| 1. |
- Abou-Hachem, Maher, et al.
(författare)
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Calcium binding and thermostability of carbohydrate binding module CBM4-2 of Xyn10A from Rhodothermus marinus.
- 2002
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:18, s. 5720-5729
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Tidskriftsartikel (refereegranskat)abstract
- Calcium binding to carbohydrate binding module CBM4-2 of xylanase 10A (Xyn10A) from Rhodothermus marinus was explored using calorimetry, NMR, fluorescence, and absorbance spectroscopy. CBM4-2 binds two calcium ions, one with moderate affinity and one with extremely high affinity. The moderate-affinity site has an association constant of (1.3 +/- 0.3) x 10(5) M(-1) and a binding enthalpy DeltaH(a) of -9.3 +/- 0.4 kJ x mol(-1), while the high-affinity site has an association constant of approximately 10(10) M(-1) and a binding enthalpy DeltaH(a) of -40.5 +/- 0.5 kJ x mol(-1). The locations of the binding sites have been identified by NMR and structural homology, and were verified by site-directed mutagenesis. The high-affinity site consists of the side chains of E11 and D160 and backbone carbonyls of E52 and K55, while the moderate-affinity site comprises the side chain of D29 and backbone carbonyls of L21, A22, V25, and W28. The high-affinity site is in a position analogous to the calcium site in CBM4 structures and in a recent CBM22 structure. Binding of calcium increases the unfolding temperature of the protein (T(m)) by approximately 23 degrees C at pH 7.5. No correlation between binding affinity and T(m) change was noted, as each of the two calcium ions contributes almost equally to the increase in unfolding temperature.
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| 2. |
- Abou Hachem, Maher, et al.
(författare)
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Carbohydrate-binding modules from a thermostable Rhodothermus marinus xylanase : Cloning, expression and binding studies
- 2000
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Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021. ; 345:1, s. 53-60
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Tidskriftsartikel (refereegranskat)abstract
- The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichia coli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na+ and Ca2+ ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and β-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.
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| 3. |
- Abou-Hachem, Maher, et al.
(författare)
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The modular organisation and stability of a thermostable family 10 xylanase
- 2003
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Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1024-2422 .- 1029-2446. ; 21:5-6, s. 253-260
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Tidskriftsartikel (refereegranskat)abstract
- The thermophilic marine bacterium Rhodothermus marinus produces a modular family 10 xylanase (Xyn10A). It consists of two N-terminal family 4 carbohydrate binding modules (CBMs) followed by a domain of unknown function (D3), and a catalytic module (CM) flanked by a small fifth domain (D5) at its C-terminus. Several truncated mutants of the enzyme have been produced and characterised with respect to biochemical properties and stability. Multiple calcium binding sites are shown to be present in the two N-terminal CBMs and recent evidence suggests that the third domain of the enzyme also has the ability to bind the same metal ligand. The specific binding of Ca2+ was demonstrated to have a pronounced effect on thermostability as shown by differential scanning calorimetry and thermal inactivation studies. Furthermore, deletion mutants of the enzyme were less stable than the full-length enzyme suggesting that module interactions contributed to the stability of the enzyme. Finally, recent evidence indicates that the fifth domain of Xyn10A is a novel type of module mediating cell-attachment.
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| 4. |
- Blücher, Anna, et al.
(författare)
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Substrate-dependent production and some properties of a thermostable, α-galactosidase from Rhodothermus marinus
- 2000
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Ingår i: Biotechnology Letters. - 0141-5492. ; 22:8, s. 663-669
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Tidskriftsartikel (refereegranskat)abstract
- α-Galactosidase activity in Rhodothermus marinus is dependent on the composition of the growth media. A maximum of 46 μU g-1 cell dry weight was obtained using minimal medium with galactooligo- or polysaccharides as single carbon source. An enzyme hydrolysing both high and low molecular weight galacto-saccharides was partly purified from the cell fractions. The molecular weight was 200 kDa (native) and 50 kDa (monomer). It was optimally active at 85°C, with a half-life of 2 h at 75°C, and had a broad pH range (4-8).
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| 5. |
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| 6. |
- Cicortas Gunnarsson, Lavinia, et al.
(författare)
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Evolution of a carbohydrate binding module into a protein-specific binder
- 2006
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Ingår i: Biomolecular Engineering. - : Elsevier BV. - 1389-0344 .- 1878-559X. ; 23:2-3, s. 111-117
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Tidskriftsartikel (refereegranskat)abstract
- A carbohydrate binding module, CBM4-2, derived front the xylanase (Xyn 10A) of Rhodothermus marinus has been used as a scaffold for molecular diversification. Its binding specificity has been evolved to recognise a quite different target, a human monoclonal IgG4. In order to understand the basis for this drastic change in specificity we have further investigated the target recognition of the IgG4-specific CBMs. Firstly, we defined that the structure target recognised by the selected CBM-variants was the protein and not the carbohydrates attached to the glycoprotein. We also identified key residues involved in the new specificity and/or responsible for the swap in specificity, from xylan to human IgG4. Specific changes present in all these CBMs included mutations not introduced in the design of the library from which the specific clones were selected. Reversion of such mutations led to a complete loss of binding to the target molecule, suggesting that they are critical for the recognition of human IgG4. Together with the mutations introduced at will, they had transformed the CBM scaffold into a protein binder. We have thus shown that the scaffold of CBM4-2 is able to harbour molecular recognition for either carbohydrate or protein structures. (c) 2005 Elsevier B.V. All rights reserved.
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| 7. |
- Cicortas Gunnarsson, Lavinia, et al.
(författare)
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Molecular engineering of a thermostable carbohydrate-binding module
- 2006
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Ingår i: Biocatalysis and Biotransformation. - : Informa UK Limited. - 1029-2446 .- 1024-2422. ; 24:1-2, s. 31-37
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Konferensbidrag (refereegranskat)abstract
- Structure-function studies are frequently practiced on the very diverse group of natural carbohydrate-binding modules in order to understand the target recognition of these proteins. We have taken a step further in the study of carbohydrate-binding modules and created variants with novel binding properties by molecular engineering of one such molecule of known 3D-structure. A combinatorial library was created from the sequence encoding a thermostable carbohydrate-binding module, CBM4-2 from a Rhodothermus marinus xylanase, and phage-display technology was successfully used for selection of variants with specificity towards different carbohydrate polymers (birchwood xylan, Avicel (TM), ivory nut mannan and recently also xyloglucan), as well as towards a glycoprotein (human IgG4). Our work not only generated a number of binders with properties that would suite a range of biotechnological applications, but analysis of selected binders also helped us to identify residues important for their specificities.
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| 8. |
- de Maré, Lena, et al.
(författare)
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A cultivation technique for E. coli fed-batch cultivations operating close to the maximum oxygen transfer capacity of the reactor
- 2005
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Ingår i: Biotechnology Letters. - : Springer Science and Business Media LLC. - 0141-5492 .- 1573-6776. ; 27:14, s. 983-990
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Tidskriftsartikel (refereegranskat)abstract
- A cultivation strategy combining the advantages of temperature-limited fed-batch and probing feeding control is presented. The technique was evaluated in fed-batch cultivations with E. coli BL21(DE3) producing xylanase in a 3 liter bioreactor. A 20% increase in cell mass was achieved and the usual decrease in specific enzyme activity normally observed during the late production phase was diminished with the new technique. The method was further tested by growing E. coli W3110 in a larger bioreactor (50 l). It is a suitable cultivation technique when the O2 transfer capacity of the reactor is reached and it is desired to continue to produce the recombinant protein.
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| 9. |
- Gunnarsson, Lavinia Cicortas, 1977-, et al.
(författare)
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A carbohydrate binding module as a diversity-carrying scaffold
- 2004
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Ingår i: Protein Engineering Design & Selection. - : Oxford University Press. - 1741-0126 .- 1741-0134. ; 17:3, s. 213-221
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Tidskriftsartikel (refereegranskat)abstract
- The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large-scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4-2, from a xylanase found in Rhodothermus marinus, as a diversity-carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4-2. Despite the small size of the library (1.6 x 10(6) clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 +/- 5.3 degrees C). All our results demonstrate that the CBM4-2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.
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| 10. |
- Huang, Fang, et al.
(författare)
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Cultivation of the gut bacterium Prevotella copri DSM 18205T using glucose and xylose as carbon sources
- 2021
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Ingår i: MicrobiologyOpen. - : Wiley. - 2045-8827. ; 10:3
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Tidskriftsartikel (refereegranskat)abstract
- Prevotella copri DSM18205T is a human gut bacterium, suggested as a next-generation probiotic. To utilize it as such, it is, however, necessary to grow the species in a reproducible manner. Prevotella copri has previously been reported to be highly sensitive to oxygen, and hence difficult to isolate and cultivate. This study presents successful batch cultivation strategies for viable strain inoculations and growth in both serum bottles and a stirred tank bioreactor (STR), without the use of an anaerobic chamber, as long as the cells were kept in the exponential growth phase. A low headspace volume in the STR was important to reach high cell density. P. copri utilized xylose cultivated in Peptone Yeast Xylose medium (PYX medium), resulting in a comparable growth rate and metabolite production as in Peptone Yeast Glucose medium (PYG medium) in batch cultivations at pH 7.2.Up to 5 g/L of the carbon source was consumed, leading to the production of succinic acid, acetic acid, and formic acid, and cell densities (OD620 nm ) in the range 6-7.5. The highest yield of produced succinic acid was 0.63 ± 0.05 g/g glucose in PYG medium cultivations and 0.88 ± 0.06 g/g xylose in PYX medium cultivations.
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