| 1. |
- Gullfot, Fredrika, 1967-
(författare)
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Synthesis of xyloglucan oligo- and polysaccharides with glycosynthase technology
- 2009
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Xyloglucans are polysaccharides found as storage polymers in seeds and tubers, and as cross-linking glycans in the cell wall of plants. Their structure is complex with intricate branching patterns, which contribute to the physical properties of the polysaccharide including its binding to and interaction with other glycans such as cellulose. Xyloglucan is widely used in bulk quantities in the food, textile and paper making industries. With an increasing interest in technically more advanced applications of xyloglucan, such as novel biocomposites, there is a need to understand and control the properties and interactions of xyloglucan with other compounds, to decipher the relationship between xyloglucan structure and function, and in particular the effect of different branching patterns. However, due to the structural heterogeneity of the polysaccharide as obtained from natural sources, relevant studies have not been possible to perform in practise. This fact has stimulated an interest in synthetic methods to obtain xyloglucan mimics and analogs with well-defined structure and decoration patterns. Glycosynthases are hydrolytically inactive mutant glycosidases that catalyse the formation of glycosidic linkages between glycosyl fluoride donors and glycoside acceptors. Since its first conception in 1998, the technology is emerging as a useful tool in the synthesis of large, complex polysaccharides. This thesis presents the generation and characterisation of glycosynthases based on xyloglucanase scaffolds for the synthesis of well-defined homogenous xyloglucan oligo- and polysaccharides with regular substitution patterns.
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| 2. |
- Enoksson, Peter, 1957, et al.
(författare)
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Micro- and Nanosystems for Sensing in Medicine
- 2008
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Ingår i: Proceedings of Medicinteknikdagarna 2008, 14-15 October, Göteborg, Sweden. ; , s. 117-
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)
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| 3. |
- Sunner, Hampus, 1981, et al.
(författare)
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Fungal Ferulic Acid Esterases – Specificity and Phylogeny
- 2009
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Ingår i: Italic5 Science and Technology of Biomasses Proceedings Book, M Orlandi, C Crestine (Ed.). Italic5/COST conference, Sept 1-4 2009, Varenna, Italy.
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Ferulic Acid Esterases (FAE) is a large heterogeneous group of enzymes with activity on esters of hydroxy- and metoxy- substituted cinnamic acid derivatives, such as ferulic acid. These ester bonds occur in the cell walls of plants and are especially common in grasses. As little systematic knowledge has been collected about this group of enzymes and only a few enzymes have been biochemically characterised to date, we have explored the phylogeny of FAEs using bioinformatic tools. We can conclude that the known Ferulic Acid Esterases belong to several evolutionary distant groups, two of which have dozens of highly related sequences, and a few groups with no members other than the known enzyme. The phylogeny also suggests certain similarities of substrate specificity within groups and proposes enzymes, whose biochemical characterisation would be especially informative for our understanding of the FAE families.
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| 4. |
- Millati, Ria, 1972, et al.
(författare)
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Ethanol production from xylose and wood hydrolyzate by Mucor indicus at different aeration rates
- 2008
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Ingår i: BioResources. - : North Carolina State University. - 1930-2126 .- 1930-2126. ; 3:4, s. 1020-1029
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Tidskriftsartikel (refereegranskat)abstract
- The fungus Mucor indicus is able to produce ethanol from xylose as well as dilute-acid lignocellulosic hydrolyzates. The fungus completely assimilated 10 g/L xylose as the sole carbon and energy source within 32 to 65 h at an aeration rate of 0.1 to 1.0 vvm. The highest ethanol yield was 0.16 g/g at 0.1 vvm. Xylitol was formed intermediately with a maximum yield of 0.22 g/g at 0.5 vvm., but disappeared towards the end of experiments. During cultivation in a mixture of xylose and glucose, the fungus did not assimilate xylose as long as glucose was present in the medium. The anaerobic cultivation of the fungus in the hydrolyzate containing 20% xylose and 80% hexoses resulted in no assimilation of xylose but complete consumption of the hexoses in less than 15 h. The ethanol yield was 0.44 g/g. However, the xylose in the hydrolyzate was consumed when the media were aerated at 0.067 to 0.333 vvm. The best ethanol yield was 0.44 g/g at 0.067 vvm. The results of this study suggest that M. indicus hydrolyzate can be first fermented anaerobically for hexose assimilation and subsequently continued under oxygen-limited conditions for xylose fermentation.
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| 5. |
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| 6. |
- Hanora, Amro
(författare)
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Primary Recovery of Biologically Active Compounds Using Macroporous Monolithic Hydrogels
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The completion of sequencing of human and other mammalian genomes opens a new era for drug development. New approaches have been proposed for tackling diseases such as development of small molecule therapies based on understanding of human genome and proteome, new proteinous based therapies, where the therapeutic protein can inhibit or modify a certain metabolic pathway or DNA-based therapies delivering a gene into the cell (e.g., cancer cells) to correct or inhibit the imperfect gene function. The possibility of production of novel therapeutics (small, proteinous and DNA based substances) in biotechnological processes utilising mammalian cells or microorganisms such as bacteria or yeast, presents new challenges for the separation processes. Moreover, new methods for high throughput screening are needed to identify potentially important lead molecules within the large molecular libraries. Indeed, designing downstream process (DSP) of biologically active compounds depends on the nature and intended use of such compounds. Commodity compounds that are produced in tons need fast and cheap purification methods. However, high molecular weight molecules intended for pharmaceutical applications, need careful design of DSP to meet the legislator demands such as removal of contaminants and toxic substances. Two examples were demonstrated for the design of DSP purification process for small and large molecular weight compounds. Lactic acid was purified from cell broth using composite ion exchanger in expanded bed chromatographic format. Plasmid DNA was captured directly from cell lysate using monolithic macroporous hydrogel, so called cryogel, grafted with different polycations. Using cryogels with coupled specific ligands, bacterial endotoxin was successfully removed from protein solutions and captured from disposed cell homogenate. Fast parallel screening of complex samples containing target substance was demonstrated using affinity minicryogel columns in 96-well format plate.
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| 7. |
- Soares, Ana
(författare)
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Biodegradation of the Recalcitrant Endocrine Disruptor Nonylphenol
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Currently, over 100 high-volume chemicals have been classified as endocrine disrupters. One of these is nonylphenol, which was used as the model compound in this study. This substance originates mainly from the degradation of non-ionic surfactants (nonylphenol ethoxylates) which are widely used in industrial and domestic applications. Hence, nonylphenol is widespread in the environment. Under anaerobic conditions the degradation of nonylphenol with 10 ethoxylate groups was observed to take place through sequential removal of ethoxylate groups, resulting in the formation of nonylphenol (up to 11 mg/kg dw) and nonylphenol ethoxylate with 1-3 ethoxylate groups that were accumulated on the sludge. Due to its harmful effects and recalcitrance, the investigation of organisms that can metabolize nonylphenol is important. This was performed by acclimatization and enrichment of selected inocula with nonylphenol as the only carbon source. Suitable bacteria were only found in soil contaminated with ethoxylated non-ionic surfactants collected in an industrial area in Gothenburg, Sweden. The bacteria isolated were classified as Stenotrophomonas sp., Pseudomonas mandelii and Pseudomonas veronii, and were able to grow at low temperatures, with an optimum at 10?C for the two Pseudomonas spp. and they were thus classified as cold-adapted microorganisms. These are, to the best of our knowledge, the first reported cold-adapted organisms that can degrade nonylphenol.. The enriched culture was used to inoculate a lab-scale packed-bed bioreactor fed with mineral salt medium saturated with nonylphenol in order to simulate contaminated water/groundwater. Nonylphenol was degraded by 99-100%, at rates of 43 mg/l?day at 10?C and 22 mg/l?day at temperatures of 5.5?C and 15?C. Removal of endocrine-disrupting activity was also observed. The same type of bioreactor was inoculated with a pure strain of Sphingomonas sp. TTPN3, known to biodegrade nonylphenol and classified as a mesophilic organism. The degradation rate achieved at reactor removal yields of 99-100% was 33 mg nonylphenol/l?day at room temperature (22?2?C), which was lower than the rate obtained at 10?C. These results indicate that the low degradation rates usually observed at low temperatures, common in most of the industrialized countries, can be overcome by the use of adapted organisms. This underlines the relevance of studying cold-adapted organisms which are poorly known, according to FISH analysis performed, in favour of mesophilic organisms. Besides bacteria, white-rot fungi, Trametes versicolor and Bjerkandera sp. BOL13 were screened, among other fungi, for their ability to remove nonylphenol from liquid matrices. Removal rates of 6.6 mg nonylphenol/l?day and 6.1 mg nonylphenol/l?day were observed after 25 days of incubation by Trametes versicolor and Bjerkandera sp. BOL13, respectively. These rates could be increased to 9.7 mg nonylphenol/l?day for Bjerkandera sp. BOL13 by raising the concentration of active biomass in the inoculum and reducing mass transfer limitations by agitating the cultures. However, the removal of nonylphenol was not clearly correlated with the production of extracellular enzymes. For Trametes versicolor, nonylphenol removal was closely related with laccase production. This fungus was inhibited at nonylphenol concentrations above 15 mg/l, indicating the toxic effects of nonylphenol on this fungal strain. Nonylphenol removal from soil was also attempted by washing artificially contaminated soil at 40?C. The leachate was then fed into a packed-bed bioreactor where nonylphenol was successfully degraded. However, when real contaminated soil was used, the removal of nonylphenol from the soil decreased, showing the limitation of this method in aged soils where adsorption restricted the extraction process. White-rot fungi are potential soil colonizers with high surface growth rates, and are able to reach pollutants in soil in ways bacteria can not. Trametes versicolor and Bjerkandera sp. BOL13 degraded approximately 430 mg nonylphenol/kg in artificially contaminated soil after 5 weeks of incubation. These results could not be repeated with real contaminated soil due to the high pH of the soil used and the presence of other contaminants which interfered with the growth of the fungi.
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| 8. |
- Waara, Sylvia, et al.
(författare)
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Performance of a constructed wetland system for treatment of landfill leachate.
- 2008
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Ingår i: Waste 2008. Waste and Resource management-A shared responsibility. - Stanton-on-the-Wolds : Waste Conference Ltd.. ; , s. 655-667, s. 655-667
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Konferensbidrag (refereegranskat)abstract
- The performance of a constructed wetland for treatment of landfill leachate has been evaluated based upon data obtained during 4 years (2003-2006). It consists of a series of 10 ponds with a total capacity of 52.000 m3 covering 8 ha. Using univariate and multivariate statistics (PCA) the reduction pattern of a large number of chemical parameters including heavy metals has been investigated in 3 parts of the wetland with equal volume. Analyses show that many parameters are removed to the greatest extent in the first part of the system (e.g. many heavy metals, total suspended solids) or the second part of the system (N-NH4) while other parameters such as total nitrogen are more gradually reduced (10 ton/year removed). Toxicity testing with 5 bioassays showed that toxicity was sometimes observed at the inlet but no toxicity was observed at the outlet for 4 of the test species. The data presented will be used for optimizing the treatment process as well as to improve the monitoring program.
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| 9. |
- Dahlin, Andreas, 1980
(författare)
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Nanoplasmonic Biosensors compatible with Artificial Cell Membranes
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Within life science, there is currently an intense search for novel techniques that enable efficient and reliable analysis of biomolecular interactions. Such methods have future applications within medical diagnostics and drug development, as well as within proteomic research in general. Lately, several concepts have emerged that are based on monitoring molecular binding to surfaces via optical, mechanical or electrical signal transduction. In particular, the plasmons associated with metallic nanoparticles are of interest since they offer a convenient way to monitor biomolecular interactions, also in a miniaturized format, by optical spectroscopy.This thesis describes the development of a biosensor based on the optical properties of nanoscale apertures in continuous metal films. The fabrication and characterization of the nanostructure is described, as well as surface modification protocols based on thiol chemistry for material-specific functionalization. In addition, an experimental setup for spectroscopy is presented together with data analysis algorithms for minimizing noise.It is emphasized that, from an experimental sensing perspective, nanoholes and nanoparticles have essentially the same plasmonic properties. However, the nanoholes offer several advantages because of the fact that the structure is physically different. In particular, it is shown how various artificial cell membranes can be spontaneously formed inside nanoholes. This makes the sensor compatible with studies of processes related to biological membranes. In this context, membrane-bound proteins are of special interest since they constitute a third of our genome and represent the target of half of the most common medical drugs. Potential future applications of the artificial membranes on the plasmonic nanostructures are discussed, with focus on probing transport across the membrane.
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| 10. |
- Löfdahl, Per-Åke, 1959-
(författare)
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On bacterial formats in protein library technology
- 2009
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Millions of years of evolution have resulted in an immense number of different proteins, which participate in virtually every process within cells and thus are of utmost importance for allknown forms of life. In addition, there are several examples of natural proteins which have found use in applications outside their natural environment, such as the use of enzymes infood industry and washing powders or the use of antibodies in diagnostic, bioseparation or therapeutic applications. To improve the performance of proteins in such applications, anumber of techniques, all collectively referred to as ‘protein engineering’, are performed in thelaboratory.Traditionally, methods involving ‘rational design’, where a few alterations are introduced atspecific protein locations to hopefully result in expected improvements have been applied.However, the use of more recent techniques involving a simultaneous construction of a large number of candidate variants (protein libraries) by various diversification principles, fromwhich rare clones showing enhanced properties can be isolated have contributed greatly to thefield of protein engineering.In the present thesis, different protein traits of biotechnological importance have beenaddressed for improvements by the use of such methods, in which there is a crucial need tomaintain a clonal link between the genotype and the phenotype to allow an identification of protein library members isolated by virtue of their functional properties. In all protein library investigations included in this thesis this coupling has been obtained by Escherichia coli bacterialcell-membrane compartmental confinement.In a first study, a combination of error prone PCR and gene-shuffling was applied to the Tobacco Etch Virus (TEV)-protease gene in order to produce collections from which genesencoding variants showing an enhanced soluble expression of the enzyme frequently used inbiotechnology to cleave fusion proteins were identified. Using Green Fluorescence Protein(GFP)-based cell fluorescence analysis, a clone with a five-fold increase in the yield of solubly produced protein was successfully isolated. In a second study, a novel and different GFPbased selection system, in addition also involving targeted in vivo protein degradation principles,was employed for investigations of the substrate sequence space of the same protease. In two additional studies, a selection system denoted Protein Fragment Complementation Assay(PCA), based on the affinity driven structural complementation of a genetically split β-lactamase enzyme was used to identify variants having desired target protein binding abilities,including both specificity and affinity. Using Darwinian principles concerning clonal growth advantages, affibody binding proteins showing sub-nanomolar dissociation constants to thehuman cytokine TNF-α were isolated. Taken together, these studies have shown that the bacterial format is very well suited for use in various aspects of protein library selection.
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