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- Becker, D, et al.
(författare)
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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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Ingår i: Biochemical Journal. ; 356, s. 19-30
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Tidskriftsartikel (refereegranskat)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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| 2. |
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| 3. |
- Rodstedth, M, et al.
(författare)
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Chloride imbalances in soil lysimeters
- 2003
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Ingår i: Chemosphere. - 0045-6535 .- 1879-1298. ; 52:2, s. 381-389
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Tidskriftsartikel (refereegranskat)abstract
- The assumption that soil neither acts as a source or a sink of chloride is evaluated by incubating soil cores in lysimeters in a climate chamber under controlled conditions. Some of the lysimeters acted as a sink while others acted as a source of chloride. Considerable amounts of organic chlorine were lost by leaching. The loss by leaching of organic chlorine could only explain part of the discrepancy in the lysimeters where the soil acted as a sink and it could certainly not explain the cases where the soil acted as a source. The storage of organic chlorine was four times larger than the storage of chloride and comparably small changes in the organic chlorine storage will thus have a considerable influence on the chloride budget. However, the soil was too heterogeneous to determine whether a change in the storage had taken place or not. It is concluded that the observed chloride surplus and also, at least to some extent, the observed chloride deficit, most likely was caused by net-changes in the storage of organic chlorine in soil. An inverse correlation was found between the initial chloride content of the soil and the imbalance in the chloride budget. Dry deposition of chloride is generally assumed to equal the run-off minus the wet deposition. Extrapolation to the field situation suggests that the output of organic chlorine by soil leachate is at risk to cause an underestimation of the dry deposition by about 25%. (C) 2003 Elsevier Science Ltd. All rights reserved.
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| 4. |
- Wirestam, Ronnie, et al.
(författare)
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Perfusion-related parameters in intravoxel incoherent motion MR imaging compared with CBV and CBF measured by dynamic susceptibility-contrast MR technique
- 2001
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Ingår i: Acta Radiologica. - : SAGE Publications. - 1600-0455 .- 0284-1851. ; 42:2, s. 123-128
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: Perfusion-related parameters obtained by intravoxel incoherent motion (IVIM) MR imaging (MRI) were compared with cerebral blood volume and flow (CBV and CBF), retrieved by dynamic susceptibility-contrast (DSC) MRI. MATERIAL AND METHODS: Twenty-eight volunteers (average age 68.5 years) were investigated. Spin-echo echo-planar imaging with IVIM-encoding gradients was employed (36 different b values, 0-1200 s/mm2). The perfusion fraction and the pseudo-diffusion coefficient were calculated for regions in thalamus gray matter and frontal white matter, using asymptotic and full fitting. In DSC-MRI, a Gd-DTPA-BMA contrast-agent bolus was monitored using simultaneous-dual FLASH. Deconvolution of the measured tissue concentration-versus-time curve with an arterial input function from the carotid artery was applied, and maps of CBV and CBF were calculated. RESULTS: The correlation between the perfusion fraction and CBV was r=0.56 (p<0.0000006) using asymptotic fitting, and r=0.35 (p<0.0004) when full fitting was applied. Average CBF was 41.5 ml/(min 100 g), to be compared with the IVIM-based value of 63.6 ml/(min 100 g), obtained from the median value of the pseudo-diffusion coefficient in combination with assumptions about capillary network structure. CONCLUSION: The IVIM concept provided results that agreed reasonably with conventional CBV and CBF. The non-linear fitting to noisy signal data was problematic, in accordance with previously presented simulations.
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