Atypical Glycolysis in Clostridium Thermocellum

Cofactor specificities of glycolytic enzymes in Clostridium thermocellum were studied with cellobiose-grown cells from batch cultures. Intracellular glucose was phosphorylated by glucokinase using GTP rather than ATP. Although phosphofructokinase typically uses ATP as a phosphoryl donor, we found only pyrophosphate (PPi)-linked activity. Phosphoglycerate kinase used both GDP and ADP as phosphoryl acceptors. In agreement with the absence of a pyruvate kinase sequence in the C. thermocellum genome, no activity of this enzyme could be detected. Also, the annotated pyruvate phosphate dikinase (ppdk) is not crucial for the generation of pyruvate from phosphoenolpyruvate (PEP), as deletion of the ppdk gene did not substantially change cellobiose fermentation. Instead pyruvate formation is likely to proceed via a malate shunt with GDP-linked PEP carboxykinase, NADH-linked malate dehydrogenase, and NADP-linked malic enzyme. High activities of these enzymes were detected in extracts of cellobiose-grown cells. Our results thus show that GTP is consumed while both GTP and ATP are produced in glycolysis of C. thermocellum. The requirement for PP i in this pathway can be satisfied only to a small extent by biosynthetic reactions, in con- trast to what is generally assumed for a PPi-dependent glycolysis in anaerobic heterotrophs. Metabolic network analysis showed that most of the required PPi must be generated via ATP or GTP hydrolysis exclusive of that which happens during biosynthesis. Experimental proof for the necessity of an alternative mechanism of PPi generation was obtained by studying the glycolysis in washed-cell suspensions in which biosynthesis was absent. Under these conditions, cells still fermented cellobiose to ethanol

Physiological roles of pyruvate ferredoxin oxidoreductase and pyruvate formate-lyase in Thermoanaerobacterium saccharolyticum JW/SL-YS485

Background: Thermoanaerobacter saccharolyticum is a thermophilic microorganism that has been engineered to produce ethanol at high titer (30–70 g/L) and greater than 90 % theoretical yield. However, few genes involved in pyruvate to ethanol production pathway have been unambiguously identified. In T. saccharolyticum, the products of six putative pfor gene clusters and one pfl gene may be responsible for the conversion of pyruvate to acetyl-CoA. To gain insights into the physiological roles of PFOR and PFL, we studied the effect of deletions of several genes thought to encode these activities. Results: It was found that pyruvate ferredoxin oxidoreductase enzyme (PFOR) is encoded by the pforA gene and plays a key role in pyruvate dissimilation. We further demonstrated that pyruvate formate-lyase activity (PFL) is encoded by the pfl gene. Although the pfl gene is normally expressed at low levels, it is crucial for biosynthesis in T. saccharolyticum. In pforA deletion strains, pfl expression increased and was able to partially compensate for the loss of PFOR activity. Deletion of both pforA and pfl resulted in a strain that required acetate and formate for growth and produced lactate as the primary fermentation product, achieving 88 % theoretical lactate yield. Conclusion: PFOR encoded by Tsac_0046 and PFL encoded by Tsac_0628 are only two routes for converting pyruvate to acetyl-CoA in T. saccharolyticum. The physiological role of PFOR is pyruvate dissimilation, whereas that of PFL is supplying C1 units for biosynthesis

Revisiting the Intriguing Electronic Features of the BeOBeC Carbyne and Some Isomers: A Quantum‐Chemical Assessment

Extensive high‐level quantum‐chemical calculations reveal that the rod‐shaped molecule BeOBeC, which was recently generated in matrix experiments, exists in two nearly isoenergetic states, the 5Σ quintet (56) and the 3Σ triplet (36). Their IR features are hardly distinguishable at finite temperature. The major difference concerns the mode of spin coupling between the terminal beryllium and carbon atoms. Further, the ground‐state potential‐energy surface of the [2Be,C,O] system at 4 K is presented and differences between the photochemical and thermal behaviors are highlighted. Finally, a previously not considered, so far unknown C2v‐symmetric rhombus‐like four‐membered ring 3[Be(O)(C)Be] (35) is predicted to represent the global minimum on the potential‐energy surface.TU Berlin, Open-Access-Mittel – 2020DFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat

Design of a Seat Mounting with a Rail Vehicle Frame

V předkládané diplomové práci, kterou jsem vypracoval ve spolupráci s VŠB – TU Ostrava a společností Siemens s.r.o., se věnuji návrhu upevnění sedadel k boční stěně hrubé stavby kolejového vozidla. Práce souvisí s projektem výstavby metra ve městě Riyadh. Hlavními částmi této práce jsou variantní návrh upevnění pomocí tzv. cantileveru, rozpracování zvoleného řešení, pevnostně deformační analýza a kontrola šroubového spoje. Součástí práce je také teoretický rozbor norem a vyhlášek souvisejících s návrhem sedadlových systémů v kolejových vozidlech.In the presented master´s thesis, which I developed in cooperation with VŠB – TU Ostrava and company Siemens s.r.o., I designed variant Fastening of a Seats to the side wall of Rail Vehicle Frame. Thesis related with project Metro Riyadh. The main parts of this thesis are variant design of cantilever, Fastening of a seats, stress – strain analysis and check of bolted connection. Creation of drawing documentation is another part.347 - Katedra částí a mechanismů strojůvýborn

追加1. 胃切除術後の鼻腔ゾンデ早期抜去について(シンポジウム 中山式切除術をめぐって,第473回千葉医学会例会,第4回佐藤外科例会)

Additional file 4. Proteomic data of strains wild-type, AG553, AG553, AG601 and LL1210 of Clostridium thermocellum

Methodology to improve design of accelerated life tests in civil engineering projects.

For reliability testing an Energy Expansion Tree (EET) and a companion Energy Function Model (EFM) are proposed and described in this paper. Different from conventional approaches, the EET provides a more comprehensive and objective way to systematically identify external energy factors affecting reliability. The EFM introduces energy loss into a traditional Function Model to identify internal energy sources affecting reliability. The combination creates a sound way to enumerate the energies to which a system may be exposed during its lifetime. We input these energies into planning an accelerated life test, a Multi Environment Over Stress Test. The test objective is to discover weak links and interactions among the system and the energies to which it is exposed, and design them out. As an example, the methods are applied to the pipe in subsea pipeline. However, they can be widely used in other civil engineering industries as well. The proposed method is compared with current methods

A Markerless Gene Deletion and Integration System for Thermoanaerobacter Ethanolicus

Background: Thermoanaerobacter ethanolicus produces a considerable amount of ethanol from a range of carbohydrates and is an attractive candidate for applications in bioconversion processes. A genetic system with reusable selective markers would be useful for deleting acid production pathways as well as other genetic modifications. Results: The thymidine kinase (tdk) gene was deleted from T. ethanolicus JW200 to allow it to be used as a selectable marker, resulting in strain X20. Deletion of the tdk gene reduced growth rate by 20 %; however, this could be reversed by reintroducing the tdk gene (strain X20C). The tdk and high-temperature kanamycin (htk) markers were tested by using them to delete lactate dehydrogenase (ldh). During positive selection of ldh knockouts in strain X20 on kanamycin agar plates, six out of seven picked colonies were verified transformants. Deletion of ldh reduced lactic acid production by 90 %. The tdk and 5-fluoro-2\u27-deoxyuridine (FUDR) combination worked reliably as demonstrated by successful tdk removal in all 21 colonies tested. Conclusion: A gene deletion and integration system with reusable markers has been developed for Thermoanaerobacter ethanolicus JW200 with positive selection on kanamycin and negative selection on FUDR. Gene deletion was demonstrated by ldh gene deletion and gene integration was demonstrated by re-integration of the tdk gene. Transformation via a natural competence protocol could use DNA PCR products amplified directly from Gibson Assembly mixture for efficient genetic modification

Cyclodextrin Inclusion Complexes and Their Application in Food Safety Analysis: Recent Developments and Future Prospects

Food safety issues are a major threat to public health and have attracted much attention. Therefore, exploring accurate, efficient, sensitive, and economical detection methods is necessary to ensure consumers’ health. In this regard, cyclodextrins (CDs) are promising candidates because they are nontoxic and noncaloric. The main body of CDs is a ring structure with hydrophobic cavity and hydrophilic exterior wall. Due to the above characteristics, CDs can encapsulate small guest molecules into their cavities, enhance their stability, avoid agglomeration and oxidation, and, at the same time, interact through hydrogen bonding and electrostatic interactions. Additionally, they can selectively capture the target molecules to be detected and improve the sensitivity of food detection. This review highlights recent advances in CD inclusion technology in food safety analysis, covering various applications from small molecule and heavy metal sensing to amino acid and microbial sensing. Finally, challenges and prospects for CDs and their derivatives are presented. The current review can provide a reference and guidance for current research on CDs in the food industry and may inspire breakthroughs in this field

Curcumin encapsulation and protection based on lysozyme nanoparticles

Curcumin possesses antioxidant, anti‐inflammatory, and other properties. However, this compound exhibits low bioavailability because of its poor solubility and stability. In this paper, lysozyme nanoparticles were fabricated through solvent evaporation, and then, the solubilization and protection capability of curcumin were investigated. Lysozyme nanoparticles were characterized by dynamic light scattering technique, atomic force microscopy, transmission electron microscopy, and Fourier transform infrared spectroscopy. The load capacity and stability in thermal environment were further explored. Results showed that the lysozyme nanoparticle displayed a spherical structure (127.9 ± 2.12 nm) with favorable distribution. The solubility of curcumin can increase to 22 μg/mL. After encapsulation by lysozyme nanoparticles, the retentive curcumin can reach up to 67.9% and 30.25% at 25°C and 50°C, respectively, significantly higher than that of free curcumin. Meanwhile, experiments on DPPH free radicals indicated the curcumin loaded by lysozyme nanoparticle possessed higher free radical scavenging activity than that of free curcumin with same treatments. The results confirmed that lysozyme nanoparticles exhibit potential applications in solubilizing and protecting the environment‐sensitive hydrophobic functional components