| 1. |
- Bennett, Neil A., et al.
(författare)
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Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Thermomyces lanuginosus ATCC 46882
- 1998
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Ingår i: Carbohydrate Research. - 0008-6215 .- 1873-426X. ; 306:3, s. 445-455
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Tidskriftsartikel (refereegranskat)abstract
- An endoxylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) from the culture filtrates of T. lanuginosus ATCC 46882 was purified to homogeneity by DEAE-Sepharose and Bio-Gel P-30 column chromatographies. The purified endoxylanase had a specific activity of 888.8 mumol min-1 mg-1 protein and accounted for approximately 30% of the total protein secreted by this fungus. The molecular mass of native (non-denatured) and denatured endoxylanase were 26.3 and 25.7 kD as, respectively. Endoxylanase had a pI of 3.7 and was optimally active between pH 6.0-6.5 and at 75 degrees C. The enzyme showed > 50% of its original activity between pH 5.5-9.0 and at 85 degrees C. The pH and temperature stability studies revealed that this endoxylanase was almost completely stable between pH 5.0-9.0 and up to 60 degrees C for 5 h and at pH 10.0 up to 55 degrees C for 5 h. Thin-layer chromatography (TLC) analysis showed that endoxylanase released mainly xylose (Xyl) and xylobiose (Xyl2) from beechwood 4-O-methyl-D-glucuronoxylan, O-acetyl-4-O-methyl-D-glucuronoxylan and rhodymenan (a beta-(1-->3)-beta(1-->4)-xylan). Also, the enzyme released an acidic xylo-oligosaccharide from 4-O-methyl-D-glucuronoxylan, and an isomeric xylotetraose and an isomeric xylopentaose from rhodymenan. The enzyme hydrolysed [1-3H]-xylo-oligosaccharides in an endofashion, but the hydrolysis of [1-3H]-xylotriose appeared to proceed via transglycosylation. since the xylobiose was the predominant product. Endoxylanase was not active on pNPX and pNPC at 40 and 100 mM for up to 6 h, but showed some activity toward pNPX at 100 mM after 20-24 h. The results suggested that the endoxylanase from T. lanuginosus belongs to family 11.
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| 2. |
- Christakopoulos, Paul, et al.
(författare)
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Purification and characterisation of a major xylanase with cellulase and transferase activities from Fusarium oxysporum
- 1996
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Ingår i: Carbohydrate Research. - : Elsevier BV. - 0008-6215 .- 1873-426X. ; 289, s. 91-104
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Tidskriftsartikel (refereegranskat)abstract
- A major xylanase from Fusarium oxysporum was purified to homogeneity by gel filtration, affinity, and ion-exchange chromatographies. It has a molecular mass of 60.2 kDa and pl of 6.6 and was optimally active at pH 7.4 and at 50 °C. The enzyme was stable over the pH range 5.8–8.2 at 40 °C for 24 h and lost 45% of its original activity at pH 9.0 under the identical conditions. The enzyme rapidly hydrolysed xylans from oat spelts (husks) and birchwood, but the activities on carboxymethylcellulose (CMC), filter paper, and Avicel were very low. Determination of kcat/Km revealed that the enzyme hydrolysed oat spelts and birchwood xylans, 15–30 times more efficiently than CMC. In a 24 h incubation, at pH 7.0 and 9.0, the enzyme hydrolysed oat spelts and birchwood xylans by 75 and 65%, respectively. However, at pH 7.0, the enzyme released almost equal amounts of xylose and xylobiose from both xylans, whereas at pH 9.0, the concentration of xylobiose was twice as muchi as that of xylose and xylotriose. Xylanase attacked preferentially the internal glycosidic bonds of xylo- and 4-methylumbelliferyl cello-oligosaccharides [MeUmb(Glc)n]. The enzyme catalysed transglycosylation reaction with xylotriose, xylotetraose, and xylopentaose as donors and 4-methylumbelliferyl β-d-glucoside (MeUmbGlc) as an acceptor.
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| 3. |
- Christakopoulos, Paul, et al.
(författare)
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Purification and Characterisation of an Extracellular β-Glucosidase with Transglycosylation and Exo-glucosidase Activities from Fusarium oxysporum
- 1994
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 224:2, s. 379-385
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Tidskriftsartikel (refereegranskat)abstract
- An extracellular β-glucosidase from Fusarium oxysporum was purified to homogeneity by gel-filtration and ion-exchange chromatographies. The enzyme, a monomeric protein of 110 kDa, was maximally active at pH 5.0–6.0 and at 60°C. It hydrolysed 1→4-linked aryl-β-glucosides and 1→4-linked, 1→3-linked and 1→6–linked β-glucosides. The apparent Km and kcat values for p -nitrophenyl β-d-glucopyranoside (4-NpGlcp) and cellobiose were 0.093 (Km), 1.07 mM (kcat) and 1802 (Km), 461.5 min-1 (kcat), respectively. Glucose and gluconolactone inhibited the enzyme competitively with Ki values of 2.05 mM and 3.03 μM, respectively. Alcohols activated the enzyme; butanol showed maximum effect (2.2-fold at 0.5 M) while methanol increased the activity by 1.4-fold at 1 M. The enzyme catalysed the synthesis of methylglucosides, ethylglucoside and propylglucosides, as well as trisaccharides in the presence of different alcohols and disaccharides, respectively. In addition, the enzyme hydrolysed the unsubstituted and methylumbelliferyl cello-oligosaccharides [MeUmb(Glc)n] but the rate of hydrolysis decreased with increasing chain length. Analysis of products released from MeUmb(Glc)n as a function of time revealed that the enzyme attacked these substrates in a stepwise manner and from both ends. Thus, β-glucosidase from F. oxysporum, with the above interesting properties, could be of commercial interest.
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| 4. |
- Christakopoulos, Paul, et al.
(författare)
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Purification and mode of action of an alkali-resistant endo-1,4-β-glucanase from Bacillus pumilus
- 1999
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 364:1, s. 61-66
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Tidskriftsartikel (refereegranskat)abstract
- Alkaline endo-1,4-β-d-glucanase was secreted byBacillus pumilusgrown in submerged culture on a combination of oat spelt xylan and corn starch as carbon sources. The enzyme was purified to homogeneity by Sephacryl S-200 and Q-Sepharose column chromatography. The protein corresponded to molecular mass and pIvalues of 67 kDa and 3.7, respectively. The enzyme was optimally active at pH 7.0–8.0 and 60°C and retained 50% of its optimum activity at pH 12. The most notable characteristic of the endoglucanase was its high stability up to pH 12 for 20 h at 30°C. The enzyme hydrolyzed carboxymethylcellulose (CMC) and cello-oligosaccharides but was inactive on cellobiose, cellotriose, Avicel, xylan, 4-nitrophenyl-β-d-glucoside, 4-nitrophenyl-β-d-cellobioside, and 4-nitrophenyl-β-d-xyloside. Analysis of reaction mixtures by HPLC revealed that the enzyme produced almost exclusively cellotriose when acted on CMC and appeared to hydrolyze cello-oligosaccharides by successively releasing cellotriose. The use of 4-methylumbelliferyl cello-oligosaccharides and the determination of bond cleavage frequency revealed that the enzyme preferentially hydrolyzed the third glycosidic bond adjacent to the glycon. The enzyme mediated a decrease in the viscosity of CMC associated with a release of only small amounts of reducing sugar. The enzyme activity was not inhibited by metal ions, surfactants, and chelating agents used as components of laundry detergents.
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| 5. |
- Christakopoulos, Paul, et al.
(författare)
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The alkaline xylanase III from Fusarium oxysporum F3 belongs to family F/10
- 1997
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Ingår i: Carbohydrate Research. - 0008-6215 .- 1873-426X. ; 302:3-4, s. 191-195
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Tidskriftsartikel (refereegranskat)abstract
- Xylanase III from Fusarium oxysporum F3 was purified to homogeneity by ion-exchange chromatography and gel filtration. The enzyme has a molecular mass of 38 kDa, an isoelectric point of 9.5, and is maximally active on oat spelt xylan at pH 7 and 45 °C with a Km of 0.8 mg/mL. The xylanase displays remarkable stability at pH 9.0. It is not active on xylotriose but hydrolyzes the 4-methylumbelliferyl glycosides of β-xylobiose and --- , and to a lower extent 4-methylumbelliferyl β-cellobioside. When acted on xylooligosaccharides and xylan, analysis of reaction mixtures by high-pressure liquid chromatography shows preferred internal glycoside cleavage. Thus the purified enzyme appears to be a true endo-β-1,4-xylanase. Partial amino acid analysis of xylanase III shows high sequence homology with xylanases of family F/10.Xylanase III from Fusarium oxysporum F3 was purified to homogeneity by ion-exchange chromatography and gel filtration, and was functionally characterised. The enzyme displays remarkable stability at pH 9.0, appears to be a true endo-β-1,4-xylanase, and shows high sequence homology with xylanases of family F/10.
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| 6. |
- Desmet, Tom, et al.
(författare)
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Synthesis and Evaluation of 2-Deoxy-2-amino-beta-cellobiosides as Cellulase Inhibitors
- 2010
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Ingår i: Journal of carbohydrate chemistry. - : Informa UK Limited. - 0732-8303 .- 1532-2327. ; 29:4, s. 164-180
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Tidskriftsartikel (refereegranskat)abstract
- The cellulase mixture of Hypocrea jecorina (formerly Trichoderma reesei) contains a variety of exo- and endoglucanases that belong to different structural families. As such, these enzymes form an interesting model system to study the enzyme-ligand interactions in glycoside hydrolases. The nucleophilic carboxylate of retaining -glycosidases is believed to form a hydrogen bond with the 2-hydroxyl group of their substrate. Consequently, replacing this hydroxyl group with an amino group should result in a stronger electrostatic interaction and thus an increased affinity for the ligand. In this study, several modified cellobiosides were synthesized and evaluated as cellulase inhibitors. The introduction of an amino group was found to have an unpredictable effect on the inhibitory power of the ligands. However, the enzymes display a very high affinity for the corresponding 2-azido compounds, precursors in the synthetic route. The new ligand m-iodobenzyl 2-deoxy-2-azido--cellobioside even is the strongest inhibitor of cellobiohydrolase I known to date (KI= 1 M).
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| 7. |
- Hägglund, Per, et al.
(författare)
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A cellulose-binding module of the Trichoderma reesei @b-mannanase Man5A increases the mannan-hydrolysis of complex substrates
- 2003
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Ingår i: Journal of Biotechnology. - 1873-4863. ; 101:1, s. 37-48
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Tidskriftsartikel (refereegranskat)abstract
- Endo-@b-1,4-d-mannanases (@b-mannanase; EC 3.2.1.78) are endohydrolases that participate in the degradation of hemicellulose, which is closely associated with cellulose in plant cell walls. The @b-mannanase from Trichoderma reesei (Man5A) is composed of an N-terminal catalytic module and a C-terminal carbohydrate-binding module (CBM). In order to study the properties of the CBM, a construct encoding a mutant of Man5A lacking the part encoding the CBM (Man5A@DCBM), was expressed in T. reesei under the regulation of the Aspergillus nidulans gpdA promoter. The wild-type enzyme was expressed in the same way and both proteins were purified to electrophoretic homogeneity using ion-exchange chromatography. Both enzymes hydrolysed mannopentaose, soluble locust bean gum galactomannan and insoluble ivory nut mannan with similar rates. With a mannan/cellulose complex, however, the deletion mutant lacking the CBM showed a significant decrease in hydrolysis. Binding experiments using activity detection of Man5A and Man5A@DCBM suggests that the CBM binds to cellulose but not to mannan. Moreover, the binding of Man5A to cellulose was compared with that of an endoglucanase (Cel7B) from T. reesei.
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| 8. |
- Katapodis, Petros, et al.
(författare)
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Biochemical and catalytic properties of an endoxylanase purified from the culture filtrate of Sporotrichum thermophile
- 2003
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Ingår i: Carbohydrate Research. - 0008-6215 .- 1873-426X. ; 338:18, s. 1881-1890
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Tidskriftsartikel (refereegranskat)abstract
- An endo-β-1,4-xylanase (1,4-β-d-xylan xylanoxydrolase, EC 3.2.1.8) present in culture filtrates of Sporotrichum thermophile ATCC 34628 was purified to homogeneity by Q-Sepharose and Sephacryl S-200 column chromatographies. The enzyme has a molecular mass of 25,000 Da, an isoelectric point of 6.7, and is optimally active at pH 5 and at 70 °C. Thin-layer chromatography (TLC) analysis showed that endo-xylanase liberates mainly xylose (Xyl) and xylobiose (Xyl2) from beechwood 4-O-methyl-d-glucuronoxylan, O-acetyl-4-O-methylglucuronoxylan and rhodymenan (a β-(1→4)-β(1→3)-xylan). Also, the enzyme releases an acidic xylo-oligosaccharide from 4-O-methyl-d-glucuronoxylan, and an isomeric xylotetraose and an isomeric xylopentaose from rhodymenan. Analysis of reaction mixtures by high performance liquid chromatography (HPLC) revealed that the enzyme cleaves preferentially the internal glycosidic bonds of xylooligosaccharides, [1-3H]-xylooligosaccharides and xylan. The enzyme also hydrolyses the 4-methylumbelliferyl glycosides of β-xylobiose and β-xylotriose at the second glycosidic bond adjacent to the aglycon. The endoxylanase is not active on pNPX and pNPC. The enzyme mediates a decrease in the viscosity of xylan associated with a release of only small amounts of reducing sugar. The enzyme is irreversibly inhibited by series of ω-epoxyalkyl glycosides of d-xylopyranose. The results suggest that the endoxylanase from S. thermophile has catalytic properties similar to the enzymes belonging to family 11.
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| 9. |
- Katapodis, Petros, et al.
(författare)
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Purification and characterization of a thermostable intracellular β-xylosidase from the thermophilic fungus Sporotrichum thermophile
- 2006
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Ingår i: Process Biochemistry. - : Elsevier BV. - 1359-5113 .- 1873-3298. ; 41:12, s. 2402-2409
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Tidskriftsartikel (refereegranskat)abstract
- An intracellular beta-xylosidase from the thermophilic fungus Sporotricum thermophile strain ATCC 34628 was purified to homogeneity by Q-Sepharose and Mono-Q column chromatographies. The protein properties correspond to molecular mass and pI values of 45 kDa and 4.2, respectively. The enzyme is optimally active at pH 7.0 and 50 degrees C. The purified beta-xylosidase is fully stable at pH 6.0-8.0 and temperatures up to 50 degrees C and retained over 58% of its activity after 1 h at 60 degrees C. The enzyme hydrolyzes beta-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 6, releasing xylose from the non-reducing end, but is inactive against xylan substrates. The apparent K-m and V-max values from p-nitrophenyl beta-D-xylopyranoside are 1.1 mM and 114 mu mol p-nitrophenol min(-1) mg(-1), respectively. Alcohols inactivate the enzyme, ethanol at 10% (v/v) yields a 30% decrease of its activity. The enzyme is irreversibly inhibited by 2,3-epoxypropyl beta-D-xylobioside while alkyl epoxides derived from D-Xylose were not inhibitors of the enzyme. The enzyme catalyses the condensation reaction using high donor concentration, up to 60% (w/v) xylose. (c) 2006 Elsevier Ltd. All rights reserved
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| 10. |
- Koivula, Anu, et al.
(författare)
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The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175.
- 2002
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Ingår i: J Am Chem Soc. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 124:34, s. 10015-24
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Tidskriftsartikel (refereegranskat)abstract
- Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other. Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve a catalytic base. The value of (D2O)(V) of 1.16 +/- 0.14 for hydrolysis of cellotriose suggests that the large direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond between D221 and D175 before catalysis.
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