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Aboulnaga, El-Hussiny ; Pinkenburg, Olaf ; Schiffels, Johannes ; El-Refai, Ahmed ; Buckel, Wolfgang ; Selmer, Thorsten
Cupriavidus necator H16 gains increasing attention in microbial research and biotechnological application due to its diverse metabolic features. Here we present a tightly controlled gene expression system for C. necator including the pBBR1-vector that contains hybrid promoters originating from C. necator native tolC-promoter in combination with a synthetic tetO-operator. The expression of the reporter gene from these plasmids relies on the addition of the exogenous inducer doxycycline (dc). The novel expression system offers a combination of advantageous features as; (i) high and dose-dependent recombinant protein production, (ii) tight control with a high dynamic range (On/Off ratio), which makes it applicable for harmful pathways or for toxic protein production, (iii) comparable cheap inducer (doxycycline, dc), (iv) effective at low inducer concentration, that makes it useful for large scale application, (v) rapid, diffusion controlled induction, and (vi) the inducer does not interfere within the cell metabolism. As applications of the expression system in C. necator H16, the growth ability on glycerol was enhanced by constitutively expressing the E. coli glpk gene-encoding for glycerol kinase. Likewise, we used the system to overcome the expression toxicity of mevalonate pathway in C. necator H16. With this system, the mevalonate-genes were successfully introduced in the host and the recombinant strains could produce about 200 mg/l mevalonate.
Abulnaga, El-Hussiny ; Pinkenburg, Olaf ; Schiffels, Johannes ; E-Refai, Ahmed ; Buckel, Wolfgang ; Selmer, Thorsten
With the prevalence of glucosamine- and chondroitin-containing dietary supplements for people with osteoarthritis in the marketplace, it is important to have an accurate and reproducible analytical method for the quantitation of these compounds in finished products. NMR spectroscopic method based both on low- (80 MHz) and high- (500–600 MHz) field NMR instrumentation was established, compared and validated for the determination of chondroitin sulfate and glucosamine in dietary supplements. The proposed method was applied for analysis of 20 different dietary supplements. In the majority of cases, quantification results obtained on the low-field NMR spectrometer are similar to those obtained with high-field 500–600 MHz NMR devices. Validation results in terms of accuracy, precision, reproducibility, limit of detection and recovery demonstrated that the developed method is fit for purpose for the marketed products. The NMR method was extended to the analysis of methylsulfonylmethane, adulterant maltodextrin, acetate and inorganic ions. Low-field NMR can be a quicker and cheaper alternative to more expensive high-field NMR measurements for quality control of the investigated dietary supplements. High-field NMR instrumentation can be more favorable for samples with complex composition due to better resolution, simultaneously giving the possibility of analysis of inorganic species such as potassium and chloride.
Electronic cigarettes (e-cigarettes) have become popular worldwide with the market growing exponentially in some countries. The absence of product standards and safety regulations requires urgent development of analytical methodologies for the holistic control of the growing diversity of such products. An approach based on low-field nuclear magnetic resonance (LF-NMR) at 80 MHz is presented for the simultaneous determination of key parameters: carrier solvents (vegetable glycerine (VG), propylene glycol (PG) and water), total nicotine as well as free-base nicotine fraction. Moreover, qualitative and quantitative determination of fourteen weak organic acids deliberately added to enhance sensory characteristics of e-cigarettes was possible. In most cases these parameters can be rapidly and conveniently determined without using any sample manipulation such as dilution, extraction or derivatization steps. The method was applied for 37 authentic e-cigarettes samples. In particular, eight different organic acids with the content up to 56 mg/mL were detected. Due to its simplicity, the method can be used in routine regulatory control as well as to study release behaviour of nicotine and other e-cigarettes constituents in different products.
Several unconnected laboratory experiments are usually offered for students in instrumental analysis lab. To give the students a more rational overview of the most common instrumental techniques, a new laboratory experiment was developed. Marketed pain relief drugs, familiar consumer products with one to three active components, namely, acetaminophen (paracetamol), acetylsalicylic acid (ASA), and caffeine, were selected. Common analytical methods were compared regarding the performance of qualitative and quantitative analysis of unknown tablets: UV–visible (UV–vis), infrared (IR), and nuclear magnetic resonance (NMR) spectroscopies, as well as high-performance liquid chromatography (HPLC). The students successfully uncovered the composition of formulations, which were divided into three difficulty categories. Students were shown that in addition to simple mixtures handled in theoretical classes, the composition of complex drug products can also be uncovered. By comparing the performance of different techniques, students deepen their understanding and compare the efficiency of analytical methods in the context of complex mixtures. The laboratory experiment can be adjusted for graduate level by including extra tasks such as method optimization, validation, and 2D spectroscopic techniques.
Aggarwal, Pranav ; Dhiman, Shashi K. ; Kumar, G. ; Scherer, Ulrich W. ; Singla, M. L. ; Srivastava, Alok
Thin films of poly(ethyleneterephthalate) [PET]were exposed to radiation dose ranging from 10 to 30 kGy by using gamma rays in the range 12.8-177.8 MGy using swift light ions of hydrogen. There was no effect of the radiation dose on the optical behaviour of PET as a result of exposure to radiation dose up to 30 kGy brought about by gamma rays but a significant decrease in the optical band gap values was observed when PET was exposed to swift light ions of hydrogen. The data obtained are discussed in terms of optical studies carried out on PET using swift heavy ions.
Aggarwal, Prerna ; Singh, Virendra ; Singh, Arjun ; Scherer, Ulrich W. ; Singh, Tejvir ; Singla, Madan L. ; Srivastava, Alok
Al-Kaidy, Huschyar ; Duwe, Anna ; Huster, Manuel ; Muffler, Kai ; Schlegel, Christin ; Sieker, Tim ; Stadtmüller, Ralf ; Tippkötter, Nils ; Ulber, Roland
Biotechnologie und die mit ihr verbundenen technischen Prozesse prägen seit Jahrtausenden die Entwicklung der Menschheit. Ausgehend von empirischen Verfahren, insbesondere zur Herstellung von Lebensmitteln und täglichen Gebrauchsgütern, haben sich diese Disziplinen zu einem der innovativsten Zukunftsfelder entwickelt. Durch das immer detailliertere Verständnis zellulärer Vorgänge können mittlerweile Produktionsstämme gezielt optimiert werden. Im Zusammenspiel mit moderner Prozesstechnik können so eine Vielzahl von Bulk- und Feinchemikalien sowie Pharmazeutika effizient hergestellt werden. In diesem Artikel werden exemplarisch einige der aktuellen Trends vorgestellt.
Al-Kaidy, Huschyar ; Duwe, Anna ; Huster, Manuel ; Muffler, Kai ; Schlegel, Christin ; Tim, Sieker ; Stadtmüller, Ralf ; Tippkötter, Nils ; Ulber, Roland
For several thousand years, biotechnology and its associated technical processes have had a great impact on the development of mankind. Based on empirical methods, in particular for the production of foodstuffs and daily commodities, these disciplines have become one of the most innovative future issues. Due to the increasing detailed understanding of cellular processes, production strains can now be optimized. In combination with modern bioprocesses, a variety of bulk and fine chemicals as well as pharmaceuticals can be produced efficiently. In this article, some of the current trends in biotechnology are discussed.