03 Fakultät Chemie
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Item Open Access Impact of remote mutations on metallo-beta-lactamase substrate specificity : implications for the evolution of antibiotic resistance(2005) Ölschläger, Peter; Mayo, Stephen L.; Pleiss, JürgenMetallo-beta-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of beta-lactam antibiotics. The G262S point mutation distinguishing the metallo-beta-lactamase IMP 1 from IMP 6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants, with IMP-1 being the most active. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket and lead to enhanced activity. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion and increased sensitivity to mutations. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally.Item Open Access Targeted methylation of the epithelial cell adhesion molecule (EpCAM) promoter to silence its expression in ovarian cancer cells(2014) Nunna, Suneetha; Reinhardt, Richard; Ragozin, Sergey; Jeltsch, AlbertThe Epithelial Cell Adhesion Molecule (EpCAM) is overexpressed in many cancers including ovarian cancer and EpCAM overexpression correlates with decreased survival of patients. It was the aim of this study to achieve a targeted methylation of the EpCAM promoter and silence EpCAM gene expression using an engineered zinc finger protein that specifically binds the EpCAM promoter fused to the catalytic domain of the Dnmt3a DNA methyltransferase. We show that transient transfection of this construct increased the methylation of the EpCAM promoter in SKOV3 cells from 4–8% in untreated cells to 30%. Up to 48% methylation was observed in stable cell lines which express the chimeric methyltransferase. Control experiments confirmed that the methylation was dependent on the fusion of the Zinc finger and the methyltransferase domains and specific for the target region. The stable cell lines with methylated EpCAM promoter showed a 60–80% reduction of EpCAM expression as determined at mRNA and protein level and exhibited a significantly reduced cell proliferation. Our data indicate that targeted methylation of the EpCAM promoter could be an approach in the therapy of EpCAM overexpressing cancers.Item Open Access Microbial P450 enzymes in biotechnology(2004) Urlacher, Vlada B.; Lutz-Wahl, Sabine; Schmid, Rolf D.Oxidations are key reactions in chemical syntheses. Biooxidations using fermentation processes have already conquered some niches in industrial oxidation processes, since they allow the introduction of oxygen even into non-activated carbon atoms in a sterically and optically selective manner which is difficult or impossible to achieve by synthetic organic chemistry. Biooxidation using isolated enzymes is limited to oxidases and dehydrogenases. Surprisingly, cytochrome P450 monooxygenases (CYPs) have scarcely been studied for use in biooxidations, although they are one of the largest known superfamilies of enzyme proteins. Their gene sequences have been identified in various organisms such as humans, bacteria, algae, fungi and plants. The reactions catalyzed by P450s are quite diverse and range from biosynthetic pathways (e.g. those of animal hormones and secondary plant metabolites) to the activation or biodegradation of hydrophobic xenobiotic compounds (e. g. those of various drugs in the liver of higher animals). From a practical point of view, the great potential of P450s is limited by their functional complexity, low activity, and limited stability. In addition, P450-catalyzed reactions require a constant supply of NAD(P)H which makes continuous cell-free processes very expensive. Quite recently, several groups have started to investigate cost-efficient ways which could allow the continuous supply of electrons to the heme iron. These include, for example, the use of electron mediators, direct electron supply from electrodes and enzymatic approaches. In addition, methods of protein design and directed evolution have been applied in an attempt to enhance the activity of the enzymes and improve their selectivity. The promising application of bacterial P450s as catalyzing agents in biocatalytic reactions and recent progress made in this field are covered in this review.Item Open Access Whisker formation on Sn thin films(2010) Sobiech, Matthias Lukas; Mittemeijer, Eric Jan (Prof. Dr. Ir.)The system Sn on Cu will usually be applied for interconnection of modern electronic systems, i.e. for mechanical, thermal and electrical “integration” of electronic components (e.g. composed of Cu) on rigid substrates (i.e. printed circuit boards) by (e.g. Sn) solder-joint technology. Nowadays Sn is the material of choice for this purpose because the up to now commonly and successfully used SnPb alloys for soldering and coating applications are prohibited by law since 1st July 2006 due to environmental concerns (Pb-free and “green” legislation). However, it is well known since nearly 60 years that pure Sn thin films deposited on Cu substrates are very prone to spontaneous formation of needle-like Sn single-crystals, called whiskers, during ageing at room temperature. Such filamentary Sn whiskers exhibiting growth rates of about 1 Å/sec constitute an issue of great technological relevance for Sn coated leadframe legs of modern microelectronic devices because whisker-induced short-circuit failures of various electronic devices have resulted in enormous financial damage including breakdowns of satellites, computer centres and military and medical devices. Unfortunately, profound knowledge on this controversially discussed phenomenon of whisker-growth is still lacking. Therefore, particularly in recent years, the electronic industry promotes scientific activities to arrive at fundamental understanding of Sn whisker formation in order to implement industrially reliable (accelerated) whisker tests and/or mitigation strategies. Against the above background, the present thesis focuses in particular on revealing the driving force for Sn whiskering in the system Sn on Cu during room temperature ageing and thus to devise a coherent understanding of the processes leading to the formation and growth of Sn whiskers. The obtained fundamental interrelations of microstructural evolution, phase formation, residual stress development and the associated whiskering of Sn thin films electro- and sputter-deposited on Cu as well as of SnPb thin films electrodeposited on Cu during ageing at room temperature have lead to a qualitative understanding of whisker growth in terms of localized Coble-creep. On this basis, whisker mitigation strategies can be proposed.Item Open Access Durch Überexpression in der Hefe Pichia pastoris zu erhöhter Enantioselektivität : ein neues Kapitel in der Anwendung von Schweineleberesterase(2001) Musidlowska, Anna; Lange, Stefan; Bornscheuer, Uwe TheoLipases and Esterases can be used as efficient biocatalysts for the preparation of a wide variety of optically pure compounds. Whereas a range of lipases - especially of microbial origin - are commercially available, only a few esterases can be obtained for the kinetic resolution of racemates or desymmetrization. In the majority of publications, pig liver esterase (PLE) is used, which is isolated from pig liver by extraction. Although it could be demonstrated, that this preparation can convert a broad range of compounds at partially very high stereoselectivity, its application is encountered with a number of disadvantages.Item Open Access "In gel patch electrophoresis" : a new method for environmental DNA purification(2005) Roh, Changhyun; Villatte, Francois; Kim, Byung-Gee; Schmid, Rolf D.Most of the microorganism species are largely untapped and could represent an interesting reservoir of genes useful for biotechnological applications. Unfortunately, a major difficulty associated with the methods used to isolate environmental DNA is related to the contamination of the extracted material with humic substances. These polyphenolic compounds inhibit the DNA processing reactions and severely impede cloning procedures. In this work, we describe a rapid, simple and efficient method for the purification of genomic DNA from environmental samples: we added a chromatography step directly embedded into an agarose gel electrophoresis. This strategy enabled the DNA extraction from various environmental samples and it appeared that the purity grade was compatible with digestion by restriction enzymes and PCR amplifications.Item Open Access Development, validation, and application of an acetylcholinesterase-biosensor test for the direct detection of insecticide residues in infant food(2002) Schulze, Holger; Scherbaum, Ellen; Anastassiades, Michelangelo; Vorlová, Sandra; Schmid, Rolf D.; Bachmann, Till T.A highly sensitive and rapid food-screening test based on disposable screen-printed biosensors was developed, which is suitable for monitoring infant food. The exposure of infants and children to neurotoxic organophosphates and carbamates is of particular concern because of their higher susceptibility to adverse effects. The European Union has therefore set a very low limit for pesticides in infant food which must not contain concentrations exceeding 10 μg/kg for any given pesticide. The maximum residue limit (MRL) has been set to be near the determination threshold that is typically achieved for pesticides with traditional analytical methods. The biosensor method could detect levels lower than 5 μg/kg and thus clearly fulfills the demands of the EU. To substantiate these measurements, recovery rates were determined and amounted on average to 104 % in food. Matrix effects were eliminated by the introduction of a special electrode treatment. The test was compared with two traditional pesticide multiresidue analysis methods (GC/MS, LC/MS) using 26 fruit and vegetable samples from local markets and 23 samples of processed infant food from Germany, Spain, Poland and the USA. Three infant food samples exceeded the MRL of 10 μg/kg when analyzed by either biosensor test or multiresidue methods.Item Open Access Directed evolution of a bacterial alpha-amylase : towards enhanced pH-performance and higher specific activity(2003) Bessler, Cornelius; Schmitt, Jutta; Maurer, Karl-Heinz; Schmid, Rolf D.Alpha-Amylases, in particular, microbial Alpha-amylases are used widely in industrial processes such as starch liquefaction and pulp processes and more recently in detergency. Following the need for Alpha-amylases adapted to latter, we enhanced the alkali-activity of the Alpha-amylase from Bacillus amyloliquefaciens (BAA). The genes coding for the wild type BAA and the mutants BAA S201N and BAA N297D were subjected to error prone PCR and gene shuffling. For the screening of mutants we developed a novel, reliable assay suitable for high throughput screening based on the Phadebas® assay. One mutant (BAA 42) has an optimal activity at pH 7, corresponding to a shift of one pH unit compared to the wild type. BAA 42 is active over a broader pH-range than the wild type resulting in a fivefold higher activity at pH 10. In addition, the activity in periplasmic extracts and the specific activity increased 4 and 1.5 fold, respectively. Another mutant (BAA 29) possesses a wild type like pH-profile but reveals a 40-fold higher activity in periplasmic extracts and a nine fold higher specific activity. The comparison of the amino acid sequences of these two mutants with other homologous microbial Alpha-amylases revealed the mutation of the highly conserved residues W194R, S197P and A230V. In addition, three further mutations were found K406R, N414S and E356D, the latter being present in other bacterial Alpha-amylases.Item Open Access A comparative study of extraction and purification methods for environmental DNA from soil and sludge samples(2006) Roh, Changhyun; Villatte, Francois; Kim, Byung-Gee; Schmid, Rolf D.An important prerequisite for a successful metagenome library construction is an efficient extraction procedure for DNA out of environmental samples. In this study we compared three indirect and four direct extraction methods, including a commercial kit, in terms of DNA yield, purity and time requirement. A special focus was set on methods which are appropriate for the extraction of environmental DNA (eDNA) from very limited sample sizes (0.1 g) to enable a highly parallel approach. Direct extraction procedures yielded on average 100-fold higher DNA amounts than indirect ones. A drawback of direct extraction was the small fragment size of appr. 12 kb. The quality of the extracted DNA was evaluated by the ability of different restriction enzymes to digest the environmental DNA. Only the commercial kit and a direct extraction method using freeze-thaw cell lysis in combination with an in gel patch electrophoresis with hydroxyapatite to remove humic acid substances yielded DNA, which was completely digested by all restriction enzymes. Moreover, only DNA extracted by these two procedures could be used as template for the amplification of fragments of several 16S rDNA, 18S rDNA groups under standard PCR conditions.Item Open Access Molecular modelling of family GH16 glycoside hydrolases : potential roles for xyloglucan endotransglucosylases/hydrolases in cell wall modification in the Poaceae(2004) Strohmeier, Marco; Hrmova, Maria; Fischer, Markus; Harvey, Andrew J.; Pleiss, Jürgen; Fincher, Geoffrey B.Family GH16 glycoside hydrolases can be assigned to five sub-groups according to their substrate specificities, including xyloglucan endotransglucosylases/hydrolases (XTHs), (1,3)-β- galactanases, (1,4)-β-galactanases/κ-carrageenases, “non-specific” (1,3/1,3;1,4)-β-D-glucan endohydrolases and (1,3;1,4)-β-D-glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni-stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modelling of seven family GH16 glycoside hydrolases, based on structural coordinates for (1,3;1,4)-β-D-glucan endohydrolases and a κ-carrageenase. In combination with multiple sequence alignments, the models predict the three-dimensional dispositions of amino acid residues in the substrate-binding and catalytic sites of XTHs and (1,3/1,3;1,4)-β-D-glucan endohydrolases, for which no structural information is available. Furthermore, they reveal similarities with the active sites of family GH11 (1,4)-β-D-xylan endohydrolases. From a biological viewpoint, the classification and molecular modelling establish structural and evolutionary connections between XTHs, (1,3;1,4)-β-D-glucan endohydrolases and xylan endohydrolases, and raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)-β-D-glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)-β-D-glucan and arabinoxylan modification would be consistent with the apparent over-representation of XTH sequences in cereal EST databases.