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In humans, 75% of all drugs are metabolized by the cytochrome P450-dependent monooxygenase system. Enzymes encoded by the CYP2C, CYP2D, and CYP3A gene clusters account for ∼80% of this activity. There are profound species differences in the multiplicity of cytochrome P450 enzymes, and the use of mouse models to predict pathways of drug metabolism is further complicated by overlapping substrate specificity between enzymes from different gene families. To establish the role of the hepatic and extrahepatic P450 system in drug and foreign chemical disposition, drug efficacy, and toxicity, we created a unique mouse model in which 30 cytochrome P450 genes from the Cyp2c, Cyp2d, and Cyp3a gene clusters have been deleted. Remarkably, despite a wide range of putative important endogenous functions, Cyp2c/2d/3a KO mice were viable and fertile, demonstrating that these genes have evolved primarily as detoxification enzymes. Although there was no overt phenotype, detailed examination showed Cyp2c/2d/3a KO mice had a smaller body size (15%) and larger livers (20%). Changes in hepatic morphology and a decreased blood glucose (30%) were also noted. A five-drug cocktail of cytochrome P450 isozyme probe substrates were used to evaluate changes in drug pharmacokinetics; marked changes were observed in either the pharmacokinetics or metabolites formed from Cyp2c, Cyp2d, and Cyp3a substrates, whereas the metabolism of the Cyp1a substrate caffeine was unchanged. Thus, Cyp2c/2d/3a KO mice provide a powerful model to study the in vivo role of the P450 system in drug metabolism and efficacy, as well as in chemical toxicity.
Developing a new production host from a blueprint: Bacillus pumilus as an industrial enzyme producer
(2014)
Üblicherweise werden biotechnologische Reaktionssysteme im mikrofluidischen Maßstab in vorstrukturierten Bauteilen oder mit auf Wellplatten basierenden Robotersystemen realisiert. In dem hier vorgestellten System werden chemische oder biologische Reaktionen mit magnetischen Mikroreaktoren (MR) durchgeführt, bei denen hydrophobe magnetische Mikropartikel einen wässrigen Kern umschließen. Solche MR bieten eine gute Kontrolle der Reaktionsbedingungen, eine verbesserte Sicherheit und Portabilität. Die neue Plattformtechnologie ermöglicht die zweidimensionale Bewegung der magnetischen MR auf einer planaren Ebene. Oberhalb oder unterhalb der Plattform werden Magnetfeldgradienten zum Manipulieren und Bewegen eines oder mehrerer magnetischer MR erzeugt. Die optimal auf die MR wirkenden magnetischen Kräfte werden experimentell ermittelt und simuliert. Die Aktivierung der Magnetfelder wird automatisiert durch elektrische Spulen mit Eisenkern bzw. Neodymmagnet gesteuert. Angewendet wurde das System beim reversiblen Öffnen von MR, um z. B. Reaktionspartner in den wässrigen Kern zu injizieren oder Proben zu entnehmen. Ferner wurde Lac-case A und b-Glucosidase auf einer Quarzglasoberfläche immobilisiert und mit einem MR zum Reagieren gebracht. Weiterhin wurden MR fusioniert und so ein wässriger Kern bestehend aus Laccase mit einem aus dem entsprechenden Substrat Syringaldazin vereint.
The development of a cost-effective hydrolysis for crude cellulose is an essential part of biorefinery developments. To establish such high solid hydrolysis, a new solid state reactor with static mixing is used. However, concentrations >10% (w/w) cause a rate and yield reduction of enzymatic hydrolysis. By optimizing the synergetic activity of cellulolytic enzymes at solid concentrations of 9%, 17% and 23% (w/w) of crude Organosolv cellulose, glucose concentrations of 57, 113 and 152 g L⁻¹ are reached. However, the glucose yield decreases from 0.81 to 0.72gg⁻¹ at 17% (w/w). Optimal conditions for hydrolysis scale-up under minimal enzyme addition are identified. As result, at 23% (w/w) crude cellulose the glucose yield increases from 0.29 to 0.49gg⁻¹. As proof of its applicability, biobutanol, succinic and itaconic acid are produced with the crude hydrolysate. The potential of the substrate is proven e.g. by a high butanol yield of 0.33gg⁻¹.
Organic anion transporting polypeptide (Oatp) 1a/1b knockout and OATP1B1 and -1B3 humanized mouse models are promising tools for studying the roles of these transporters in drug disposition. Detailed characterization of these models will help to better understand their utility for predicting clinical outcomes. To advance this approach, we carried out a comprehensive analysis of these mouse lines by evaluating the compensatory changes in mRNA expression, quantifying the amounts of OATP1B1 and -1B3 protein by liquid chromatography–tandem mass spectrometry, and studying the active uptake in isolated hepatocytes and the pharmacokinetics of some prototypical substrates including statins. Major outcomes from these studies were 1) mostly moderate compensatory changes in only a few genes involved in drug metabolism and disposition, 2) a robust hepatic expression of OATP1B1 and -1B3 proteins in the respective humanized mouse models, and 3) functional activities of the human transporters in hepatocytes isolated from the humanized models with several substrates tested in vitro and with pravastatin in vivo. However, the expression of OATP1B1 and -1B3 in the humanized models did not significantly alter liver or plasma concentrations of rosuvastatin and pitavastatin compared with Oatp1a/1b knockout controls under the conditions used in our studies. Hence, although the humanized OATP1B1 and -1B3 mice showed in vitro and/or in vivo functional activity with some statins, further characterization of these models is required to define their potential use and limitations in the prediction of drug disposition and drug-drug interactions in humans.
1. Drug metabolizing enzymes and transporters play important roles in the absorption, metabolism, tissue distribution and excretion of various compounds and their metabolites and thus can significantly affect their efficacy and safety. Furthermore, they can be involved in drug–drug interactions which can result in adverse responses, life-threatening toxicity or impaired efficacy. Significant species differences in the interaction of compounds with drug metabolizing enzymes and transporters have been described.
2. In order to overcome the limitation of animal models in accurately predicting human responses, a large variety of mouse models humanized for drug metabolizing enzymes and to a lesser extent drug transporters have been created.
3. This review summarizes the literature describing these mouse models and their key applications in studying the role of drug metabolizing enzymes and transporters in drug bioavailability, tissue distribution, clearance and drug–drug interactions as well as in human metabolite testing and risk assessment.
4. Though such humanized mouse models have certain limitations, there is great potential for their use in basic research and for testing and development of new medicines. These limitations and future potentials will be discussed.
Der zunehmende Bedarf an fossilen Rohstoffen bei gleichzeitig abnehmender Versorgungssicherheit führt zu einer intensiven Suche nach erneuerbaren Ressourcen. Ein vielversprechendes Ausgangsmaterial mit einer weltweiten Verfügbarkeit stellt Gras dar. In 2012 wurden in Deutschland 33 Millionen Tonnen (Heugewicht) Gras auf 4,82 Millionen Hektar Ackerland produziert, davon wurden 60,5 % siliert. Durch die Silierung kann Gras als Substrat zeitlich uneingeschränkt verfügbar sein, ohne dem Risiko des schnellen Verderbs ausgesetzt zu sein. Eine Schlüsselrolle im Rahmen des Silierprozesses nimmt die Produktion von Milchsäure ein. Milchsäure ist einbedeutendes biotechnologisches Produkt für die Lebensmittel- und die chemische Industrie. Im Rahmen dieser Arbeit wird die vollständige Umwandlung der fermentierbaren Zucker in der Silage zu Milchsäure angestrebt, um die maximale Ausbeute der organischen Säure zu erreichen. Im ersten Verfahrensschritt wird die Silage gepresst und der erhaltene Presskuchen einer Liquid-Hot-Water-Behandlung unterzogen. Durch diese einfache Vorbehandlung können hohe Glucoseausbeuten im nachfolgenden SSF-Schritt bei gleichzeitig geringem Enzymeinsatz und Chemikalienverbrauch realisiert werden. Zur Aufreinigung der Milchsäure wurden extraktive und chromatographische Methoden untersucht.
Clostridium propionicum is the only organism known to ferment β-alanine, a constituent of coenzyme A (CoA) and the phosphopantetheinyl prosthetic group of holo-acyl carrier protein. The first step in the fermentation is a CoA-transfer to β-alanine. Subsequently, the resulting β-alanyl-CoA is deaminated by the enzyme β-alanyl-CoA:ammonia lyase (Acl) to reversibly form ammonia and acrylyl-CoA. We have determined the crystal structure of Acl in its apo-form at a resolution of 0.97 Å as well as in complex with CoA at a resolution of 1.59 Å. The structures reveal that the enyzme belongs to a superfamily of proteins exhibiting a so called “hot dog fold” which is characterized by a five-stranded antiparallel β-sheet with a long α-helix packed against it. The functional unit of all “hot dog fold” proteins is a homodimer containing two equivalent substrate binding sites which are established by the dimer interface. In the case of Acl, three functional dimers combine to a homohexamer strongly resembling the homohexamer formed by YciA-like acyl-CoA thioesterases. Here, we propose an enzymatic mechanism based on the crystal structure of the Acl·CoA complex and molecular docking. Proteins 2014; 82:2041–2053. © 2014 Wiley Periodicals, Inc.