03 Fakultät Chemie

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Author: search.filters.author.Schmid, Rolf D.

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    A model of the pressure dependence of the enantioselectivity of Candida rugosa lipase towards (±)-menthol
    (2001) Kahlow, Ulrich; Schmid, Rolf D.; Pleiss, Jürgen
    Transesterification of (±)-menthol using propionic acid anhydride and Candida rugosa lipase was performed in chloroform and water at different pressures (1, 10, 50, and 100 bar) to study the pressure dependence of enantioselectivity E. As a result, E significantly decreased with increasing pressure from E=55 (1 bar) to E=47 (10 bar), E=37 (50 bar), and E=9 (100 bar). In order to rationalize the experimental findings, molecular dynamics simulations of Candida rugosa lipase were carried out. Analyzing the lipase geometry at 1, 10, 50, and 100 bar revealed a cavity in the Candida rugosa lipase. The cavity leads from a position on the surface distinct from the substrate binding site to the core towards the active site and is limited by F415 and the catalytic H449. In the crystal structure of the Candida rugosa lipase, this cavity is filled with 6 water molecules. The number of water molecules in this cavity gradually increased with increasing pressure: 6 molecules in the simulation at 1 bar, 10 molecules at 10 bar, 12 molecules at 50 bar, and 13 molecules at 100 bar. Likewise, the volume of the cavity progressively increased from about 1864 ų in the simulation at 1 bar to 2529 ų at 10 bar, 2526 ų at 50 bar, and 2617 ų at 100 bar. At 100 bar, one water molecule slipped between F415 and H449, displacing the catalytic histidine side chain and thus opening the cavity to form a continuous water channel. The rotation of the side chain leads to a decreased distance between the H449-N and the (+)-menthyl-oxygen (non-preferred enantiomer) in the acyl enzyme intermediate, a factor determining the enantioselectivity of the lipase. While the geometry of the preferred enantiomer is similar in all simulations, the geometry of the non-preferred enantiomer gets gradually more reactive. This observation correlates with the gradually decreasing enantioselectivity E.
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    Rapid detection of neurotoxic insecticides in food using disposable acetylcholinesterase-biosensors and simple solvent extraction
    (2002) Schulze, Holger; Schmid, Rolf D.; Bachmann, Till T.
    The extensive use of pesticides to protect agricultural crops necessitates reliable tools for the detection of residues in food and water, thus ensuring environmental protection and consumer safety. Neuroinhibitors such as organophosphates and carbamates in particular, represent a potential hazard to human health. These compounds are frequently found in food but conventional methods of analysis are limited as they are either time consuming or not sufficiently sensitive. As a result, a rapid and sensitive biosensor test based on AChE-inhibition was developed. The disposable AChE-biosensor was directly applied in solvent extracts of food samples using isooctane as extraction solvent. A complete assay could be performed in less than 2 hours. Recovery rates of 84 % were obtained in tests with spiked orange juice samples. Tests in food samples with a lower water content resulted in reduced recovery rates (44 % for peach pap baby food). Phosphorothionate insecticides could be detected after direct oxidation in food with N-bromosuccinimide and solvent extraction. The assay displayed a detection limit of 2 μg/kg paraoxon which was sufficient for the monitoring of maximum residue limits in food according to EU regulations.
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    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.
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    Cloning, expression, and characterization of a self-sufficient cytochrome P450 monooxygenase from Rhodococcus ruber DSM 44319
    (2006) Liu, Luo; Schmid, Rolf D.; Urlacher, Vlada B.
    A new member of class IV of cytochrome P450 monooxygenases was identified in Rhodococcus ruber strain DSM 44319. As the genome of Rhodococcus ruber has not been sequenced, a P450-like gene fragment was amplified using degenerated primers. The flanking regions of the P450-like DNA fragment were identified by directional genome walking using PCR. The primary protein structure suggests a natural self-sufficient fusion protein consisting of a ferredoxin, flavin-containing reductase and P450 monooxygenase. The only flavin found within the enzyme was FMN. The enzyme was successfully expressed in Escherichia coli and purified and characterized. In the presence of NADPH, the P450 monooxygenase showed hydroxylation activity towards polycyclic aromatic hydrocarbons naphthalene, indene, acenaphthene, toluene, fluorene, m-xylene and ethyl benzene. The conversion of naphthalene, acenaphthene and fluorene resulted in respective ring monohydroxylated metabolites. Alkyl aromatics like toluene, m-xylene and ethyl benzene were hydroxylated exclusively at the side chains. The new enzyme’s ability to oxidize such compounds makes it a potential candidate for biodegradation of pollutants and an attractive biocatalyst for synthesis.
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    Catalytic hydroxylation in biphasic systems using CYP102A1 mutants
    (2005) Maurer, Steffen Christian; Kühnel, Katja; Kaysser, Leonard A.; Eiben, Sabine; Schmid, Rolf D.; Urlacher, Vlada B.
    Cytochrome P450 monooxygenases are biocatalysts that hydroxylate or epoxidise a wide range of hydrophobic organic substrates. To date their technical application is limited to a small number of whole-cell biooxidations. The use of the isolated enzymes is believed to be impractical due to the low stability of this enzyme class, to the stochiometric need of the expensive cofactor NADPH, and due to the low solubility of most substrates in aqueous media. To overcome these problems we have investigated the application of a bacterial monooxygenase (mutants of CYP102A1) in a biphasic reaction system supported by cofactor recycling with NADP+-dependent formate dehydrogenase from Pseudomonas sp 101. Using this experimental setup, cyclohexane, octane and myristic acid were hydroxylated. To reduce the process costs a novel NADH-dependent double mutant of CYP102A1 was designed. For recycling of NADH during myristic acid hydroxylation in a biphasic system NAD+-dependent FDH was used. Stability of the monooxygenase under the reaction conditions is quite high as revealed by total turnover numbers of up to 12850 in NADPH-dependent cyclohexane hydroxylation and up to 30000 in NADH-dependent myristic acid oxidation.
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    "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.
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    Insight into the mechanism of the IMP-1 metallo-beta-lactamase by molecular dynamics simulations
    (2003) Ölschläger, Peter; Schmid, Rolf D.; Pleiss, Jürgen
    Two models, a purely nonbonded model and a cationic dummy atom approach, were examined for the modeling of the binuclear zinc-containing IMP-1 metallo-beta-lactamase in complex with a mercaptocarboxylate inhibitor. The cationic dummy atom approach had substantial advantages as it maintained the initial, experimentally determined geometry of the metal-containing active site during molecular dynamics simulations in water. The method was extended to the modeling of the free enzyme and the enzyme in complex with a cephalosporin substrate docked in an intermediate structure. For all three systems, the modeled complexes and the tetrahedral coordination of the zinc ions were stable. The average zinc-zinc distance increased by about 1 Å in the substrate complex compared to the inhibitor complex and the free enzyme in which a hydroxide ion acts as a bridging ligand. Thus, the zinc ions are predicted to undergo a back and forth movement upon the cycle of hydrolysis. In contrast to previous assumptions, no interaction of the Asn167 side chain with the bound cephalosporin substrate was observed. Our observations are in agreement with quantum-mechanical calculations and experimental data and indicate that the cationic dummy atom approach is useful to model zinc-containing metallo-beta-lactamases as free proteins, in complex with inhibitors and in complex with substrates.
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    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.
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    High-yield expression of the recombinant, atrazine-specific Fab fragment K411B by the methylotrophic yeast Pichia pastoris
    (2001) Lange, Stefan; Schmitt, Jutta; Schmid, Rolf D.
    In this report, we describe the high-yield secretory expression (~ 40 mg l-1) of pure, atrazine-specific Fab fragments (K411B) from P. pastoris that was achieved by co-integration of the genes encoding the heavy and light chains (both under the control of the alcohol oxidase promoter) into the genome of the yeast cells. Antibody-expressing clones were selected by SDS-PAGE and ELISA and fed-batch fermentations were carried out in a 5 l scale. Both chains of the Fab were successfully expressed upon methanol induction and almost no other proteins were secreted into the media. Approximately 30 % of the two chains formed the active Fab fragment containing the intermolecular disulphide bond, as determined by Western blot analysis under non-reducing conditions. Crude culture supernatant was used to study the binding properties of the Fab fragment toward different s-triazines by means of competitive ELISA: the IC50 value for the detection of atrazine was determined from the standard curve as 3 µg l-1, which is one magnitude higher than the value obtained with the parental mAb K4E7 but equals that obtained when the same Fab fragment was expressed in E. coli cells. In addition, the cross-reactivity pattern of the Fab from Pichia is comparable to that of E. coli and the parental mAb K4E7.
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    Structural basis of stereoselectivity in Candida rugosa lipase-catalyzed hydrolysis of secondary alcohols
    (2001) Schulz, Tanja; Schmid, Rolf D.; Pleiss, Jürgen
    Lipases are widely applied catalysts for highly enantioselective resolution of chiral secondary alcohols. While stereopreference is determined predominantly by the substrate structure, stereoselectivity (enantioselectivity and diastereoselectivity) depends on atomic details of interactions between substrate and lipase. Experimentally obtained stereoselectivity and activity in the hydrolysis of butanoic acid esters of two secondary alcohols with two neighbouring stereocenters by Candida rugosa lipase have been investigated by computer-aided molecular modeling of tetrahedral substrate intermediates in complex with the lipase. Breakdown of this intermediate is considered to be the rate-limiting step. Sterical interactions of stereo isomers with the side chain of catalytic histidine led to different orientations of the imidazole. The distance d(HNε-Oalc) between HNε of the imidazole side chain of catalytic histidine and the alcohol oxygen of the substrate was identified to correlate with the experimentally determined reactivity order of the four stereo isomers. Modelled distances d(HNε-Oalc) were short (≤ 1.8 Å) for RR stereo isomers, which were also experimentally found to be hydrolyzed most rapidly; distances d(HNε-Oalc) were about 2 Å for SS and SR stereo isomers, which were converted at similar rates but at lower rate than RR stereo isomers; finally, distances d(HNε-Oalc) for SR stereo isomers were greater than 4 Å, in accordance with very slow conversion of SR stereo isomers.