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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. - 0264-6021 .- 1470-8728. ; 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 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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| 2. |
- Selber, K, et al.
(författare)
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Large-scale separation and production of engineered proteins, designed for facilitated recovery in detergent-based aqueous two-phase extraction systems
- 2004
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Ingår i: Process Biochemistry. - 1873-3298. ; 39:7, s. 889-896
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Tidskriftsartikel (refereegranskat)abstract
- The feasibility and scalability of extraction in detergent-based aqueous two-phase systems for the separation of proteins from culture broth is demonstrated. At the same time the large-scale production of a fusion protein and the influence of cultivation scale on the efficiency of separation were investigated. An amphiphilic fusion protein EGIcore-HFBI was chosen, consisting of the catalytic core of the cellulase endoglucanase I and the small protein hydrophobin I, expressed homologously in Trichoderma reesei. Using the technical nonionic detergent Agrimul NRE 1205 the separation was successfully scaled up to 1200 l. No differences in yield or in partition coefficient were observed at 10 ml and 1200 l scale. Changes in the fermentation temperature and scale, however, can influence the properties of the protein and thus alter partition coefficient and yield. The decreased separation efficiency appears to be correlated with changes in glycosylation at lower cultivation temperatures
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| 3. |
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| 4. |
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| 5. |
- Hahn, Robert, et al.
(författare)
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Isotonic saline in elderly men: an open-labelled controlled infusion study of electrolyte balance, urine flow and kidney function
- 2016
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Ingår i: Anaesthesia. - : WILEY-BLACKWELL. - 0003-2409 .- 1365-2044. ; 71:2, s. 155-162
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Tidskriftsartikel (refereegranskat)abstract
- Isotonic saline is a widely-used infusion fluid, although the associated chloride load may cause metabolic acidosis and impair kidney function in young, healthy volunteers. We wished to examine whether these effects also occurred in the elderly, and conducted a crossover study in 13 men with a mean age of 73 years (range 66-84), who each received intravenous infusions of 1.5 l of Ringers acetate and of isotonic saline. Isotonic saline induced mild changes in plasma sodium (mean +1.5 mmol.l(-1)), plasma chloride (+3 mmol.l(-1)) and standard bicarbonate (-2 mmol.l(-1)). Three hours after starting the infusions, 68% of the Ringers acetate and 30% of the infused saline had been excreted (p < 0.01). The glomerular filtration rate increased in response to both fluids, but more after the Ringers acetate (p < 0.03). Pre-infusion fluid retention, as evidenced by high urinary osmolality (> 700 mOsmol.kg(-1)) and/or creatinine (> 7 mmol.l(-1)), was a strong factor governing the responses to both fluid loads.
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| 6. |
- Stenstrom, A., et al.
(författare)
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Genetic variation and clonal diversity in four clonal sedges (Carex) along the Arctic coast of Eurasia
- 2001
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Ingår i: Molecular Ecology. - 0962-1083 .- 1365-294X. ; 10:2, s. 497-513
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Tidskriftsartikel (refereegranskat)abstract
- We studied the structure of genetic variation (at both ramet- and genet-level) and clonal diversity within and among populations in the four closely related arctic clonal sedges Carex bigelowii, C. ensifolia, C. lugens and C. stans by use of allozyme markers. Compared to other sedges and arctic plants, the studied taxa all had high levels of genetic variation, both within populations and taxa. These taxa contained most of the total gene diversity (HT) within populations and a small part of the diversity among populations (GST ranged 0.05-0.43). Carex bigelowii had genetic variation (HS = 0.173, mean for populations) at a comparable level to other outbreeding arctic plants and to other widespread, rhizomatous and mainly outbreeding Carex species. In contrast, C. ensifolia (HS = 0.335), C. lugens (HS = 0.339) and C. stans (HS = 0.294) had within-population variations that were higher than in most other studied Carex species and for arctic plants in general. Genetic variation was not related to any tested environmental variable, but it was lower in areas deglaciated only 10 000 years BP compared to areas deglaciated 60 000 years BP or not glaciated at all during the Weichselian. All the populations were multiclonal, except for two populations of C. stans that were monoclonal. In contrast to genetic variation, clonal diversity decreased with latitude and did not differ between areas with different times of deglaciation. In accordance with previous studies, C. bigelowii and C. lugens were found to be outbreeding, while C. ensifolia and C. stans had mixed mating systems.
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