Proteins play an important role in cells, as the morphology, function and activities of the cell depend on the proteins they express. The key to understanding how different proteins function lies in an understanding of the molecular structure. The overall aim of this thesis was the determination of the structure of DWNN domains. This thesis described the preparation of samples of human DWNN suitable for structural analysis by nuclear magnetic resonance spectroscopy (NMR), as well as NMR analysis.

The development of ligands derived from natural amino acids for asymmetric transfer hydrogenation (ATH) of prochiral ketones is described herein. In the first part, reductions performed in alcoholic media are examined, where it is found that amino acid-derived hydroxamic acids and thioamides, respectively, are simple and versatile ligands that in combination with [RhCp*Cl2]2 efficiently catalyze this particular transformation. Selectivities up to 97% ee of the corresponding secondary alcohols are obtained, and it is furthermore observed that the two different ligand classes, albeit based on the same amino acid scaffold, give rise to products of opposite configuration. The highly interesting enantioswitchable nature of the two abovementioned catalysts is studied in detail by mechanistic investigations. A structure/activity correlation analysis is performed, which reveals that the diverse behavior of the catalysts arise from different interactions between the ligands and the metal. Kinetic studies furthermore stress the catalyst divergence, since a difference in the rate determining step is established from initial rate measurements. In addition, rate constants are determined for each step of the overall reduction process. In the last part, catalyst development for ATH executed in water is discussed. The applicability of hydroxamic acid ligands is further extended, and catalysts based on these compounds are found to be efficient and compatible with aqueous conditions. The structurally even simpler amino acid amide is also evaluated as a ligand, and selectivities up to 90% ee are obtained in the reduction of a number of aryl alkyl ketones. The very challenging reduction of dialkyl ketones is moreover examined in the Rh-catalyzed aqueous ATH, where a modified surfactant-resembling sulfonylated diamine is used as ligand, and the reaction is carried out in the presence of SDS-micelles. A positive effect is to some extent found on the catalyst performance upon addition of phase-transfer components, especially regarding the catalytic activity in the reduction of more hydrophobic substrates. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: In press.

No description available.

Hyaluronic acid (HA) is a high molecular weight polysaccharide that is mainly produced by animals and certain bacteria. This polymer is biocompatible and possesses desirable rheological properties that are accentuated by high molecular weight. Diverse therapeutic applications have developed which harness these features. Pharmaceutical grade HA is mostly extracted from animal tissue. The HA derived from this source is suitable for most pharmaceutical preparations but there is growing pressure to avoid animal tissue products. This has provided the incentive to expand microbial-based HA manufacturing. However, the inherent low molecular weight of the polymer derived via this route has hampered widespread acceptance of microbial HA. This thesis examined the ramifications of improving the cellular economy of the HA-producing, gram-positive bacterium, Streptococcus zooepidemicus. Improved cellular economy is believed to be a prerequisite for achieving superior HA yields and molecular weights in this microorganism. This work examined the metabolic variation that accompanied the shift to more efficient modes of carbon utilization. In particular the effect of different sugar sources, uncoupling growth and polymer formation, and changes to the cellular oxidoreduction capacity were studied in more detail. This study utilized different sugar sources to enhance the recovery of energy. Fermenting glucose, fructose and maltose produced contrasting patterns of growth and HA formation. Culturing the organism in maltose caused a shift towards energy-efficient heterofermentative metabolism. Maltose-cultured cells displayed a biphasic pattern of metabolism. The first stage corresponded to a growth phase in which biomass synthesis profited from the increased energy yield. The second stage corresponded to an arginine-deficient stationary phase where the majority of the HA was formed. The fermentation rate was slower during stationary phase but continued to support HA biosynthesis. This bisphasic metabolism proved to be beneficial. A protracted stationary phase led to higher molecular weight HA. Fructose was unable to sustain a comparable polymer yield or molecular weight as glucose or maltose. There was evidence that the arginine deiminase pathway was responsible for the premature depletion of arginine in maltose-fermenting cultures. The accumulation of biomass exhibited a concentration-dependent response to the amount of glutamine in the medium. A second arginine transporter possessing a low affinity for glutamine could explain this phenomenon. Arginine consumption was slower when the glutamine level was elevated. This may indicate competition for a common transmembrane carrier. An elevated energetic yield and ATP formation rate were features of aerobic maltose metabolism. The relative improvement in biomass and HA yields were substantially greater for cultures fermenting maltose compared to glucose. However, no improvement in molecular weight compared to glucose was observed. A major factor contributing to the success of aerobic maltose fermentation was the particularly high NADH oxidase flux. This enzyme reoxidizes reduction equivalents in a reaction that is physically decoupled from the production of reduced metabolic products. Less lactate and ethanol accumulated in the presence of high NADH oxidase levels but acetate production was stimulated leading to an improved energetic yield. This result prompted an investigation into the effect of elevating the NADH oxidase level. The native NADH oxidase gene was sequenced and cloned into an inducible expression plasmid and introduced into S. zooepidemicus. Overproduction of this enzyme led to the desired improvement in ATP yield. A significant improvement in biomass yield was demonstrated. HA yield and molecular weight were not affected. Lactate and acetate were the main fermentation products. At high induction levels the quantity of lactate and acetate approached limiting levels and pyruvate overflow was more pronounced. This was attributed to insufficient flux capacity of the pyruvate dehydrogenase enzyme complex. The application of metabolic engineering to S. zooepidemicus has provided some insight into the regulation of energy metabolism in this microorganism and its relationship to HA synthesis. This study has observed that the specific rate of HA synthesis is correlated to the sugar uptake rate but is unaffected by the ATP yield. Under present conditions the formation of HA is not limited by the availability of energy. Nonetheless, microbial HA production will benefit from maximizing energetic yield. It was demonstrated that less catabolic carbon was expended to support biomass formation if the energetic yield was high. Therefore more residual carbon was available for HA synthesis.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 7, 2008). Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Univ., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 7 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 7 uppsatser.