactions also maintain the accessory domain tightly linked to the catalytic core, which may be important for enzyme function and stability. [ABSTRACT FROM AUTHOR]
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- 2012
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222. The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus β-Xylosidase I.
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Graciano, Luciana, Corrêa, Juliana, Gandra, Rinaldo, Seixas, Flavio, Kadowaki, Marina, Sampaio, Silvio, Conceição Silva, José, Osaku, Clarice, and Simão, Rita de
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CAULOBACTER crescentus , *XYLOSIDASES , *ARABINOFURANOSIDASES , *POLYMERASE chain reaction , *CLONING , *SEPHAROSE , *AQUATIC bacteria - Abstract
The xynB1 gene (CCNA 01040) of Caulobacter crescentus that encodes a bifunctional enzyme containing the conserved β-Xylosidase and α- l-Arabinofuranosidase (β-Xyl I-α- l-Ara) domains was amplified by PCR and cloned into the vector pJet1.2Blunt. The xynB1 gene was subcloned into the vector pPROEX-hta that produces a histidine-fused translation product. The overexpression of recombinant β-Xyl I-α- l-Ara was induced with IPTG in BL21 (DE3) and the resulting intracellular protein was purified with pre-packaged nickel-Sepharose columns. The recombinant β-Xyl I-α- l-Ara exhibited a specific β-Xylosidase I activity of 1.25 U mg to oNPX and a specific α- l-Arabinofuranosidase activity of 0.47 U mg to pNPA. The predominant activity of the recombinant enzyme was its β-Xylosidase I activity, and the enzymatic characterization was focused on it. The β-Xylosidase I activity was high over the pH range 3-10, with maximal activity at pH 6. The enzyme activity was optimal at 45 °C, and a high degree of stability was verified over 240 min at this temperature. Moreover, β-Xylosidase activity was inhibited in the presence of the metals Zn and Cu, and the enzyme exhibited K and V values of 2.89 ± 0.13 mM and 1.4 ± 0.04 μM min to oNPX, respectively. The modeled structure of β-xylosidase I showed that its active site is highly conserved compared with other structures of the GH43 family. The increase in the number of contact residues responsible for maintaining the dimeric structure indicates that this dimer is more stable than the tetramer form. [ABSTRACT FROM AUTHOR]
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- 2012
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223. Trans-α-xylosidase and trans-β-galactosidase activities, widespread in plants, modify and stabilize xyloglucan structures.
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Franková, Lenka and Fry, Stephen C.
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XYLOGLUCANS , *OLIGOSACCHARIDES , *XYLOSIDASES , *GLYCOSIDASES , *PLANT cell walls , *HEMICELLULOSE , *GALACTOSIDASES - Abstract
Cell-wall components are hydrolysed by numerous plant glycosidase and glycanase activities. We investigated whether plant enzymes also modify xyloglucan structures by transglycosidase activities. Diverse angiosperm extracts exhibited transglycosidase activities that progressively transferred single sugar residues between xyloglucan heptasaccharide (XXXG or its reduced form, XXXGol) molecules, at 16 μ m and above, creating octa- to decasaccharides plus smaller products. We measured remarkably high transglycosylation:hydrolysis ratios under optimized conditions. To identify the transferred monosaccharide(s), we devised a dual-labelling strategy in which a neutral radiolabelled oligosaccharide (donor substrate) reacted with an amino-labelled non-radioactive oligosaccharide (acceptor substrate), generating radioactive cationic products. For example, 37 μ m [ Xyl-3H]XXXG plus 1 m m XXLG-NH2 generated 3H-labelled cations, demonstrating xylosyl transfer, which exceeded xylosyl hydrolysis 1.6- to 7.3-fold, implying the presence of enzymes that favour transglycosylation. The transferred xylose residues remained α-linked but were relatively resistant to hydrolysis by plant enzymes. Driselase digestion of the products released a trisaccharide (α-[3H]xylosyl-isoprimeverose), indicating that a new xyloglucan repeat unit had been formed. In similar assays, [ Gal-3H]XXLG and [ Gal-3H]XLLG (but not [ Fuc-3H]XXFG) yielded radioactive cations. Thus plants exhibit trans-α-xylosidase and trans-β-galactosidase (but not trans-α-fucosidase) activities that graft sugar residues from one xyloglucan oligosaccharide to another. Reconstructing xyloglucan oligosaccharides in this way may alter oligosaccharin activities or increase their longevity in vivo. Trans-α-xylosidase activity also transferred xylose residues from xyloglucan oligosaccharides to long-chain hemicelluloses (xyloglucan, water-soluble cellulose acetate, mixed-linkage β-glucan, glucomannan and arabinoxylan). With xyloglucan as acceptor substrate, such an activity potentially affects the polysaccharide's suitability as a substrate for xyloglucan endotransglucosylase action and thereby modulates cell expansion. We conclude that certain proteins annotated as glycosidases can function as transglycosidases. [ABSTRACT FROM AUTHOR]
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- 2012
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224. Amino acids and protein profile in floral nectar: Much more than a simple reward
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Nepi, Massimo, Soligo, Chiara, Nocentini, Daniele, Abate, Mariangela, Guarnieri, Massimo, Cai, Giampiero, Bini, Luca, Puglia, Michele, Bianchi, Laura, and Pacini, Ettore
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AMINO acids , *POLLINATORS , *XYLOSIDASES , *ALANINE , *INVERTASE , *FORAGING behavior - Abstract
Abstract: Nectar is considered a primary alimentary reward for a large variety of pollinators. Recent studies demonstrated that nectar may have other functions in addition to attracting pollinators. Mainly other two functions have been recognized: defense against microbial invasion and post-secretion modification of sugar profile. The floral nectar of Cucurbita pepo L. has been analyzed to confirm the presence of defense proteins, namely xylosidases that were identified by means of a proteomic approach in a previous study. An enzyme assay was also performed that revealed low invertase activity for which the optimal pH was determined. This invertase activity is not able to modify the sugar profile significantly during the short period of anthesis (6h). The amino acids complement of the nectar of both sexes was also determined. Sixteen of the 20 protein amino acids have been detected. Proline comprises more than 30% of total amino acid content in male and female flowers. Three non-protein amino acids (taurine, β-alanine, and GABA) represent almost one third of the total amino acid content, and of these, GABA is the most abundant (16%). Several biological activities were attributed to these amino acids and further studies are needed to assess their presumed involvement in managing the foraging behavior of insects. More recent studies, including our own, demonstrate that the various functions of nectar are much more complex than previously thought. [Copyright &y& Elsevier]
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- 2012
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225. Trans-α-xylosidase, a widespread enzyme activity in plants, introduces (1→4)-α-d-xylobiose side-chains into xyloglucan structures
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Franková, Lenka and Fry, Stephen C.
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XYLOSIDASES , *PLANT enzymes , *XYLOGLUCANS , *MOLECULAR structure , *ANGIOSPERMS , *OLIGOSACCHARIDES , *AMINATION - Abstract
Abstract: Angiosperms possess a retaining trans-α-xylosidase activity that catalyses the inter-molecular transfer of xylose residues between xyloglucan structures. To identify the linkage of the newly transferred α-xylose residue, we used [Xyl-3H]XXXG (xyloglucan heptasaccharide) as donor substrate and reductively-aminated xyloglucan oligosaccharides (XGO–NH2) as acceptor. Asparagus officinalis enzyme extracts generated cationic radioactive products ([3H]Xyl·XGO–NH2) that were Driselase-digestible to a neutral trisaccharide containing an α-[3H]xylose residue. After borohydride reduction, the trimer exhibited high molybdate-affinity, indicating xylobiosyl-(1→6)-glucitol rather than a di-xylosylated glucitol. Thus the trans-α-xylosidase had grafted an additional α-[3H]xylose residue onto the xylose of an isoprimeverose unit. The trisaccharide was rapidly acetolysed to an α-[3H]xylobiose, confirming the presence of an acetolysis-labile (1→6)-bond. The α-[3H]xylobiitol formed by reduction of this α-[3H]xylobiose had low molybdate-affinity, indicating a (1→2) or (1→4) linkage. In NaOH, the α-[3H]xylobiose underwent alkaline peeling at the moderate rate characteristic of a (1→4)-disaccharide. Finally, we synthesised eight non-radioactive xylobioses [α and β; (1↔1), (1→2), (1→3) and (1→4)] and found that the [3H]xylobiose co-chromatographed only with (1→4)-α-xylobiose. We conclude that Asparagus trans-α-xylosidase activity generates a novel xyloglucan building block, α-d-Xylp-(1→4)-α-d-Xylp-(1→6)-d-Glc (abbreviation: ‘V’). Modifying xyloglucan structures in this way may alter oligosaccharin activities, or change their suitability as acceptor substrates for xyloglucan endotransglucosylase (XET) activity. [Copyright &y& Elsevier]
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- 2012
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226. Screening for xylanase and β-xylosidase production from wood-inhabiting Penicillium strains for potential use in biotechnological applications.
- Author
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Lee, Jaejung, Jang, Yeongseon, Lee, Hanbyul, Lee, Sangjoon, Kim, Gyu-Hyeok, and Kim, Jae-Jin
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- *
XYLANASES , *XYLOSIDASES , *PENICILLIUM , *WOOD-pulp , *BIOTECHNOLOGY , *CELLULASE , *ETHANOL as fuel - Abstract
Experiments were performed to find potential sources for enzyme production for the pulp and paper industry and for biological ethanol production by screening the cellulase, xylanase and β-xylosidase activities of 36 species of Penicillium isolated from various wood materials in Korea. Rice straw powder (RiceP), birchwood xylan (BirchX), and beechwood xylan (BeechX) were supplied as individual carbon sources for the Penicillium species. All Penicillium species tested in this study showed little cellulase activity, but some species exhibited remarkably high xylanase and β-xylosidase activities, as determined by a filter paper assay. P. oxalicum showed the greatest xylanase activity on RiceP (158.70 U ml-1). On the other hand, P. brevicompactum produced the highest active β-xylosidase on BirchX (6.25 U ml-1). [ABSTRACT FROM AUTHOR]
- Published
- 2012
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227. Engineering Escherichia coli for succinate production from hemicellulose via consolidated bioprocessing.
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BACTERIAL genetics , *ESCHERICHIA coli , *CELLULASE , *SUCCINATES , *BEECH , *XYLOSIDASES - Abstract
The article presents a research in which researchers engineered escherichia coli strains capable of exporting three hemicellulases to the broth for the succinate production directly from beechwood xylan through consolidated bioprocessing. It is reported that, the researchers characterized and tested twelve variant OsmY fused endoxylanase-xylosidase combinations.
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- 2012
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228. High-level expression of a novel multifunctional GH3 family β-xylosidase/α-arabinosidase/β-glucosidase from Dictyoglomus turgidum in Escherichia coli
- Author
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Qi Li, Jianjun Pei, Zhipeng Qi, Daiyi Zheng, Xinyi Tong, and Linguo Zhao
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Glycoside Hydrolases ,medicine.disease_cause ,01 natural sciences ,Biochemistry ,Hydrolysis ,Drug Discovery ,Escherichia coli ,medicine ,Glycoside hydrolase ,Enzyme kinetics ,Molecular Biology ,Epimedium ,chemistry.chemical_classification ,Bacteria ,biology ,010405 organic chemistry ,Chemistry ,beta-Glucosidase ,Organic Chemistry ,biology.organism_classification ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,Xylosidases ,Enzyme ,Yield (chemistry) ,Dictyoglomus turgidum - Abstract
A novel β-xylosidase Dt-2286 from Dictyoglomus turgidum was cloned and overexpressed in Escherichia coli BL21 (DE3). Dt-2286 belonging to glycoside hydrolase (GH) family 3 encodes a polypeptide with 762 amino acid residues with a molecular weight of 85.1 kDa. By optimization of the growth and induction conditions, the activity of β-xylosidase reached 273 U/mL, which is the highest yield reported to date from E. coli in a shake-flask. The optimal activities of the purified Dt-2286 were found at pH 5.0 and 98 °C. It also shows excellent thermostable/haloduric/organic solvent-tolerance. Dt-2286 was revealed to be a multifunctional enzyme with β-xylosidase, α-arabinofuranoside, α-arabinopyranoside and β-glucosidase activities, and Kcat/Km was 5245.316 mM−1 s−1, 2077.353 mM−1 s−1, 1626.454 mM−1 s−1, and 470.432 mM−1 s−1 respectively. Dt-2286 showed significant synergistic effects on the degradation of xylans, releasing more reduced sugars (up to 15.08 fold) by simultaneous addition with endoxylanase. Moreover, this enzyme has good activity in the hydrolysis of epimedium B, demonstrating its versatility in practical applications.
- Published
- 2021
229. Wheat Domestication Accelerated Evolution and Triggered Positive Selection in the β-Xylosidase Enzyme of Mycosphaerella graminicola.
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MYCOSPHAERELLA graminicola , *WHEAT diseases & pests , *PHYTOPATHOGENIC microorganisms , *NUCLEOTIDES , *NUCLEIC acids , *PLANT cell walls , *PLANT enzymes , *BIOLOGICAL evolution , *XYLOSIDASES - Abstract
The article presents information on a study which investigates whether plant cell wall degrading enzymes (PCWDEs) of plant pathogens like fungal wheat pathogen Mycosphaerella graminicola have been positively selected by analyzing ratios of non-synonymous and synonymous nucleotide changes in the genes, involved in production of these enzymes. PCWDEs have the potential to trigger defense reactions in plants. In this study, five PCWDEs were analyzed and it was found that one (b-xylosidase) had been under strong positive selection and went through an accelerated rate of evolution, and PCWDEs played an important role in the emergence of a host-specialized pathogen.
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- 2009
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230. Characterization of an ionic liquid-tolerant β-xylosidase from a marine-derived fungal endophyte
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Arthur Broadstock, Si Yu Tan, Trichur S. Suryanarayanan, Angela Zabala, Anindita Sengupta, and Venkat Gopalan
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0106 biological sciences ,0301 basic medicine ,Ionic Liquids ,Lignocellulosic biomass ,Sodium Chloride ,01 natural sciences ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Organophosphorus Compounds ,010608 biotechnology ,Endophytes ,Organic chemistry ,Hemicellulose ,Enzyme Inhibitors ,Cellulose ,Molecular Biology ,Trichoderma ,Dose-Response Relationship, Drug ,Imidazoles ,Fungal endophyte ,Cell Biology ,Xylosidases ,030104 developmental biology ,chemistry ,Ionic liquid - Abstract
Ionic liquids (ILs) are used in lignocellulosic biomass (LCB) pretreatment because of their ability to disrupt the extensive hydrogen-bonding network in cellulose and hemicellulose, and thereby decrease LCB recalcitrance to subsequent enzymatic degradation. However, this approach necessitates the development of cellulases and hemicellulases that can tolerate ∼20% (w/v) IL, an amount that either co-precipitates with the sugar polymers after the initial pretreatment or is typically used in single-pot biomass deconstructions. By investigating the secretomes from 4 marine-derived fungal endophytes, we identified a β-xylosidase derived from Trichoderma harzianum as the most promising in terms of tolerating 1-ethyl-3-methylimidazolium-dimethyl phosphate (EMIM-DMP), an IL. When tested with p-nitrophenyl-β-d-xyloside, this extracellular xylosidase retained ∼50% activity even in 1.2 mol·L–1 (20% w/v) EMIM-DMP after incubation for 48 h. When tested on the natural substrate xylobiose, there was ∼85% of the initial activity in 1.2 mol·L–1 EMIM-DMP after incubation for 9 h and ∼80% after incubation for 48 h. Despite previous findings associating thermostability and IL tolerance, our findings related to the mesophilic T. harzianum β-xylosidase(s) emphasize the need to include the marine habitat in the bioprospecting dragnet for identification of new IL-tolerant LCB-degrading enzymes.
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- 2017
231. Recycling of a selectable marker with a self-excisable plasmid in Pichia pastoris
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Xinying Zhang, Xueyun Zheng, Qingyan Yuan, Ying Lin, Xihao Liao, Nuo Pang, Cheng Li, Yuanyuan Huang, and Shuli Liang
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0301 basic medicine ,Genetic Markers ,Genetic Vectors ,Gene Dosage ,Heterologous ,Gene Expression ,lcsh:Medicine ,Pichia ,Article ,Pichia pastoris ,03 medical and health sciences ,Plasmid ,Gene Order ,Vector (molecular biology) ,Lipase ,lcsh:Science ,Selectable marker ,Genetics ,Recombination, Genetic ,6-Phytase ,Multidisciplinary ,biology ,lcsh:R ,biology.organism_classification ,030104 developmental biology ,Xylosidases ,Biochemistry ,Genetic marker ,biology.protein ,Xylanase ,lcsh:Q ,Plasmids - Abstract
Pichia pastoris is a widely used heterologous protein production workhorse. However, with its multiple genetic modifications to solve bottlenecks for heterologous protein productivity, P. pastoris lacks selectable markers. Existing selectable marker recycling plasmids have drawbacks (e.g., slow growth and conditional lethality). Here, zeocin-resistance marker recycling vectors were constructed using the Cre/loxP recombination system. The vectors were used to (i) knock in heterologous phytase, xylanase and lipase expression cassettes, (ii) increase the phytase, xylanase and lipase gene copy number to 13, 5, and 5, respectively, with vector introduction and (iii) engineer the secretion pathway by co-overexpressing secretion helper factors (Sly1p and Sec1p) without introducing selectable markers, giving a phytase field of 0.833 g/L. The vectors allow selectable marker recycling and would be a useful tool to engineer P. pastoris for high heterologous protein productivity.
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- 2017
232. Characterization of the arabinoxylan-degrading machinery of the thermophilic bacterium Herbinix hemicellulosilytica—Six new xylanases, three arabinofuranosidases and one xylosidase
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F. Krabichler, Daniela E. Koeck, Jannis Broeker, Wolfgang H. Schwarz, Vladimir V. Zverlov, B. Roessler, Matthias Mechelke, and Wolfgang Liebl
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0106 biological sciences ,0301 basic medicine ,Hot Temperature ,Glycoside Hydrolases ,Oligosaccharides ,Glucuronates ,Bioengineering ,01 natural sciences ,Applied Microbiology and Biotechnology ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Polysaccharides ,010608 biotechnology ,Glycoside hydrolase family 10 ,Arabinoxylan ,Glycoside hydrolase ,Cloning, Molecular ,Triticum ,Clostridiales ,Endo-1,4-beta Xylanases ,Chemistry ,Beta-glucosidase ,Hydrolysis ,beta-Glucosidase ,Thermophile ,General Medicine ,Hydrogen-Ion Concentration ,Xylosidases ,Xylan ,Recombinant Proteins ,Enzyme Activation ,030104 developmental biology ,Biochemistry ,Xylanase ,Carbohydrate Metabolism ,Xylans ,Biotechnology - Abstract
Herbinix hemicellulosilytica is a newly isolated, gram-positive, anaerobic bacterium with extensive hemicellulose-degrading capabilities obtained from a thermophilic biogas reactor. In order to exploit its potential as a source for new industrial arabinoxylan-degrading enzymes, six new thermophilic xylanases, four from glycoside hydrolase family 10 (GH10) and two from GH11, three arabinofuranosidases (1x GH43, 2x GH51) and one β-xylosidase (GH43) were selected. The recombinantly produced enzymes were purified and characterized. All enzymes were active on different xylan-based polysaccharides and most of them showed temperature-vs-activity profiles with maxima around 55-65°C. HPAEC-PAD analysis of the hydrolysates of wheat arabinoxylan and of various purified xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) was used to investigate their substrate and product specificities: among the GH10 xylanases, XynB showed a different product pattern when hydrolysing AXOS compared to XynA, XynC, and XynD. None of the GH11 xylanases was able to degrade any of the tested AXOS. All three arabinofuranosidases, ArfA, ArfB and ArfC, were classified as type AXH-m,d enzymes. None of the arabinofuranosidases was able to degrade the double-arabinosylated xylooligosaccharides XA2+3XX. β-Xylosidase XylA (GH43) was able to degrade unsubstituted XOS, but showed limited activity to degrade AXOS.
- Published
- 2017
233. Isolation of highly thermostable β-xylosidases from a hot spring soil microbial community using a metagenomic approach
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Daisuke Shibata, Masaru Sato, Asuka Nishimura, Jiro Okuma, Yasuhiko Kondo, Migiwa Suda, Tomohiko Kato, and Yoshitsugu Hirose
- Subjects
DNA, Bacterial ,0301 basic medicine ,Sequence Homology ,Biology ,medicine.disease_cause ,Hot Springs ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Escherichia coli ,Genetics ,medicine ,Amino Acid Sequence ,Molecular Biology ,Gene ,Peptide sequence ,Phylogeny ,Soil Microbiology ,chemistry.chemical_classification ,Contig ,Gene Expression Regulation, Bacterial ,General Medicine ,Full Papers ,Xylosidases ,thermostable enzyme, β-xylosidase, metagenome, xylan-degradation ,030104 developmental biology ,Enzyme ,Amino Acid Substitution ,Biochemistry ,chemistry ,Metagenomics ,DNA - Abstract
The DNA extracted from a high-temperature environment in which micro-organisms are living will be a good source for the isolation of thermostable enzymes. Using a metagenomic approach, we aimed to isolate thermostable β-xylosidases that will be exploited for biofuel production from lignocellulosic biomass. DNA samples obtained from the soil near a spout of a hot spring (70°C, pH7.2) were subjected to sequencing, which generated a total of 84.2 Gbp with 967,925 contigs of >500 bp in length. Similarity search for β-xylosidase in the contigs revealed the presence of 168 candidate sequences, each of which may have arisen from more than one gene. Individual genes were amplified by PCR using sequence-specific primers. The resultant DNA fragments were cloned and introduced into Escherichia coli BL21 Star(DE3). Consequently, 269 proteins were successfully expressed in the E. coli cells and then examined for β-xylosidase activity. A total of 82 proteins exhibited β-xylosidase activity at 50°C, six of which retained the activity even at 90°C. Out of the six, three proteins were originated from a single candidate sequence, AR19M-311. An amino acid sequence comparison suggested the amino acid residues that appeared to be crucial for thermal stability of the enzymes.
- Published
- 2017
234. Heterologous Expression and Characterization of an Acidic GH11 Family Xylanase from Hypocrea orientalis
- Author
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Minnan Long, Hongli Wu, Fengjiao Jiang, Yong Xue, Lihui Gan, Hailong Li, Jian Liu, and Jinlian Wu
- Subjects
0106 biological sciences ,0301 basic medicine ,Glycosylation ,Hot Temperature ,Hypocrea ,Bioengineering ,Biology ,Xylose ,01 natural sciences ,Applied Microbiology and Biotechnology ,Biochemistry ,High-performance liquid chromatography ,Substrate Specificity ,Pichia pastoris ,03 medical and health sciences ,chemistry.chemical_compound ,010608 biotechnology ,Glycoside hydrolase family 11 ,Cloning, Molecular ,Molecular Biology ,Chromatography, High Pressure Liquid ,Hydrolysis ,General Medicine ,Hydrogen-Ion Concentration ,biology.organism_classification ,Xylan ,Recombinant Proteins ,Culture Media ,Kinetics ,Xylosidases ,030104 developmental biology ,chemistry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Xylanase ,Chromatography, Thin Layer ,Heterologous expression ,Biotechnology - Abstract
A gene encoding glycoside hydrolase family 11 xylanase (HoXyn11B) from Hypocrea orientalis EU7-22 was expressed in Pichia pastoris with a high activity (413 IU/ml). HoXyn11B was partly N-glycosylated and appeared two protein bands (19-29 kDa) on SDS-PAGE. The recombinant enzyme exhibited optimal activity at pH 4.5 and 55 °C, and retained more than 90% of the original activity after incubation at 50 °C for 60 min. The determined apparent K m and V max values using beechwood xylan were 10.43 mg/ml and 3246.75 IU/mg, respectively. The modes of action of recombinant HoXyn11B on xylo-oligosaccharides (XOSs) and beechwood xylan were investigated by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), which indicated that the modes of action of HoXyn11B are different from HoXyn11A since it is able to release a significant amount of xylose from various substrates. This study provides an opportunity to better understand the hydrolysis mechanisms of xylan by xylanases from Trichoderma.
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- 2017
235. Synergistic hydrolysis of xylan using novel xylanases, β-xylosidases, and an α-l-arabinofuranosidase from Geobacillus thermodenitrificans NG80-2
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Yanfei Qi, Kexin Yang, Yingying Xu, Di Huang, Lu Feng, and Jia Liu
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0301 basic medicine ,Glycoside Hydrolases ,Lignocellulosic biomass ,Biomass ,Xylose ,Applied Microbiology and Biotechnology ,03 medical and health sciences ,Hydrolysis ,chemistry.chemical_compound ,Thermostability ,Endo-1,4-beta Xylanases ,Geobacillus ,General Medicine ,Hydrogen-Ion Concentration ,Xylosidases ,Arabinose ,Xylan ,030104 developmental biology ,chemistry ,Biochemistry ,Xylanase ,Xylans ,Biotechnology - Abstract
Lignocellulosic biomass from various types of wood has become a renewable resource for production of biofuels and biobased chemicals. Because xylan is the major component of wood hemicelluloses, highly efficient enzymes to enhance xylan hydrolysis can improve the use of lignocellulosic biomass. In this study, a xylanolytic gene cluster was identified from the crude oil-degrading thermophilic strain Geobacillus thermodenitrificans NG80-2. The enzymes involved in xylan hydrolysis, which include two xylanases (XynA1, XynA2), three β-xylosidases (XynB1, XynB2, XynB3), and one α-L-arabinofuranosidase (AbfA), have many unique features, such as high pH tolerance, high thermostability, and a broad substrate range. The three β-xylosidases were highly resistant to inhibition by product (xylose) accumulation. Moreover, the combination of xylanase, β-xylosidase, and α-L-arabinofuranosidase exhibited the largest synergistic action on xylan degradation (XynA2, XynB1, and AbfA on oat spelt or beechwood xylan; XynA2, XynB3, and AbfA on birchwood xylan). We have demonstrated that the proposed enzymatic cocktail almost completely converts complex xylan to xylose and arabinofuranose and has great potential for use in the conversion of plant biomass into biofuels and biochemicals.
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- 2017
236. Efficient Biotransformation of Astragaloside IV to Cycloastragenol by Bacillus sp. LG-502
- Author
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Liming Wang and Yan Chen
- Subjects
0301 basic medicine ,Magnetic Resonance Spectroscopy ,Sapogenins ,Bacillus ,Bioengineering ,Sapogenin ,Applied Microbiology and Biotechnology ,Biochemistry ,High-performance liquid chromatography ,Mass Spectrometry ,03 medical and health sciences ,chemistry.chemical_compound ,Biotransformation ,Cycloastragenol ,Molecular Biology ,Chromatography ,biology ,General Medicine ,Saponins ,Xylosidases ,Triterpenes ,Thin-layer chromatography ,030104 developmental biology ,chemistry ,biology.protein ,Fermentation ,Glucosidases ,Biotechnology - Abstract
Cycloastragenol (CA), an exclusive telomerase activator, was derived from the Astragali Radix which is widely distributed in Turkey. Until now, there is no report to produce CA with effective and environment-friendly methods. Biotransformation is considered to be a promising technology. Thus, the present study was aimed to establish a biotransformation technology that could efficiently produce CA. In this paper, a microorganism, LG-502, was used to successfully transform astragaloside IV (ASI) to CA by analysis of thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC). The phylogenetic analysis of the 16S rRNA indicated that this strain belongs to Bacillus sp. Three metabolites were separated during the fermentation and characterized to be cyclogaleginoside B, CA, and 20R, 24S-epoxy-6α, 16β, 25-trihydroxy-9, 19-cycloartan-3-one based on NMR and MS spectroscopic analyses. The conversion rate of ASI and yield rate of CA were achieved as high as 89 and 84%, respectively, under optimized conditions. Enzymatic analysis showed that the glycosidases were mainly located inside the bacterial body, and the activities of glucosidases were much higher than the xylosidases under the experimental conditions. This study provides a feasible, effective, and eco-friendly way to prepare CA from ASI, which might greatly contribute to the applications of ASI.
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- 2017
237. Growth and expression of relevant metabolic genes of Clostridium thermocellum cultured on lignocellulosic residues
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Cirano José Ulhoa, Brenda R. de Camargo, Betania Ferraz Quirino, Andrei Stecca Steindorff, Pedro R. V. Hamann, Marcelo Valle de Sousa, Vanessa Oliveira Leitão, Eliane Ferreira Noronha, and Carlos Roberto Felix
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0301 basic medicine ,Lignocellulosic biomass ,Bioengineering ,Cellulosomes ,Cellulase ,Lignin ,Applied Microbiology and Biotechnology ,Microbiology ,Clostridium thermocellum ,03 medical and health sciences ,Animals ,Biomass ,Food science ,biology ,Chemistry ,Goats ,food and beverages ,Gene Expression Regulation, Bacterial ,biology.organism_classification ,Xylosidases ,030104 developmental biology ,Cellulosic ethanol ,Biofuel ,Fermentation ,biology.protein ,Xylanase ,Biotechnology - Abstract
The plant cell wall is a source of fermentable sugars in second-generation bioethanol production. However, cellulosic biomass hydrolysis remains an obstacle to bioethanol production in an efficient and low-cost process. Clostridium thermocellum has been studied as a model organism able to produce enzymatic blends that efficiently degrade lignocellulosic biomass, and also as a fermentative microorganism in a consolidated process for the conversion of lignocellulose to bioethanol. In this study, a C. thermocellum strain (designated B8) isolated from goat rumen was characterized for its ability to grow on sugarcane straw and cotton waste, and to produce cellulosomes. We also evaluated C. thermocellum gene expression control in the presence of complex lignocellulosic biomasses. This isolate is capable of growing in the presence of microcrystalline cellulose, sugarcane straw and cotton waste as carbon sources, producing free enzymes and residual substrate-bound proteins (RSBP). The highest growth rate and cellulase/xylanase production were detected at pH 7.0 and 60 °C, after 48 h. Moreover, this strain showed different expression levels of transcripts encoding cellulosomal proteins and proteins with a role in fermentation and catabolic repression.
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- 2017
238. Endoglucanase and xylanase production byBacillussp. AR03 in co-culture
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Cintia Mariana Romero, Maria Alejandra Martinez, Johan S. Hero, Nora Ines Perotti, and José Horacio Pisa
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0301 basic medicine ,Cellobiose ,Bacillus ,Achromobacter ,INGENIERÍAS Y TECNOLOGÍAS ,Cellulase ,CELLULOSE DEGRADATION ,Biochemistry ,XYLANASE ,Biotecnología Industrial ,Cell wall ,Industrial Microbiology ,03 medical and health sciences ,Hydrolysis ,chemistry.chemical_compound ,medicine ,Cellulose ,biology ,BACILLUS SP. AR03 ,Chemistry ,Substrate (chemistry) ,General Medicine ,Coculture Techniques ,Carboxymethyl cellulose ,Xylosidases ,030104 developmental biology ,Carboxymethylcellulose Sodium ,BIOREFINERY ,Xylanase ,biology.protein ,CO-CULTURE ,ENDOGLUCANASE ,Biotechnology ,medicine.drug - Abstract
The behavior of three isolates retrieved from different cellulolytic consortia, Bacillus sp. AR03, Paenibacillus sp. AR247 and Achromobacter sp. AR476-2, were examined individually and as co-cultures in order to evaluate their ability to produce extracellular cellulases and xylanases. Utilizing a peptone-based medium supplemented with carboxymethyl cellulose (CMC), an increase estimation of 1.30 and 1.50 times was obtained by the co-culture containing the strains AR03 and AR247, with respect to enzyme titles registered by their individual cultivation. On the contrary, the extracellular enzymatic production decreased during the co-cultivation of strain AR03 with the non-cellulolytic Achromobacter sp. AR476-2. The synergistic behavior observed through the combined cultivation of the strains AR03 and AR247 might be a consequence of the consumption by Paenibacillus sp. AR247 of the products of the CMC hydrolysis (i.e., cellobiose and/or cello-oligosaccharides), which were mostly generated by the cellulase producer Bacillus sp. AR03. The effect observed could be driven by the requirement to fulfill the nutritional supply from both strains on the substrate evaluated. These results would contribute to a better description of the degradation of the cellulose fraction of the plant cell walls in nature, expected to an efficient utilization of renewable sources. Fil: Hero, Johan Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; Argentina Fil: Pisa, José Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; Argentina Fil: Perotti, Nora Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; Argentina. Universidad Nacional de Tucumán; Argentina Fil: Romero, Cintia Mariana. Universidad Nacional de Tucumán; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; Argentina Fil: Martinez, Maria Alejandra. Universidad Nacional de Tucumán; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Planta Piloto de Procesos Industriales Microbiologicos; Argentina
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- 2017
239. Crystal structure of metagenomic β-xylosidase/ α-l-arabinofuranosidase activated by calcium
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Katsuro Yaoi, Tomohiko Matsuzawa, Zui Fujimoto, Naomi Kishine, and Satoshi Kaneko
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Models, Molecular ,0301 basic medicine ,Glycoside Hydrolases ,Sodium ,Metal ions in aqueous solution ,030106 microbiology ,chemistry.chemical_element ,Protonation ,Calcium ,Crystallography, X-Ray ,Biochemistry ,Catalysis ,Ion ,03 medical and health sciences ,Molecule ,Enzyme kinetics ,Molecular Biology ,Binding Sites ,General Medicine ,Crystallography ,Xylosidases ,030104 developmental biology ,chemistry ,Biocatalysis - Abstract
The crystal structure of metagenomic β-xylosidase/α-l-arabinofuranosidase CoXyl43, activated by calcium ions, was determined in its apo and complexed forms with xylotriose or l-arabinose in the presence and absence of calcium. The presence of calcium ions dramatically increases the kcat of CoXyl43 for p-nitrophenyl β-d-xylopyranoside and reduces the Michaelis constant for p-nitrophenyl α-l-arabinofuranoside. CoXyl43 consists of a single catalytic domain comprised of a five-bladed β-propeller. In the presence of calcium, a single calcium ion was observed at the centre of this catalytic domain, behind the catalytic pocket. In the absence of calcium, the calcium ion was replaced with one sodium ion and one water molecule, and the positions of these cations were shifted by 1.3 Å. The histidine-319 side chain, which coordinates to the 2-hydroxyl oxygen atom of the bound xylose molecule in the catalytic pocket, also coordinates to the calcium ion, but not to the sodium ion. The calcium-dependent increase in activity appears to be caused by the structural change in the catalytic pocket induced by the tightly bound calcium ion and coordinating water molecules, and by the protonation state of glutamic acid-268, the catalytic acid of the enzyme. Our findings further elucidate the complex relationship between metal ions and glycosidases.
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- 2017
240. Trichoderma reesei xylanase 5 is defective in the reference strain QM6a but functional alleles are present in other wild-type strains
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Martina Marchetti-Deschmann, Verena Seidl-Seiboth, Bernhard Seiboth, and Jonas Ramoni
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0301 basic medicine ,Genotype ,In silico ,Trichoderma reesei ,030106 microbiology ,Molecular Conformation ,Biology ,Applied Microbiology and Biotechnology ,Microbiology ,03 medical and health sciences ,Cellulase ,Gene Expression Regulation, Fungal ,XYN1 ,Glycoside hydrolase ,Biotechnologically Relevant Enzymes and Proteins ,Alleles ,chemistry.chemical_classification ,Trichoderma ,Endo-1,4-beta Xylanases ,Strain (chemistry) ,Xylanase ,GH11 family ,Gene Expression Profiling ,Wild type ,General Medicine ,Sequence Analysis, DNA ,Hydrogen-Ion Concentration ,biology.organism_classification ,Recombinant protein production ,Biorefinery ,Kinetics ,Enzyme ,Xylosidases ,Biochemistry ,chemistry ,Biofuels ,Biotechnology - Abstract
Trichoderma reesei is a paradigm for the regulation and industrial production of plant cell wall-degrading enzymes. Among these, five xylanases, including the glycoside hydrolase (GH) family 11 XYN1 and XYN2, the GH10 XYN3, and the GH30 XYN4 and XYN6, were described. By genome mining and transcriptome analysis, a further putative xylanase, encoded by xyn5, was identified. Analysis of xyn5 from the genome-sequenced reference strain T. reesei QM6a shows that it encodes a non-functional, truncated form of XYN5. However, non-truncated orthologues are present in other genome sequenced Trichoderma spp., and sequencing of xyn5 in other T. reesei wild-type isolates shows that they harbor a putative functional xyn5 allele. In silico analysis and 3D modeling revealed that the encoded XYN5 has significant structural similarities to xylanases of the GH11 family, including a GH-typical substrate binding groove and a carboxylate pair in the active site. The xyn5 of wild-type strain TUCIM1282 was recombinantly expressed in a T. reesei strain with a (hemi)cellulase-free background and the corresponding protein purified to apparent homogeneity. The pH and temperature optima and the kinetic parameters of the purified XYN5 were pH 4, 50 °C, and V max = 2646 nkat/mg with a K m of 9.68 mg/ml. This functional xyn5 allele was used to replace the mutated version which led to an overall increase of the xylanolytic activity. These findings are of particular importance as GH11 xylanases are of high biotechnological relevance, and T. reesei is one of the main industrial producers of such lignocellulose-degrading enzymes. Electronic supplementary material The online version of this article (doi:10.1007/s00253-017-8161-4) contains supplementary material, which is available to authorized users.
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- 2017
241. Use of lignocellulose biomass for endoxylanase production by Streptomyces termitum
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Alenir Naves de Sales, Disney Ribeiro Dias, Rosane Freitas Schwan, Viridiana Santana Ferreira-Leitão, Angélica Cristina de Souza, and Rondinele de Oliveira Moutta
- Subjects
0106 biological sciences ,0301 basic medicine ,Biomass ,Lignocellulosic biomass ,Cellulase ,Lignin ,01 natural sciences ,Biochemistry ,Streptomyces ,Husk ,Industrial Microbiology ,03 medical and health sciences ,010608 biotechnology ,Streptomyces termitum ,Botany ,Yeast extract ,Food science ,Cellulose ,Endo-1,4-beta Xylanases ,biology ,Chemistry ,food and beverages ,General Medicine ,biology.organism_classification ,Saccharum ,Actinobacteria ,Xylosidases ,030104 developmental biology ,biology.protein ,Xylans ,Bagasse ,Biotechnology - Abstract
Actinobacteria isolates from Brazilian Cerrado soil were evaluated for their ability to produce enzymes of the cellulolytic and xylanolytic complex using lignocellulose residual biomass. Preliminary semiquantitative tests, made in Petri plates containing carboxymethylcellulose and beechwood xylan, indicated 11 potential species producing enzymes, all belonging to the genus Streptomyces. The species were subsequently grown in pure substrates in submerged fermentation and analyzed for the production of enzymes endoglucanase, β-glucosidase, endoxylanase, and β-xylosidase. The best results were obtained for endoxylanase enzyme production with Streptomyces termitum(UFLA CES 93). The strain was grown on lignocellulose biomass (bagasse, straw sugarcane, and cocoa pod husk) that was used in natura or acid pretreated. The medium containing sugarcane bagasse in natura favored the production of the endoxylanase that was subsequently optimized through an experimental model. The highest enzyme production 0.387 U mL-1, (25.8 times higher), compared to the lowest value obtained in one of the trials, was observed when combining 2.75% sugar cane bagasse and 1.0 g L-1 of yeast extract to the alkaline medium (pH 9.7). This is the first study using S. termitum as a producer of endoxylanase.
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- 2017
242. Engineering disulfide bonds in Selenomonas ruminantium β-xylosidase by experimental and computational methods
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Khosro Khajeh, Sako Mirzaie, Ehsan Dehnavi, Mehrnoosh Fathi-Roudsari, Seyed Omid Ranaei Siadat, and Seyed Shahriar Arab
- Subjects
0301 basic medicine ,Stereochemistry ,Protein subunit ,Molecular Dynamics Simulation ,Biochemistry ,Substrate Specificity ,03 medical and health sciences ,Residue (chemistry) ,Structural Biology ,Enzyme Stability ,Disulfides ,Selenomonas ruminantium ,Molecular Biology ,Thermostability ,chemistry.chemical_classification ,030102 biochemistry & molecular biology ,Chemistry ,Hydrolysis ,Point mutation ,Mutagenesis ,Temperature ,General Medicine ,Protein Structure, Tertiary ,Kinetics ,Xylosidases ,030104 developmental biology ,Enzyme ,Mutation ,Mutagenesis, Site-Directed ,Selenomonas ,Cysteine - Abstract
Homotetrameric β-xylosidase from Selenomonas ruminantium (SXA) is one of the most efficient enzymes known for the hydrolysis of cell wall hemicellulose. SXA shows a rapid rate of activity loss at temperatures above 50°C. In this study, we have introduced two inter-subunit disulfide bridges with one point mutation. Lys237 was chosen to be replaced with cysteine since it interacts with the same residue in the opposite subunit. While pH optimum, temperature profile and catalytic efficiency of the mutated variant were similar to the native enzyme, the mutated enzyme showed about 40% increase in thermal stability at 55°C. Our results showed that introduction of a single residue mutation in structure of SXA results in appearance of two disulfide bonds at dimer-dimer interface of the enzyme. Coarse-grained molecular dynamics (CG-MD) simulations also proved lower amounts of root mean square fluctuation (RMSF) for position 237 and potential energy for mutated SXA. Based these results, we suggest that choosing a correct residue for mutation in multi subunit proteins results in multiple site conversions which equals to several simultaneous mutations. Furthermore, CG-MD simulation in agreement with experimental methods showed higher thermostability of mutated SXA which proved applicability of this simulation for thermostability analysis.
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- 2017
243. Ethanol production by Escherichia coli from Arundo donax biomass under SSF, SHF or CBP process configurations and in situ production of a multifunctional glucanase and xylanase
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Sima Schein, Inés Loaces, and Francisco Noya
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Environmental Engineering ,020209 energy ,Biomass ,Bioengineering ,02 engineering and technology ,Cellulase ,Poaceae ,Hydrolysis ,Botany ,Escherichia coli ,0202 electrical engineering, electronic engineering, information engineering ,Cellulases ,Ethanol fuel ,Food science ,Waste Management and Disposal ,Ethanol ,biology ,Renewable Energy, Sustainability and the Environment ,Chemistry ,food and beverages ,Arundo donax ,General Medicine ,biology.organism_classification ,Xylosidases ,Biofuel ,Fermentation ,Xylanase ,biology.protein - Abstract
Diluted acid or liquid hot water (LHW) pretreated Arundo donax biomass was converted into ethanol under separated hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF) using Escherichia coli as the fermentative organism. Up to 0.26gL-1h-1 and 25.0gL-1 of ethanol were obtained with diluted acid pretreated biomass under SSF compared to 0.17gL-1h-1 and 24gL-1 under SHF. LHW pretreated biomass elicited 25% lower yields on average. Saccharification was carried out with Cellic CTec2 cocktail. Alternatively, under a consolidated bioprocess (CBP) where the ethanologenic bacteria was complemented with a novel multifunctional glucanase and xylanase, ethanol concentration was 7.6gL-1 and 7.2gL-1 after 96h for dilute acid or LHW pretreated biomass, respectively, without any prior saccharification step. According to these results, a bacterial fermentative host combined with in situ enzyme expression can improve ethanol production from A. donax biomass.
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- 2017
244. Crystallization and preliminary crystallographic analysis of a family 43 β- d-xylosidase from Geobacillus stearothermophilus T-6.
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Brüx, Christian, Niefind, Karsten, Ben-David, Alon, Leon, Maya, Shoham, Gil, Shoham, Yuval, and Schomburg, Dietmar
- Subjects
- *
CRYSTALLOGRAPHY , *CRYSTALLIZATION , *CELLULASE , *BACILLUS (Bacteria) , *THERMOPHILIC bacteria , *MOLECULAR weights - Abstract
β- d-Xylosidases (EC 3.2.1.37) are hemicellulases that cleave single xylose units from the nonreducing end of xylooligomers. In this study, the crystallization and preliminary X-ray analysis of a β- d-xylosidase from Geobacillus stearothermophilus T-6 (XynB3), a family 43 glycoside hydrolase, is described. XynB3 is a 535-amino-acid protein with a calculated molecular weight of 61 891 Da. Purified recombinant native and catalytic inactive mutant proteins were crystallized and cocrystallized with xylobiose in two different space groups, P21212 (unit-cell parameters a = 98.32, b = 99.36, c = 258.64 Å) and P41212 (or the enantiomorphic space group P43212; unit-cell parameters a = b = 140.15, c = 233.11 Å), depending on the detergent. Transferring crystals to cryoconditions required a very careful protocol. Orthorhombic crystals diffract to 2.5 Å and tetragonal crystals to 2.2 Å. [ABSTRACT FROM AUTHOR]
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- 2005
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245. Xylan-degrading enzymes and arabinoxylan solubilisation in barley cultivars of differing malting quality
- Author
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Sungurtas, J., Swanston, J.S., Davies, H.V., and McDougall, G.J.
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XYLANS , *ENZYMES , *ARABINOGALACTAN , *GALACTOSE - Abstract
The levels of xylanolytic activities ((1→4)-β-endoxylanases, β-d-xylosidases and α-l-arabinofuranosidases) and the levels of water extractable arabinoxylans (WEAX) in hot water extracts of malts were compared during malting of four barley cultivars of differing quality. Endoxylanase activity increased from day 1 after steeping showing a slight lag compared with the development of (1→3,1→4)-β-glucanase activity. The levels of endoxylanase activity were different between the cultivars. For example, at day 4, which would be a suitable endpoint for malting of good quality cultivars, total endoxylanase activities of Static and Chariot malts were twice that of Hart and Optic. This variation in level of endoxylanase between cultivars did not correlate with the content of water extractable arabinoxylans in hot water extracts of the malts. However, those cultivars that accumulated most water extractable arabinoxylan had a different set of endoxylanase isoforms. This suggests that expression of specific endoxylanase isoforms may influence the extent of arabinoxylan solubilisation and/or degradation.The β-xylosidases and α-arabinosidases increased from low levels after steep to a plateau of activity at day 6 or 7 of germination. Total xylosidase was always higher than the arabinosidase activity. There were differences between cultivars but these were small compared with the variation in endoxylanase activity. (1→4)-β-d-Endomannanase activity was detected in all four cultivars at 2 days germination then rose to a maximum around 5 days. [Copyright &y& Elsevier]
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- 2004
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246. Characterization of a neutral recombinant xylanase from Thermoactinospora rubra YIM 77501T
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Feng Zhang, Xiao-Yang Zhi, Min Xiao, Qing-Wen Hu, Yi-Rui Yin, Hong Ming, En-Min Zhou, Wen-Jun Li, and Wen-Dong Xian
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DNA, Bacterial ,0301 basic medicine ,Glycoside Hydrolases ,Biology ,medicine.disease_cause ,Microbiology ,law.invention ,03 medical and health sciences ,Hydrolysis ,law ,Enzyme Stability ,Glycoside hydrolase family 10 ,Escherichia coli ,medicine ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,Endo-1,4-beta Xylanases ,Sequence Homology, Amino Acid ,General Medicine ,Recombinant Proteins ,Protein tertiary structure ,Actinobacteria ,Enzyme Activation ,Xylosidases ,030104 developmental biology ,Enzyme ,Biochemistry ,chemistry ,Xylanase ,Recombinant DNA ,Xylans - Abstract
A xylanase gene (TrXyn10) from Thermoactinospora rubra YIM 77501T was cloned and expressed in Escherichia coli. The amino acid sequence displayed 78% homology with Microbispora mesophila xylanase (WP_062413927.1). The recombinant xylanase (TrXyn10), with MW 46.1 kDa, could hydrolyse beechwood, birchwood and oatspelt xylan. Based on the sequence, enzymatic properties and tertiary structure of the protein, TrXyn10 belongs to glycoside hydrolase family 10 (GH10). The optimal pH and temperature for the recombinant enzyme were determined to be 7.0 and 55 °C, respectively. TrXyn10 was stable over a wide pH range, and it retained more than 45% of the total activity at pH 6.0–12.0 for 12 h. In addition, the activity was greatly promoted, by approximately 200% of the initial activity, after incubation at pH 6.0 and 7.0 for 12 h. Based on enzymatic properties and product analysis, we showed that TrXyn10 is a neutral endoxylanase.
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- 2016
247. Mechanistic Study of β-Xylosidase from Trichoderma koningii G-391.
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Li, Yaw-Kuen, Yao, Hsin-Jan, and Pan, I-Hong
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XYLOSIDASES ,HYDROLYSIS ,ENZYMES ,TRICHODERMA ,HYDROGEN isotopes - Abstract
The catalytic mechanism of the β-xylosidase purified from the culture filtrate of Trichoderma koningii G-39 was investigated. By NMR spectroscopy, the stereochemistry of the enzyme catalyzing the hydrolysis of 2,4-dinitrophenyl and p-nitrophenyl-β-D-xylosides was found unequivocally to involve retention of the anomeric configuration. Based on the kCRt values of a series of arylxylosides with leaving group p.Kas in the range of 4-10, an extended Bronsted plot was constructed with a slope (β1g) near zero. Enzymatic hydrolysis of aryl-β-D-xylosides in acetate buffer (pH 4.0) containing 3 or 5% methanol showed a constant product ratio (methylxyloside/xylose), indicating the presence of a common intermediate, probably the xylosyl-enzyme intermediate. In the presence of DTT, the kcat values of p-cyanophenyl-β-D-xylopyranoside and p-nitrophenyl-β-D-xylopyra-noside increased greatly. A two-step mechanism involving the formation and breakdown of the xylosyl-enzyme intermediate was therefore proposed. The rate-limiting step is the breakdown of the intermediate. The secondary deuterium kinetic isotope effect (kHkD) measured for 2,4-dinitrophenyl-β-D-xyloside was 1.02±0.01, suggesting that the transition state for breakdown of the xylosyl-enzyme intermediate is SN2-like [ABSTRACT FROM AUTHOR]
- Published
- 2000
248. A novel multifunctional GH9 enzyme from Paenibacillus curdlanolyticus B-6 exhibiting endo/exo functions of cellulase, mannanase and xylanase activities
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Vanarat Phakeenuya, Khanok Ratanakhanokchai, Akihiko Kosugi, and Chakrit Tachaapaikoon
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Glycoside Hydrolases ,Oligosaccharides ,Cellulase ,Cellobiose ,Applied Microbiology and Biotechnology ,Substrate Specificity ,03 medical and health sciences ,chemistry.chemical_compound ,Hydrolysis ,Bacterial Proteins ,Polysaccharides ,Catalytic Domain ,Mannosidases ,Escherichia coli ,Glycoside hydrolase ,Enzyme kinetics ,Cellulose ,030304 developmental biology ,0303 health sciences ,biology ,030306 microbiology ,food and beverages ,General Medicine ,Xylan ,Multifunctional Enzymes ,Recombinant Proteins ,Molecular Docking Simulation ,Kinetics ,Xylosidases ,chemistry ,Biochemistry ,Mutation ,biology.protein ,Xylanase ,Paenibacillus ,Biotechnology - Abstract
PcMulGH9, a novel glycoside hydrolase family 9 (GH9) from Paenibacillus curdlanolyticus B-6, was successfully expressed in Escherichia coli. It is composed of a catalytic domain of GH9, two domains of carbohydrate-binding module family 3 (CBM3) and two domains of fibronectin type 3 (Fn3). The PcMulGH9 enzyme showed broad activity towards the β-1,4 glycosidic linkages of cellulose, mannan and xylan, including cellulose and xylan contained in lignocellulosic biomass, which is rarely found in GH9. The enzyme hydrolysed substrates with bifunctional endo-/exotypes cellulase, mannanase and xylanase activities, but predominantly exhibited exo-activities. This enzyme released cellobiose as a major product from cellohexaose, while mannotriose and xylotriose were major hydrolysis products from mannohexaose and xylohexaose, respectively. Moreover, PcMulGH9 could hydrolyse untreated corn hull and rice straw into xylo- and cello-oligosaccharides. Enzyme kinetics, site-directed mutagenesis and molecular docking revealed that Met394, located at the binding subsite + 2, was involved in broad substrate specificity of PcMulGH9 enzyme. This study offers new knowledge of the multifunctional cellulase/mannanase/xylanase in GH9. The PcMulGH9 enzyme showed a novel function of GH9, which increases its potential for saccharification of lignocellulosic biomass into value-added products, especially oligosaccharides.
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- 2019
249. Isolation methods of high glycosidase-producing mutants of
- Author
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Kazuya, Tomimoto, Yukio, Osafune, Dararat, Kakizono, Jinshun, Han, and Nobuhiko, Mukai
- Subjects
Ethanol ,beta-Glucosidase ,Genes, Fungal ,Cathepsin A ,Oryza ,alpha-Glucosidases ,Cell Separation ,Fructose ,Saccharomyces cerevisiae ,Deoxyglucose ,Citric Acid ,Aspergillus ,Glucose ,Phenotype ,Xylosidases ,Codon, Nonsense ,Drug Resistance, Fungal ,Fermentation ,Glucokinase ,Fermented Foods ,Ipomoea batatas ,alpha-Amylases - Abstract
High glycosidase-producing strains of
- Published
- 2019
250. Aroma enhancement of instant green tea infusion using β-glucosidase and β-xylosidase
- Author
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Hui Ni, Qing Biao Li, Ting Li, Ting Zhang, Ke Fang, Feng Chen, and Li Jun Li
- Subjects
Nonanal ,01 natural sciences ,Gas Chromatography-Mass Spectrometry ,Analytical Chemistry ,chemistry.chemical_compound ,0404 agricultural biotechnology ,Food science ,Aroma ,biology ,Tea ,beta-Glucosidase ,010401 analytical chemistry ,food and beverages ,04 agricultural and veterinary sciences ,General Medicine ,Decanal ,Green tea ,biology.organism_classification ,040401 food science ,0104 chemical sciences ,Xylosidases ,chemistry ,Odor ,Odorants ,Geraniol ,Methyl salicylate ,Food Science ,Instant - Abstract
β-Glucosidase and β-xylosidase were investigated for their ability to improve the aroma of instant green tea. The aroma and corresponding contributors were analyzed by sensory evaluation, gas chromatography-mass spectrometry, and odor activity value. Their specific contributions to aroma attributes were further examined by aroma reconstruction and omission experiments. The β-glucosidase treatment significantly enhanced floral and grassy notes, on account of the increases of geraniol, nonanal, and cis-3-hexen-1-ol, and weakened the caramel note, attributable to the increases of nonanal, cis-3-hexen-1-ol, geraniol, methyl salicylate, and decanal. The co-treatment with β-glucosidase and β-xylosidase further enhanced the grassy note, with further increase in nonanal and cis-3-hexen-1-ol, and further weakened the caramel note, with additional increase in nonanal, cis-3-hexen-1-ol, methyl salicylate, and decanal. The synergistic action of β-glucosidase and β-xylosidase provides new clues to the production of instant green tea infusions with high aroma quality.
- Published
- 2019
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