| 1. |
- Bäcklund, Emma
(författare)
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Impact of glucose uptake rate on recombinant protein production in Escherichia coli
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Escherichia coli (E. coli) is an attractive host for production of recombinant proteins, since it generally provides a rapid and economical means to achieve high product quantities. In this thesis, the impact of the glucose uptake rate on the production of recombinant proteins was studied, aiming at improving and optimising production of recombinant proteins in E. coli. E. coli can be cultivated to high cell densities in bioreactors by applying the fed-batch technique, which offers a means to control the glucose uptake rate. One objective of this study was to find a method for control of the glucose uptake rate in small-scale cultivation, such as microtitre plates and shake flasks. Strains with mutations in the phosphotransferase system (PTS) where used for this purpose. The mutants had lower uptake rates of glucose, resulting in lower growth rates and lower accumulation of acetic acid in comparison to the wild type. By using the mutants in batch cultivations, the formation of acetic acid to levels detrimental to cell growth could be avoided, and ten times higher cell density was reached. Thus, the use of the mutant strains represent a novel, simple alternative to fed-batch cultures. The PTS mutants were applied for production of integral membrane proteins in order to investigate if the reduced glucose uptake rate of the mutants was beneficial for their production. The mutants were able to produce three out of five integral membrane proteins that were not possible to produce by the wild-type strain. The expression level of one selected membrane protein was increased when using the mutants and the expression level appeared to be a function of strain, glucose uptake rate and acetic acid accumulation. For production purposes, it is not uncommon that the recombinant proteins are secreted to the E. coli periplasm. However, one drawback with secretion is the undesired leakage of periplasmic products to the medium. The leakage of the product to the medium was studied as a function of the feed rate of glucose in fed-batch cultivations and they were found to correlate. It was also shown that the amount of outer membrane proteins was affected by the feed rate of glucose and by secretion of a recombinant product to the periplasm. The cell surface is another compartment where recombinant proteins can be expressed. Surface display of proteins is a potentially attractive production strategy since it offers a simple purification scheme and possibilities for on-cell protein characterisation, and may in some cases also be the only viable option. The AIDA-autotransporter was applied for surface display of the Z domain of staphylococcal protein A under control of the aidA promoter. Z was expressed in an active form and was accessible to the medium. Expression was favoured by growth in minimal medium and it seemed likely that expression was higher at higher feed rates of glucose during fed-batch cultivation. A repetitive batch process was developed, where relatively high cell densities were achieved whilst maintaining a high expression level of Z.
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| 2. |
- Patel, Ami, et al.
(författare)
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Evidence for xylooligosaccharides utilization in Weissella strains isolated from Indian fermented foods and vegetables.
- 2013
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Ingår i: FEMS Microbiology Letters. - : Oxford University Press (OUP). - 1574-6968 .- 0378-1097. ; 346:1, s. 20-28
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Six strains isolated from fermented food were by 16S rDNA sequencing identified as Weissella species, clustering with the species-pair W. confusa/ W. cibaria. The strains were analysed for growth on glucose, xylose and xylooligosaccharides (XOS). All strains were xylose positive using the API CHL 50 test. Growth on XOS was observed for strain 85, 92, 145 and AV1, firstly by optical density measurements in microtiter plates and secondly in batch cultures also confirming concomitant decrease in pH. Analysis of XOS before and after growth established consumption in the DP2 - DP5 range in the four XOS-fermenting strains. XOS were consumed simultaneously with glucose, while xylose was consumed after glucose depletion. Cell-associated β-xylosidase activity was detected in the XOS fermenting strains. Analysis of genomic data suggests this activity to be linked to genes encoding glycoside hydrolases from family 3, 8 or 43. No endo-β-xylanase activity was detectable. Main fermentation end products were lactate and acetate. A higher ratio of acetic acid/lactic acid was produced during growth on XOS compared to growth on glucose. This is the first report on utilization of XOS in Weissella, indicating an increased probiotic potential for XOS-utilizing strains from the species pair W. confusa/ W. cibaria, but also showing that XOS utilization is strain-dependent for these species. This article is protected by copyright. All rights reserved.
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| 3. |
- 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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| 4. |
- 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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| 5. |
- 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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| 6. |
- Adlercreutz, Patrick, et al.
(författare)
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Characterization of Gluconobacter oxydans immobilized in calcium alginate
- 1985
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Ingår i: Applied Microbiology and Biotechnology. - 0175-7598. ; 22:1, s. 1-7
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Tidskriftsartikel (refereegranskat)abstract
- Gluconobacter oxydans cells were immobilized in calcium alginate and the preparation was used for the oxidation of glycerol to dihydroxyacetone. The characterization was done according to the guidelines given by the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology. The pH optimum of the preparation was found to be 5.0 and the temperature optimum was 40°C. However, the operational stability was better at 30°C. The glycerol concentration required to obtain half the maximal reaction rate was about 5 mM for both immobilized and free cells. At low concentrations of glycerol and high concentrations of dihydroxyacetone a slight inhibition was noted. No loss of activity of the immobilized preparation was observed after storage for 68 days at +4°C. Investigation of the operational stability revealed a half-life of 5 days. Studies of the influence of particle size and cell densities as well as that of oxygen concentration revealed that the oxygen supply was the rate limiting step.
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| 7. |
- Asplund, Maria, 1978-, et al.
(författare)
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Composite biomolecule/PEDOT materials for neural electrodes
- 2008
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Ingår i: Biointerphases. - NY : American Institute of Physics. - 1559-4106 .- 1934-8630. ; 3:3, s. 83-93
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Tidskriftsartikel (refereegranskat)abstract
- Electrodes intended for neural communication must be designed to meet boththe electrochemical and biological requirements essential for long term functionality. Metallic electrode materials have been found inadequate to meet theserequirements and therefore conducting polymers for neural electrodes have emergedas a field of interest. One clear advantage with polymerelectrodes is the possibility to tailor the material to haveoptimal biomechanical and chemical properties for certain applications. To identifyand evaluate new materials for neural communication electrodes, three chargedbiomolecules, fibrinogen, hyaluronic acid (HA), and heparin are used ascounterions in the electrochemical polymerization of poly(3,4-ethylenedioxythiophene) (PEDOT). The resultingmaterial is evaluated electrochemically and the amount of exposed biomoleculeon the surface is quantified. PEDOT:biomolecule surfaces are also studiedwith static contact angle measurements as well as scanning electronmicroscopy and compared to surfaces of PEDOT electrochemically deposited withsurfactant counterion polystyrene sulphonate (PSS). Electrochemical measurements show that PEDOT:heparinand PEDOT:HA, both have the electrochemical properties required for neuralelectrodes, and PEDOT:heparin also compares well to PEDOT:PSS. PEDOT:fibrinogen isfound less suitable as neural electrode material.
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| 8. |
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| 9. |
- 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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| 10. |
- 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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