Kiel, Univ., Diss., 2004. / Computerdatei im Fernzugriff.

München, Techn. Univ., Diss., 2003. / Computerdatei im Fernzugriff.

Regensburg, Univ., Diss., 2008

München, Techn. Univ., Diss., 2003.

Techn. Universiẗat, Diss., 2003--München.

Universiẗat, Diss., 2004--Kiel.

<p>Members of the thioredoxin superfamily of proteins catalyze disulfide bond reduction and oxidation using the active site C-X-X-C sequence. In hyperthermophilic organisms, cysteine side chains were expected in low abundance since they were not believed to endure the high temperatures under which they grow. Recently it has been found that disulfide bonds in hyperthermophiles are more frequent, the higher the growth temperature of the organism. This is perhaps used as an adaptation to high temperature in order to stabilize proteins under harsh conditions. A protein with sequence and structural similarities to mesophilic members of the thioredoxin superfamily, called protein disulfide oxidoreductases (PDO), has been found in the genomes of recently sequenced hyperthermophilic genomes. In this study PDOs from the hyperthermophiles Aquifex aeolicus (AaPDO) and Pyrococcus furiosus (PfPDO) have been investigated. The molecular weight is about 26 kDa and their structures are comprised of two homologous thioredoxin folds, referred to as the N-unit and the C-unit, each containing a C-X-X-C motif. The sequence identity between the two units and the two proteins is low, but they are still structurally very similar. The function of these proteins in vivo is unknown. As a first step in characterizing the activity of these proteins, the redox characteristics of these domains will be investigated. During this project, the genes for AaPDO and PfPDO have been cloned into overexpression vectors, expressed in E. coli and purified to homogeneity. To allow for individual study of the activities of two units, mutated proteins were prepared in which the cysteine residues of the N-unit (AaPDOnm and PfPDOnm) and of the C-unit (AaPDOcm and PfPDOcm) and purified. Circular dichroism spectra recorded of the wild type and mutants indicate that all purified proteins are folded and that the N- and C-unit active site mutants are structurally similar to the corresponding wild type proteins.</p>

Members of the thioredoxin superfamily of proteins catalyze disulfide bond reduction and oxidation using the active site C-X-X-C sequence. In hyperthermophilic organisms, cysteine side chains were expected in low abundance since they were not believed to endure the high temperatures under which they grow. Recently it has been found that disulfide bonds in hyperthermophiles are more frequent, the higher the growth temperature of the organism. This is perhaps used as an adaptation to high temperature in order to stabilize proteins under harsh conditions. A protein with sequence and structural similarities to mesophilic members of the thioredoxin superfamily, called protein disulfide oxidoreductases (PDO), has been found in the genomes of recently sequenced hyperthermophilic genomes. In this study PDOs from the hyperthermophiles Aquifex aeolicus (AaPDO) and Pyrococcus furiosus (PfPDO) have been investigated. The molecular weight is about 26 kDa and their structures are comprised of two homologous thioredoxin folds, referred to as the N-unit and the C-unit, each containing a C-X-X-C motif. The sequence identity between the two units and the two proteins is low, but they are still structurally very similar. The function of these proteins in vivo is unknown. As a first step in characterizing the activity of these proteins, the redox characteristics of these domains will be investigated. During this project, the genes for AaPDO and PfPDO have been cloned into overexpression vectors, expressed in E. coli and purified to homogeneity. To allow for individual study of the activities of two units, mutated proteins were prepared in which the cysteine residues of the N-unit (AaPDOnm and PfPDOnm) and of the C-unit (AaPDOcm and PfPDOcm) and purified. Circular dichroism spectra recorded of the wild type and mutants indicate that all purified proteins are folded and that the N- and C-unit active site mutants are structurally similar to the corresponding wild type proteins.

Pyrococcus furiosus extracellular &alpha / -amylase is a hyperthermostable glucosyl hydrolyzing enzyme which shows unique biochemical properties that may have impact on improving starch hydrolysis process / however, it is insignificantly expressed in its native archaeal host. In this study, it was aimed to express the P. furiosus extracellular alpha-amylase (PFA) in Pichia pastoris, which is a well-recognized overexpression host used in production of heterologous proteins. In this context, first, P. furiosus was grown under anaerobic conditions in capped bottles for t= 12 h at T=90&deg / C and then its genomic DNA was isolated. PFA coding cDNA frame was amplified using two specifically designed oligonucleotides and cloned into pPICZ&alpha / A expression vector. Then wild type P. pastoris X-33 cells were transfected with pPICZ&alpha / A::PFA construct. In shake flask production medium, existence of recombinant PFA activity was tested and biochemical characterization of the recombinant product was done. This was the first time PFA is expressed in an eukaryotic host. Optimum working temperature and pH of the rPFA were found to be 95 &deg / C and within the range of 4.5-6.5, respectively. rPFA is independent to metal ions and inhibition by production medium of P. pastoris was observed, in presence of divalent metal ions. Although Saccharomyces cerevisiae &alpha / -factor secretion signal was fused to the N terminal of rPFA, minute amount of extracellular secretion was detected but the majority of the enzymatic activity remained in the intracellular medium. The best producer strain was selected by measuring &alpha / -amylase activity in cell extracts by DNS method. Effects of pH on cell growth and recombinant protein production were determined by shake flask experiments and maximum of 4800 U/l rPFA was detected with 7.30 g/l wet cell density in pH=6 buffered medium. In order to achieve higher rPFA production, two bioreactor experiments were designed at two different pH operation conditions, namely pH=4 and pH=5, in a working volume of 1 L. The dissolved oxygen tension was kept over 20% and predetermined exponential methanol feeding strategy was employed in order to fix specific cell growth rate, &micro / , at 0.03 h-1. At pH=4 operation, maximum of 73,400 U/l &alpha / -amylase activity was detected at the t=27 h of production phase when the wet cell density was 209 g/l.

Regensburg, Univ., Diss., 2008