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Stephen C. Fry - One of the best experts on this subject based on the ideXlab platform.
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trans α xylosidase a widespread enzyme activity in plants introduces 1 4 α d Xylobiose side chains into xyloglucan structures
Phytochemistry, 2012Co-Authors: Lenka Franková, Stephen C. FryAbstract: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.
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Trans-α-xylosidase, a widespread enzyme activity in plants, introduces (1→4)-α-d-Xylobiose side-chains into xyloglucan structures.
Phytochemistry, 2012Co-Authors: Lenka Franková, Stephen C. FryAbstract: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.
Lenka Franková - One of the best experts on this subject based on the ideXlab platform.
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trans α xylosidase a widespread enzyme activity in plants introduces 1 4 α d Xylobiose side chains into xyloglucan structures
Phytochemistry, 2012Co-Authors: Lenka Franková, Stephen C. FryAbstract: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.
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Trans-α-xylosidase, a widespread enzyme activity in plants, introduces (1→4)-α-d-Xylobiose side-chains into xyloglucan structures.
Phytochemistry, 2012Co-Authors: Lenka Franková, Stephen C. FryAbstract: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.
Peter Biely - One of the best experts on this subject based on the ideXlab platform.
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A novel GH30 xylobiohydrolase from Acremonium alcalophilum releasing Xylobiose from the non-reducing end.
Enzyme and microbial technology, 2019Co-Authors: Katarína Šuchová, Vladimír Puchart, Nikolaj Spodsberg, Kristian B. R. M. Krogh, Peter BielyAbstract:Abstract Xylanases of the GH30 family are grouped to subfamilies GH30-7 and GH30-8. The GH30-8 members are of bacterial origin and well characterized, while the GH30-7 members are from fungal sources and their properties are quite diverse. Here, a heterologous expression and characterization of the GH30-7 xylanase AaXyn30A from a cellulolytic fungus Acremonium alcalophilum is reported. From various polymeric and oligomeric substrates AaXyn30A generates Xylobiose as the main product. It was proven that Xylobiose is released from the non-reducing end of all tested substrates, thus the enzyme behaves as a typical non-reducing-end acting xylobiohydrolase. AaXyn30A is active on different types of xylan, exhibiting the highest activity on rhodymenan (linear β-1,3-β-1,4-xylan) from which also an isomeric xylotriose Xyl-β-1,3-Xyl-β-1,4-Xyl is formed. Production of Xylobiose from glucuronoxylan is at later stage accompanied by a release of aldouronic acids differing from those liberated by the bacterial GH30-8 glucuronoxylanases.
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a simple enzymatic synthesis of 4 nitrophenyl β 1 4 d xylobioside a chromogenic substrate for assay and differentiation of endoxylanases
Journal of Biotechnology, 2007Co-Authors: Vladimír Puchart, Peter BielyAbstract:Abstract A simple procedure has been elaborated for preparation of 4-nitrophenyl β- d -xylopyranosyl-1,4-β- d -xylopyranoside (NPX 2 ), a chromogenic substrate of some endo-β-1,4-xylanases. The procedure is based on a self-transfer reaction from 4-nitrophenyl β- d -xylopyranoside catalyzed by an Aureobasidium pullulans and Aspergillus niger β-xylosidases. Both enzymes catalyzed only the formation of 4-nitrophenyl glycosides of β-1,4-Xylobiose with a small admixture of 4-nitrophenyl glycoside of β-1,3-Xylobiose. The highest yields of the NPX 2 (19.4%) was obtained at pH 5.5. The removal of the β-1,3-isomer from NPX 2 is not necessary for quantification of endo-β-1,4-xylanase activity since it is not attacked by endo-β-1,4-xylanases. In contrast to GH family 5 xylanase from Erwinia chrysanthemi , which did not attack NPX 2 , all family 10 and 11 xylanases cleaved the chromogenic substrate exclusively between Xylobiose and the aromatic aglycone. Significant differences in the K m values of GH10 and GH11 xylanases suggested that activities of these enzymes could be selectively quantified in the mixtures using various concentrations of NPX 2 . Moreover, NPX 2 could serve as an ideal substrate to follow the interaction of endo-β-1,4-xylanases with various xylanase inhibitors.
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Stereochemistry of the hydrolysis of glycosidic linkage by endo‐β‐1,4‐xylanases of Trichoderma reesei
FEBS letters, 1994Co-Authors: Peter Biely, Lubomír Kremnický, Juraj Alföldi, Maija TenkanenAbstract:Abstract Methyl β- d -xylotrioside was used as a non-reducing substrate to investigate the stereochemistry of hydrolysis of, β-1,4-xylopyranosidic linkage by purified endo-β-1,4-xylanases (EC 3.2.1.8) of Trichoderma reesei , employing 1 H NMR spectroscopy. The fungus produces one acidic species (pI 4.8–5.5), designated as EXI, and one alkaline species (pI 8.5–9.0), designated as EXII. Both enzymes were found to cleave the xylotrioside predominantly to methyl β- d -xyloside and Xylobiose. Monitoring of the intensity of the H-1 signals of α- and β-Xylobiose during the time course of hydrolysis clearly showed that both enzymes liberate the β-anomer of Xylobiose, i.e. a product with anomeric configuration identical with that of the cleaved glycosidic linkage. This means that both EXI and EXII belong to the so-called retaining glycanases that utilize the double displacement reaction mechanism of hydrolysis.
Zhengqiang Jiang - One of the best experts on this subject based on the ideXlab platform.
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Purification and Properties of a Psychrotrophic Trichoderma sp. Xylanase and its Gene Sequence
Applied biochemistry and biotechnology, 2011Co-Authors: Peng Zhou, Huifang Zhu, Qiaojuan Yan, Priti Katrolia, Zhengqiang JiangAbstract:A psychrotrophic fungus identified as Trichoderma sp. SC9 produced 36.7 U/ml of xylanase when grown on a medium containing corncob xylan at 20 °C for 6 days. The xylanase was purified 37-fold with a recovery yield of 8.2%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 20.5 kDa. The enzyme had an optimal pH of 6.0, and was stable over pH 3.5-9.0. The optimal temperature of the xylanase was 42.5 °C and it was stable up to 35 °C at pH 6.0 for 30 min. The xylanase was thermolabile with a half-life of 23.9 min at 45 °C. The apparent K(m) values of the xylanase for birchwood, beechwood, and oat-spelt xylans were found to be 3, 2.1, and 16 mg/ml respectively. The xylanase hydrolyzed beechwood xylan and birchwood xylan to yield mainly Xylobiose as end products. The enzyme-hydrolysed xylotriose, xylotetraose, and xylopentose to produce Xylobiose, but it hardly hydrolysed Xylobiose. A xylanase gene (xynA) with an open reading frame of 669 nucleotide base pairs (bp), encoding 222 amino acids, from the strain was cloned and sequenced. The deduced amino acid sequence of XynA showed 85% homology with Xyn2 from a mesophilic strain of Trichoderma viride.
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Properties of a xylanase from Streptomyces matensis being suitable for xylooligosaccharides production
Journal of Molecular Catalysis B-enzymatic, 2008Co-Authors: Qiaojuan Yan, Zhengqiang Jiang, Shanshan Hao, Qian Zhai, Weiwei ChenAbstract:Abstract An extracellular xylanase produced by Streptomyces matensis DW67 was purified from the culture supernatant by ammonium sulfate precipitation, ion exchange and gel filtration chromatography and characterized. The xylanase was purified to 14.5-fold to homogeneity with a recovery yield of 14.1%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of 21.2 kDa. However, it had a very low apparent molecular mass of 3.3 kDa as determined by gel filtration chromatography. The N-terminal sequence of first 15 amino acid residues was determined as ATTITTNQTGYDGMY. The optimal temperature and pH for purified xylanase was 65 °C and pH 7.0, respectively. The enzyme was stable within the pH range of 4.5–8.0 and was up to 55 °C. The xylanase showed specific activity towards different xylans and no activity towards other substrates tested. Hydrolysis of birchwood xylan by the xylanase yielded Xylobiose and xylotriose as principal products. The enzyme hardly hydrolyzed Xylobiose and xylotriose, but it could hydrolyze xylotetraose and xylopentaose to produce mainly Xylobiose and xylotriose through transglycosylation. These unique properties of the purified xylanase make this enzyme attractive for biotechnological applications, such as bioblenching in paper and pulp industries, production of xylooligosaccharides. This is the first report of the xylanase from S. matensis.
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the recombinant xylanase b of thermotoga maritima is highly xylan specific and produces exclusively Xylobiose from xylans a unique character for industrial applications
Journal of Molecular Catalysis B-enzymatic, 2004Co-Authors: Zhengqiang Jiang, W Deng, Yunping Zhu, Y J Sheng, Kiyoshi HayashiAbstract:Abstract The xynB of a hyperthermophilic Eubacterium, Thermotoga maritima MSB8, coding xylanase B (XynB) was previously expressed in E. coli and the recombinant protein was characterized using the synthetic substrates [J. Biosci. Bioeng. 92 (2001) 423]. In this study, the same xylanase B was purified to homogeneity with a recovery yield of about 43% using heat treatment followed by the Ni-NTA affinity chromatography. The specificity of XynB towards different natural substrates was evaluated. XynB was highly specific towards xylans tested but exhibited low activities towards lichenan (19%), gellan gum (7.3%), laminarin (3.4%) and carboxymethylcellulose (CMC, 1.4%). The apparent Km values of birchwood xylan and soluble oat-spelt xylan was 0.11 and 0.079 mg/ml, respectively. The XynB hydrolyzed xylooligosaccharides to yield predominantly Xylobiose (X2) and a small amount of xylose (X1), suggesting that XynB was possibly an endo-acting xylanase. Analysis of the products from birchwood xylan degradation confirmed that the enzyme was an endo-xylanase with Xylobiose and xylose as the main degradation products. HPLC results showed that hydrolyzed products of birchwood xylan by XynB yielded up to 66% of the total reaction product as Xylobiose. These results clearly indicated that Xylobiose could be mass-produced efficiently by the recombinant hyperthermostable XynB of T. maritima. Additionally, conversion of Xylobiose (50 mM) to xylose was observed, while xylotriose (X3) and xylotetraose (X4) were detected in small amounts, indicating that the enzyme converted Xylobiose to xylose based on the transglycosylation reaction. The increased binding ability of XynB to Avicel and/or insoluble xylan was also observed indicating the possibilities of roles of surface-aromatic amino acid residues for such action. However, further investigations are required to prove this speculation.
James P. Nakas - One of the best experts on this subject based on the ideXlab platform.
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Purification and characterization of two xylanases from Trichoderma longibrachiatum.
European journal of biochemistry, 1991Co-Authors: J. C. Royer, James P. NakasAbstract:Two endoxylanases were purified from the culture medium of Trichoderma longibrachiatum. Both enzymes were highly basic, and lacked activity on carboxymethyl-cellulose. An enzyme of 21.5 kDa (xylanase A) had a specific activity of 510 U/mg protein, a Km of 0.15 mg soluble xylan/ml, possessed transglycosidase activity and generated Xylobiose and xylotriose as the major endproducts from xylan or xylose oligomers. A larger enzyme of 33 kDa (xylanase B) had a specific activity of 131 U/mg protein, a Km of 0.19 mg soluble xylan/ml, lacked detectable transglycosidase activity and generated Xylobiose and xylose as major endproducts from xylan and xylose oligomers. Xylotriose was the smallest oligomer attacked by both enzymes. In addition, xylotriose inhibited hydrolysis of xylopentanose by both enzymes, while Xylobiose appeared to inhibit xylanase B, but not xylanase A.
