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| 2. |
- Becker, D., et al.
(author)
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Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum : the pH behaviour of Trichoderma reesei CeI7A and its E223S/A224H/L225V/T226A/D262G mutant
- 2001
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In: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 356, s. 19-30
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Journal article (peer-reviewed)abstract
- The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala(224)) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves, The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 Angstrom (= 0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 Angstrom contact between N-2 and O'(1). The pH variation of k(cat)/K-m for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wildtype and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) shifting from 5.99+/-0.02 to 6.78+/-0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K-m values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) values for cellobiosides are higher for the mutant. This we attribute to reduced nonproductive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.
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| 3. |
- Clausen, Niels, et al.
(author)
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Similar bleeding phenotype in young children with haemophilia A or B : A cohort study
- 2014
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In: Haemophilia. - : Wiley. - 1351-8216. ; 20:6, s. 747-755
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Journal article (peer-reviewed)abstract
- The bleeding phenotype has been suggested to differ between haemophilia A and B. More knowledge on the bleeding phenotype at initiation of treatment is important to optimize patient care. The aim of this study was to investigate the severity of the bleeding phenotype and the variation in bleeding in children with severe or moderate haemophilia A and B. Consecutive, previously untreated patients with severe or moderate haemophilia A and B (factor VIII or IX activity <0.01 or 0.01-0.05 IU mL-1 respectively) born between January 1st 2000 and January 1st 2010 were included. Primary outcome was severity of bleeding tendency. Secondary outcome was variation in bleeding pattern. A total of 582 patients with severe haemophilia A and 76 with severe haemophilia B did not differ in age at first exposure to clotting factor (0.81 vs. 0.88 years, P = 0.20), age at first bleed (0.82 vs. 0.88 years, P = 0.36), and age at first joint bleed (1.18 vs. 1.20 years, P = 0.59). Patients with moderate haemophilia were older compared to patients with severe haemophilia. In patients with moderate haemophilia there were no clear differences between haemophilia A and B. Severity and variation in bleeding phenotype are similar during the early stage of treatment in patients with severe and moderate haemophilia A and B respectively. The findings imply that children with haemophilia B should be observed and treated as vigilantly as those with haemophilia A.
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| 4. |
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| 5. |
- Becker, D, et al.
(author)
-
Engineering of a glycosidase Family 7 cellobiohydrolase to more alkaline pH optimum: the pH behaviour of Trichoderma reesei CeI7A and its E223S/A224H/L225V/T226A/D262G mutant
- 2001
-
In: Biochemical Journal. ; 356, s. 19-30
-
Journal article (peer-reviewed)abstract
- The crystal structures of Family 7 glycohydrolases suggest that a histidine residue near the acid/base catalyst could account for the higher pH optimum of the Humicola insolens endoglucanase Cel7B, than the corresponding Trichoderma reesei enzymes. Modelling studies indicated that introduction of histidine at the homologous position in T. reesei Cel7A (Ala(224)) required additional changes to accommodate the bulkier histidine side chain. X-ray crystallography of the catalytic domain of the E223S/A224H/L225V/T226A/D262G mutant reveals that major differences from the wild-type are confined to the mutations themselves. The introduced histidine residue is in plane with its counterpart in H. insolens Cel7B, but is 1.0 A (=0.1 nm) closer to the acid/base Glu(217) residue, with a 3.1 A contact between N(epsilon2) and O(epsilon1). The pH variation of k(cat)/K(m) for 3,4-dinitrophenyl lactoside hydrolysis was accurately bell-shaped for both wild-type and mutant, with pK(1) shifting from 2.22+/-0.03 in the wild-type to 3.19+/-0.03 in the mutant, and pK(2) shifting from 5.99+/-0.02 to 6.78+/-0.02. With this poor substrate, the ionizations probably represent those of the free enzyme. The relative k(cat) for 2-chloro-4-nitrophenyl lactoside showed similar behaviour. The shift in the mutant pH optimum was associated with lower k(cat)/K(m) values for both lactosides and cellobiosides, and a marginally lower stability. However, k(cat) values for cellobiosides are higher for the mutant. This we attribute to reduced non-productive binding in the +1 and +2 subsites; inhibition by cellobiose is certainly relieved in the mutant. The weaker binding of cellobiose is due to the loss of two water-mediated hydrogen bonds.
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| 6. |
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| 7. |
- von Ossowski, I., et al.
(author)
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Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Ce17A. A comparison with Phanerochaete chrysosporium Cel7D
- 2003
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In: Journal of Molecular Biology. - 0022-2836 .- 1089-8638. ; 333:4, s. 817-829
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Journal article (peer-reviewed)abstract
- The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations. The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K-M and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3) + Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.
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| 8. |
- Christakopoulos, Paul, et al.
(author)
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Purification and characterization of a less randomly acting endo-1,4-beta-D-glucanase from the culture filtrates of Fusarium oxysporum
- 1995
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In: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861 .- 1096-0384. ; 316:1, s. 428-433
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Journal article (peer-reviewed)abstract
- An extracellular endo-1,4-β-D-glucanase from Fusarium oxysporum was purified by affinity chromatography and gel filtration. The enzyme purified in this way was homogeneous when judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing-polyacrylamide gel electrophoresis. The protein corresponded to a molecular mass and pI value of 41.7 kDa and 6.4, respectively. It was optimally active at pH 4.5 and at 55°C. The enzyme hydrolyzed carboxymethylcellulose (CMC) and unsubstituted and substituted cello-oligosaccharides but was inactive on Avicel, filter paper, xylan, cellobiose, p-nitrophenyl-β-D-glucoside, and p-nitrophenyl-β-D-xyloside. However, the enzyme effected only a small change in viscosity of CMC per unit increase of reducing sugar. When cellotriose, cellotetraose, and cellopentaose were used as substrates, the enzyme released mainly cellobiose. Use of 4-methylumbelliferyl cello-oligosaccharides and the determination of bond cleavage frequency revealed that the enzyme preferentially hydrolyzed the glycosidic bond adjacent to 4-methylumbelliferone. Thus, the purified enzyme appeared to be a less randomly acting endoglucanase.
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| 9. |
- Koivula, A, et al.
(author)
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The active site of Trichoderma reesei cellobiohydrolase II: The role of tyrosine 169
- 1996
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In: PROTEIN ENGINEERING. - 0269-2139. ; 9:8, s. 691-699
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Journal article (other academic/artistic)abstract
- Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanase cleaving primarily cellobiose units from the non-reducing end of cellulose chains. The beta-1,4 glycosidic bond is cleaved by acid catalysis with an aspartic acid, D221, as the likely proton donor, and another aspartate, D175, probably ensuring its protonation and stabilizing charged reaction intermediates. The catalytic base has not yet been identified experimentally. The refined crystal structure of CBHII also shows a tyrosine residue, Y169, located close enough to the scissile bond to be involved in catalysis. The role of this residue has been studied by introducing a mutation Y169F, and analysing the kinetic and binding behavior of the mutated CBHII. The crystal structure of the mutated enzyme was determined to 2.0 A resolution showing no changes when compared with the structure of native CBHII. However, the association constants of the mutant enzyme for cellobiose and cellotriose are increased threefold and for 4-methylumbelliferyl cellobioside over 50-fold. The catalytic constants towards cellotriose and cellotetraose are four times lower for the mutant. These data suggest that Y169, on interacting with a glucose ring entering the second subsite in a narrow tunnel, helps to distort the glucose ring into a more reactive conformation. In addition, a change in the pH activity profile was observed. This indicates that Y169 may have a second role in the catalysis, namely to affect the protonation state of the active site carboxylates, D175 and D221.
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| 10. |
- Ljung, R., et al.
(author)
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Treatment of children with haemophilia in Europe: A survey of 20 centres in 16 countries
- 2000
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In: Haemophilia. - : Wiley. - 1351-8216. ; 6, s. 619-624
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Journal article (peer-reviewed)abstract
- A survey was made of the current status of treatment of haemophilic boys at 20 centres in 16 European countries and includes approximately 1500 of the estimated 6500 haemophiliacs in the participating countries. Many mild haemophiliacs are not seen, or seen infrequently, at haemophilia centres and this requires study. Nine of 18 centres provide continuous prophylaxis to 80-100% of their patients, five centres provide it to 55-80% and the remaining four centres to 15-40% of the boys. The median dose given was 6240 U kg-1 year-1 (range 3120-7800). Four centres administered only recombinant concentrates to children with severe haemophilia A, while seven centres administered recombinant concentrates to 75-90% and the remaining centres to less than 50% of the boys (two centres <10%). When asked for the choice of concentrate for a newly diagnosed boy with severe haemophilia A, all but one centre preferred recombinant concentrate. Most boys below 6 years received concentrates via a peripheral vein but three centres preferred a central venous line for 80-100% of the boys. Thirteen of 18 centres applied home treatment to 84-100% of the boys and the remaining five centres to 57-77% of the boys.
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