<p>Laccase is a blue multi-copper oxidase. It has a broad biotechnical potential which increases the interest to study the enzyme further. A laccase-encoding gene from the white-rot fungus Trametes versicolor (lcc2) was mutated using two different methods for random mutagenesis: error-prone PCR and a method based on an E.coli strain (ES1301 mutS) that introduces random mutations. For the error-prone PCR reaction, the vector pPICZB with the lcc2 gene inserted was used as template. The E. coli strain ES1301 mutS was transformed with the vector pBluescript SKII with the lcc2 gene as insert. The mutagenesis products were cloned into the Pichia pastoris expression vector pPICZB for transformation of P. pastoris SMD1168. The transformants were spread on agar plates containing zeocin. Laccase-secreting transformants were selected by their ability to oxidize the substrates ABTS [2,2´-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] and syringaldazine [N,N´-bis(3,5-dimethoxy-4-hydroxybenzylidene)hydrazine], the products of which give green and purple colour, respectively. Around 20 transformants from each of the mutagenesis methods were transformed to plates containing 1 mM ABTS or 1 mM syringaldazine. None of the transformants produced any colour. Control transformants (pPICZB with unmutated lcc2) were also spread on plates with either ABTS or syringaldazine. The transformants gave rise to green colour after 24 hours on the ABTS plates and to purple colour after 72 hours on the syringaldazine plates. Experimets with different chromogenic substrates indicated that ABTS and syringaldazine were best suited for screening of mutants. Remazol Brilliant Blue and Phenol Red are two substrates that after optimisation can serve as alternatives for the selection of laccase-secreting transformants.</p>

<p>Proteins exist in a variety of isoforms with minor differences, mostly due to their glycosylation patterns, which can modulate their biological functions. It seems to be of clinical relevance to measure the isoform-distribution.</p><p>Thesis describes a novel technology named Membrane-Assisted Isoform ImmunoAssay (MAIIA). This technique allows rapid (< 15 min.) isoform determination. It is based on a chromatographic separation combined with immunoassay detection. These steps are performed along a thin, disposable micro-porous chip in which capillary forces maintain the flow. By using anion-exchange as a chromatographic principle the technology has been utilized for the determination of transferrin isoforms in ten minutes. In one variant (the one-dimensional), selected isoforms (carbohydrate-deficient transferrin) are quantified. In a more elaborate variant (the two-dimensional) it was possible to determine the entire isoform profile of transferrin. Isoforms differing by only 0.1 pH unit in isoelectric point could be distinguished.</p><p>The chromatography along the microporous bed of nitrocellulose showed very good separation performance with plate heights of 10-20 µm and only minor flow rate variations between individual devices. </p><p>The quantitative determination of antibody-captured molecules was performed by using antibodies labelled with carbon black particles. Combined with a detection procedure by means of a flatbed scanner, a highly sensitive and specific immunoassay with a detection limit of 0.13 pM was obtained upon using IgE as a model analyte.</p><p>This technology can thus be used to rapidly distinguish proteins with minor structure differences and specifically determine protein isoforms in complex environments, e.g., blood, down in the pM (10^-12 M) concentration range.</p>

<p>Two polysaccharide-degrading enzymes (endo-1,4-D-glucanase and β-mannanase) from blue mussel, <i>Mytilus edulis</i>, have been purified to homogeneity using a combination of several chromatographic steps. Each enzyme has been characterized with regard to its molecular weight, isoelectric point, pH and temperature stability, pH and temperature optimum and substrate specificity. The amino acid sequence of the endoglucanase has been determined at the protein level. The two enzymes are true blue mussel proteins as confirmed at the DNA level. The nucleotide sequences of synthesized cDNA from digestive gland and of genomic DNA from gill tissue were compared. Both genes contain introns, a property typical of eucaryotic organisms. Amino acid sequence based classification has revealed that the endoglucanase belongs to the glycoside hydrolase family 45, subfamily 2 while β-mannanase is a member of family 5. </p><p>Both enzymes form insoluble inclusion bodies when expressed in <i>Escherichia coli</i>. Refolding attempts were unsuccessful. However, the β-mannanase was successfully expressed in the methylotropic yeast <i>Pichia pastoris</i> with an expression level above 100 mg/l in shaking culture. Crystals of the endoglucanase were made from the native protein and a dataset was collected to 1.85 Å resolution using an in-house rotating anode x-ray source. Crystals were also produced using recombinant β-mannanase and a dataset was collected to 1.4 Å resolution at the ESRF synchrotron beamline ID14-EH1. The three dimensional structure of the endoglucanase was solved by X-ray crystallography.</p>

Brown seaweeds lack lignin and have a low cellulose content. Thus, seaweeds should be an easier material for biological degradation than land plants. However, seaweeds have a complex composition, and complete degradation of the material necessitates the presence of microorganisms with a broad substrate range. During anaerobic degradation of organic material, energy carriers such as methane and ethanol may be produced. This is a study of two particular species of brown seaweeds; Laminaria hyperborea and Ascophyllum nodosum, which are the most abundant Norwegian species and also the two species that are commercially harvested in Norway. Most of the degradation studies were carried out in batch systems at pH 7 and at 35 °C. The digestion pattern of the seaweeds were studied by measuring gas production, alginate lyase activity, remaining alginate, the concentrations of uronic acids, VS, COD, mannitol, organic acids and polyphenols. NIR spectroscopy was applied as a new method for alginate quantification. Ethanol production was carried out at 30 °C at different pH, both in batch and continuous cultures. Gas production and concentrations of mannitol, laminaran, ethanol and organic acids were measured. Methane is the end product of a mixed microbial community. However, it is the initial steps of hydrolysis and acidogenesis that are specific for the raw material. Alginate forms the major structural component of brown algae, and its degradation is catalysed by alginate lyases. Polyphenols proved to be the most important limiting factor in the biodegradation: the content of polyphenols was much higher in A. nodosum than L. hyperborea, and this led to a reduced biodegradability of A. nodosum. However, when the polyphenols were fixed with formaldehyde, this seaweed was also readily degraded. Manipulation of the content of polyphenols in L. hyperborea gave similar results. This toxic effect was probably caused by direct inhibition of the microbes, especially the methanogenic bacteria, and complexation reactions with algal material and enzymes. Generally, the guluronate content of the remaining alginate increased during biodegradation, probably due to the Ca-linked guluronate junction zones less accessible for alginate lyase. The main organic product of the acidogenesis was acetate, which was easily converted to methane. In this study, it was not attempted to optimise the methane yield. Ethanol is an intermediate in the complete digestion of organic material and is produced by specific microbial strains. Thus, ethanol production should take place under controlled conditions to prevent contamination problems. The complex composition of seaweeds makes it a difficult substrate to ferment to ethanol by one or a few strains of microbes. In this work, laminaran and mannitol extracted from L hyperborea fronds were used as substrate for ethanol production. A bacterium, Zymobacter palmae, was able to produce ethanol from mannitol, but could not utilise laminaran. However, the yeast Pichia angophorae was able to produce ethanol from both substrates simultaneously. Some supply of oxygen was necessary for the fermentation of mannitol, while a too high aeration resulted in the production of organic acids. Thus, it has been shown that both methane and ethanol can be produced from brown seaweeds. However, an optimisation of the processes will be necessary. Energy production from seaweeds will only be economic if the harvesting costs are low. It may be noted that wastes from the alginate industry may be considered a non-cost raw material for energy production.

The scope for this work was to develop rapid assays for enumerating microorganisms on gels, and to test whether fouling of gel surfaces is principally different from that of solid substrata. For this purpose, a standard set of different gels were selected, based on biocompatibility, polymer charge and gel strength. Bacterial adhesion to gels could be conveniently enumerated by first staining with SYBR Green I nucleic acid gel strain. Images were then collected using a confocal scanning laser microscope, followed by image analysis to determine the percent coverage of bacteria. Diatom adhesion could be qualified using a fluorescence scanner recording the fluorescent chlorophyll, showing a clear correlation between average fluorescence signals and cell density determined by counting. This method was successfully tested on glass, gels, a painted surface and an antifouling coated surface. Adhesion of the marine bacterium Pseudomonas sp. NCIMB 2021on gels decreased at higher shear rates. At low shear rates, adhesion varied significantly between different gels in the following descending order: alginate &gt; agarose &gt; chitosan &gt; PVA-SbQ. Lowest cell coverage at all shear rates was recorded on the most hydrophobic gel, PVA-SbQ. Earlier work has shown that this organism adhere better to solid hydrophobic than solid hydrophilic surfaces. Thus, other properties than the surface free energy may be more important for bacterial adhesion to the gels. The marine diatom Amphora coffeaeformis was applied in the different adhesion arrays under different shear conditions. At high shear, cells adhered better to highly ionic polymer gels alginate and chitosan than to the low charge polymer gels agarose and PVA-SbQ. At very low shear, A. coeffeaeformis developed a film even on agarose equivalent to that on the charged polymer gels. Adhesion to PVA-SbQ remained low at all shear rates. As observed for solid substrate, low charge density led to reduced attachment. Settlement of Balanus amphitrite cypris larvae was tested at different polymer concentrations of the hydrogels. All gels inhibited cypris settlement compared to solid polystyrene controls. Gels consisting of 2.5% PVA-SbQ or 0.5% agarose showed the most promising antifouling properties. In all gel experiments, most of the non-settled larvae were able to settle when transferred and offered a suitable solid substratum. Results indicated that the gel strength was an important factor for cyprid settlement on gels, while the surface wettability seemed to be of minor importance. A few preliminary field experiments were carried out. These tests suggested that marine bacterial biofilm development is more readily on glass than on a PVA-SbQ gel surface, in accordance with monoculture lab experiments. However, similar amounts of photosynthetic organisms adhered to gels of agorose, alginate, chitosan and PVA-SbQ tested in an outdoor seawater basin during spring bloom. Finally, barnacle settlement was delayed on PVA-SbQ gels exposed in the open sea. After incubation for a full summer season, even those gels became as covered with marine fouling organisms as any other non-toxic surface. In conclusion, no universal antifouling effects of hydrogels were found. However, this work suggests that both adhesion of a bacterium and settlement of barnacle cypris larvae on gel surfaces may be principally different from solid substrata. Diatom adhesion, on the other hand, was lower on gels with a low charge density, as observed for solid substrata. In general, the most hydrophobic gel, PVA-SbQ, was the least attractive surface for all three organisms.

Proteins exist in a variety of isoforms with minor differences, mostly due to their glycosylation patterns, which can modulate their biological functions. It seems to be of clinical relevance to measure the isoform-distribution. Thesis describes a novel technology named Membrane-Assisted Isoform ImmunoAssay (MAIIA). This technique allows rapid (&lt; 15 min.) isoform determination. It is based on a chromatographic separation combined with immunoassay detection. These steps are performed along a thin, disposable micro-porous chip in which capillary forces maintain the flow. By using anion-exchange as a chromatographic principle the technology has been utilized for the determination of transferrin isoforms in ten minutes. In one variant (the one-dimensional), selected isoforms (carbohydrate-deficient transferrin) are quantified. In a more elaborate variant (the two-dimensional) it was possible to determine the entire isoform profile of transferrin. Isoforms differing by only 0.1 pH unit in isoelectric point could be distinguished. The chromatography along the microporous bed of nitrocellulose showed very good separation performance with plate heights of 10-20 µm and only minor flow rate variations between individual devices. The quantitative determination of antibody-captured molecules was performed by using antibodies labelled with carbon black particles. Combined with a detection procedure by means of a flatbed scanner, a highly sensitive and specific immunoassay with a detection limit of 0.13 pM was obtained upon using IgE as a model analyte. This technology can thus be used to rapidly distinguish proteins with minor structure differences and specifically determine protein isoforms in complex environments, e.g., blood, down in the pM (10-12 M) concentration range.

Two polysaccharide-degrading enzymes (endo-1,4-D-glucanase and β-mannanase) from blue mussel, Mytilus edulis, have been purified to homogeneity using a combination of several chromatographic steps. Each enzyme has been characterized with regard to its molecular weight, isoelectric point, pH and temperature stability, pH and temperature optimum and substrate specificity. The amino acid sequence of the endoglucanase has been determined at the protein level. The two enzymes are true blue mussel proteins as confirmed at the DNA level. The nucleotide sequences of synthesized cDNA from digestive gland and of genomic DNA from gill tissue were compared. Both genes contain introns, a property typical of eucaryotic organisms. Amino acid sequence based classification has revealed that the endoglucanase belongs to the glycoside hydrolase family 45, subfamily 2 while β-mannanase is a member of family 5. Both enzymes form insoluble inclusion bodies when expressed in Escherichia coli. Refolding attempts were unsuccessful. However, the β-mannanase was successfully expressed in the methylotropic yeast Pichia pastoris with an expression level above 100 mg/l in shaking culture. Crystals of the endoglucanase were made from the native protein and a dataset was collected to 1.85 Å resolution using an in-house rotating anode x-ray source. Crystals were also produced using recombinant β-mannanase and a dataset was collected to 1.4 Å resolution at the ESRF synchrotron beamline ID14-EH1. The three dimensional structure of the endoglucanase was solved by X-ray crystallography.

Innovation is the fundamental driver for the advancement of societies. The advent of the Industrial Revolution in the 17th century precipitated a dramatic increase in the rate of innovation. Societies of the time struggled in how to deal with the rapid changes that resulted from these innovations and their application. Present day society is no different. Innovations in todays society have the ability to be widely adopted and the potential to affect far larger segments of the population that previous innovations. The rapid rise of genetic modification is one such innovation. This innovative technology has been widely adopted by the drug and agriculture industries and as a result, it has impacted all segments of Canadian society. This thesis examines how Canadian society has dealt with the specific innovation of agricultural biotechnology, or the genetic modification of plants. The commercialization of genetically modified plants has resulted in regulatory challenges for the government, intellectual property and liability concerns for industry and consumer acceptance issues within the general public. By researching the interaction and relationships between government regulators, private firms and consumer organizations, it is possible to identify how Canada has reacted to the challenge of regulating agricultural biotechnology. The interdisciplinary framework necessary to accomplish this requires conceptual contributions from economics, political science and sociology. In the development of the innovation, or innovative product, the regulatory approval process requires a risk analysis for all new plant varieties. This risk analysis process is comprised of risk assessment, risk management and risk communication. This thesis argues that risk and the application of risk analysis is appropriate for pre-commercialization, but once the innovative product is in the marketplace, any failure regarding this product can be viewed as a potential liability. The management of and communication about liabilities differs from that of risk management and communication. The key theme of this research is to examine how regulators in Canada have attempted to regulate post-commercialization liabilities and to identify what governance structures or institutions are essential for the regulation of post-commercialization liabilities.

Innovation is the fundamental driver for the advancement of societies. The advent of the Industrial Revolution in the 17th century precipitated a dramatic increase in the rate of innovation. Societies of the time struggled in how to deal with the rapid changes that resulted from these innovations and their application. Present day society is no different. Innovations in todays society have the ability to be widely adopted and the potential to affect far larger segments of the population that previous innovations. The rapid rise of genetic modification is one such innovation. This innovative technology has been widely adopted by the drug and agriculture industries and as a result, it has impacted all segments of Canadian society. This thesis examines how Canadian society has dealt with the specific innovation of agricultural biotechnology, or the genetic modification of plants. The commercialization of genetically modified plants has resulted in regulatory challenges for the government, intellectual property and liability concerns for industry and consumer acceptance issues within the general public. By researching the interaction and relationships between government regulators, private firms and consumer organizations, it is possible to identify how Canada has reacted to the challenge of regulating agricultural biotechnology. The interdisciplinary framework necessary to accomplish this requires conceptual contributions from economics, political science and sociology. In the development of the innovation, or innovative product, the regulatory approval process requires a risk analysis for all new plant varieties. This risk analysis process is comprised of risk assessment, risk management and risk communication. This thesis argues that risk and the application of risk analysis is appropriate for pre-commercialization, but once the innovative product is in the marketplace, any failure regarding this product can be viewed as a potential liability. The management of and communication about liabilities differs from that of risk management and communication. The key theme of this research is to examine how regulators in Canada have attempted to regulate post-commercialization liabilities and to identify what governance structures or institutions are essential for the regulation of post-commercialization liabilities.

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