TY - JOUR A1 - Niedermeier, Jana A1 - Penner, Crystal A1 - Usherovich, Samuel A1 - Bélanger-Champagne, Camille A1 - Paulßen, Elisabeth A1 - Cornelia, Hoehr T1 - Optical Fibers as Dosimeter Detectors for Mixed Proton/Neutron Fields - A Biological Dosimeter JF - electronics N2 - In recent years, proton therapy has gained importance as a cancer treatment modality due to its conformality with the tumor and the sparing of healthy tissue. However, in the interaction of the protons with the beam line elements and patient tissues, potentially harmful secondary neutrons are always generated. To ensure that this neutron dose is as low as possible, treatment plans could be created to also account for and minimize the neutron dose. To monitor such a treatment plan, a compact, easy to use, and inexpensive dosimeter must be developed that not only measures the physical dose, but which can also distinguish between proton and neutron contributions. To that end, plastic optical fibers with scintillation materials (Gd₂O₂S:Tb, Gd₂O₂S:Eu, and YVO₄:Eu) were irradiated with protons and neutrons. It was confirmed that sensors with different scintillation materials have different sensitivities to protons and neutrons. A combination of these three scintillators can be used to build a detector array to create a biological dosimeter. KW - biological dosimeter KW - Bragg peak KW - relative dosimetry KW - optical fibers KW - proton therapy KW - protons KW - neutrons Y1 - 2023 U6 - http://dx.doi.org/10.3390/electronics12020324 SN - 2079-9292 N1 - This article belongs to the Special Issue "Applications of Optical Fiber Sensors" VL - 12 IS - 2 PB - MDPI CY - Basel ER - TY - JOUR A1 - Haeger, Gerrit A1 - Grankin, Alina A1 - Wagner, Michaela T1 - Construction of an Aspergillus oryzae triple amylase deletion mutant as a chassis to evaluate industrially relevant amylases using multiplex CRISPR/Cas9 editing technology JF - Applied Research N2 - Aspergillus oryzae is an industrially relevant organism for the secretory production of heterologous enzymes, especially amylases. The activities of potential heterologous amylases, however, cannot be quantified directly from the supernatant due to the high background activity of native α-amylase. This activity is caused by the gene products of amyA, amyB, and amyC. In this study, an in vitro CRISPR/Cas9 system was established in A. oryzae to delete these genes simultaneously. First, pyrG of A. oryzae NSAR1 was mutated by exploiting NHEJ to generate a counter-selection marker. Next, all amylase genes were deleted simultaneously by co-transforming a repair template carrying pyrG of Aspergillus nidulans and flanking sequences of amylase gene loci. The rate of obtained triple knock-outs was 47%. We showed that triple knockouts do not retain any amylase activity in the supernatant. The established in vitro CRISPR/Cas9 system was used to achieve sequence-specific knock-in of target genes. The system was intended to incorporate a single copy of the gene of interest into the desired host for the development of screening methods. Therefore, an integration cassette for the heterologous Fpi amylase was designed to specifically target the amyB locus. The site-specific integration rate of the plasmid was 78%, with exceptional additional integrations. Integration frequency was assessed via qPCR and directly correlated with heterologous amylase activity. Hence, we could compare the efficiency between two different signal peptides. In summary, we present a strategy to exploit CRISPR/Cas9 for gene mutation, multiplex knock-out, and the targeted knock-in of an expression cassette in A. oryzae. Our system provides straightforward strain engineering and paves the way for development of fungal screening systems. KW - aspergillus KW - CRISPR/Cas9 KW - filamentous fungi KW - genome engineering Y1 - 2023 U6 - http://dx.doi.org/10.1002/appl.202200106 SN - 2702-4288 IS - Early View SP - 1 EP - 15 PB - Wiley-VCH ER - TY - JOUR A1 - Block, Franziska A1 - May, Alexander A1 - Wetzel, Katharina A1 - Adels, Klaudia A1 - Elbers, Gereon A1 - Schulze, Margit A1 - Monakhova, Yulia T1 - What is the best spectroscopic method for simultaneous analysis of organic acids and (poly)saccharides in biological matrices: Example of Aloe vera extracts? JF - Talanta Open N2 - Several species of (poly)saccharides and organic acids can be found often simultaneously in various biological matrices, e.g., fruits, plant materials, and biological fluids. The analysis of such matrices sometimes represents a challenging task. Using Aloe vera (A. vera) plant materials as an example, the performance of several spectro-scopic methods (80 MHz benchtop NMR, NIR, ATR-FTIR and UV–vis) for the simultaneous analysis of quality parameters of this plant material was compared. The determined parameters include (poly)saccharides such as aloverose, fructose and glucose as well as organic acids (malic, lactic, citric, isocitric, acetic, fumaric, benzoic and sorbic acids). 500 MHz NMR and high-performance liquid chromatography (HPLC) were used as the reference methods. UV–vis data can be used only for identification of added preservatives (benzoic and sorbic acids) and drying agent (maltodextrin) and semiquantitative analysis of malic acid. NIR and MIR spectroscopies combined with multivariate regression can deliver more informative overview of A. vera extracts being able to additionally quantify glucose, aloverose, citric, isocitric, malic, lactic acids and fructose. Low-field NMR measurements can be used for the quantification of aloverose, glucose, malic, lactic, acetic, and benzoic acids. The benchtop NMR method was successfully validated in terms of robustness, stability, precision, reproducibility and limit of detection (LOD) and quantification (LOQ), respectively. All spectroscopic techniques are useful for the screening of (poly)saccharides and organic acids in plant extracts and should be applied according to its availability as well as information and confidence required for the specific analytical goal. Benchtop NMR spectroscopy seems to be the most feasible solution for quality control of A. vera products. KW - Spectroscopy KW - (Poly)saccharides KW - Organic acids KW - Aloe vera KW - qNMR Y1 - 2023 U6 - http://dx.doi.org/10.1016/j.talo.2023.100220 SN - 2666-8319 VL - 7 IS - Art. No. 100220 SP - 1 EP - 9 PB - Elsevier CY - Amsterdam ER - TY - RPRT A1 - Haeger, Gerrit A1 - Bongaerts, Johannes A1 - Siegert, Petra T1 - Abschlussbericht Teil II: Eingehende Darstellung Neue biobasierte Lipopeptide aus nachhaltiger Produktion (LipoPep) Y1 - 2023 N1 - Förderkennzeichen: 13FH256PA6 Titel: FHprofUnt 2016: Neue biobasierte Lipopeptide aus nachhaltiger Produktion Laufzeit: 01.02.2019 – 31.10.2022 ER - TY - JOUR A1 - Trapp, Svenja A1 - Lammers, Tom A1 - Engudar, Gokce A1 - Hoehr, Cornelia A1 - Denkova, Antonia G. A1 - Paulßen, Elisabeth A1 - de Kruijff, Robin M. T1 - Membrane-based microfluidic solvent extraction of Ga-68 from aqueous Zn solutions: towards an automated cyclotron production loop JF - EJNMMI Radiopharmacy and Chemistry KW - Microfluidic solvent extraction KW - Ga-68 KW - Cyclotron production KW - Medical radionuclide production KW - Metal contaminants Y1 - 2023 U6 - http://dx.doi.org/10.1186/s41181-023-00195-2 SN - 2365-421X VL - 2023 IS - 8, Article number: 9 SP - 1 EP - 14 PB - Springer Nature ER - TY - JOUR A1 - Falkenberg, Fabian A1 - Voß, Leonie A1 - Bott, Michael A1 - Bongaerts, Johannes A1 - Siegert, Petra T1 - New robust subtilisins from halotolerant and halophilic Bacillaceae JF - Applied Microbiology and Biotechnology N2 - The aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465ᵀ and Alkalibacillus haloalkaliphilus DSM 5271ᵀ and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976ᵀ served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0–12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications. KW - Biotechnological application KW - Bacillaceae KW - Subtilisin KW - Subtilases KW - Halotolerant protease Y1 - 2023 U6 - http://dx.doi.org/10.1007/s00253-023-12553-w SN - 1432-0614 N1 - Corresponding author: Petra Siegert VL - 107 SP - 3939 EP - 3954 PB - Springer Nature CY - Berlin ER - TY - INPR A1 - Greiner, Lasse A1 - Jeromin, Günter Erich A1 - Sithole, Patience A1 - Petersen, Soenke T1 - Preprint: Studies on the enzymatic reduction of levulinic acid using Chiralidon-R and Chiralidon-S T2 - ChemRxiv N2 - The enzymatic reduction of levulinic acid by the chiral catalysts Chiralidon-R and Chiralidon-S which are commercially available superabsorbed alcohol dehydrogenases is described. The Chiralidon®-R/S reduces the levulinic acid to the (R,S)-4-hydroxy valeric acid and the (R)- or (S)- gamma-valerolactone. KW - Levulinic acid KW - Chiralidon-R KW - Chiralidon-S KW - 4-hydroxy valeric acid KW - (R)- or (S)- gamma-valerolactone Y1 - 2023 U6 - http://dx.doi.org/10.26434/chemrxiv-2023-jlvcv ER - TY - JOUR A1 - Adels, Klaudia A1 - Elbers, Gereon A1 - Diehl, Bernd A1 - Monakhova, Yulia T1 - Multicomponent analysis of dietary supplements containing glucosamine and chondroitin: comparative low- and high-field NMR spectroscopic study JF - Analytical Sciences N2 - 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. KW - Glucosamine KW - Chondroitin sulfate KW - Polysaccharides KW - Dietary supplements KW - High-field NMR Y1 - 2023 U6 - http://dx.doi.org/10.1007/s44211-023-00433-2 SN - 1348-2246 (Online) SN - 0910-6340 (Print) N1 - Corresponding author: Yulia Monakhova VL - 2023 PB - Springer Verlag CY - Cham ER - TY - JOUR A1 - Falkenberg, Fabian A1 - Kohn, Sophie A1 - Bott, Michael A1 - Bongaerts, Johannes A1 - Siegert, Petra T1 - Biochemical characterisation of a novel broad pH spectrum subtilisin from Fictibacillus arsenicus DSM 15822ᵀ JF - FEBS Open Bio N2 - Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822ᵀ (obtained through a data mining-based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4-nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5% (w/v) SDS and 5% H₂O₂ (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H₂O₂, suggest it has potential for biotechnological applications. KW - Bacillaceae KW - Biotechnological application KW - Broad pH spectrum KW - Subtilases KW - Subtilisin Y1 - 2023 U6 - http://dx.doi.org/10.1002/2211-5463.13701 SN - 2211-5463 N1 - Corresponding author: Petra Siegert VL - 13 IS - 11 SP - 2035 EP - 2046 PB - Wiley CY - Hoboken, NJ ER - TY - JOUR A1 - Dellmann, Sophia Florence A1 - Glorius, J. A1 - Litvinov, Yu A. A1 - Reifarth, R. A1 - Al-Khasawneh, Kafa A1 - Aliotta, M. A1 - Bott, L. A1 - Brückner, Benjamin A1 - Bruno, C. G. A1 - Chen, Ruijiu A1 - Davinson, T. A1 - Dickel, T. A1 - Dillmann, Iris A1 - Dmytriev, D. A1 - Erbacher, P. A1 - Freire-Fernández, D. A1 - Forstner, Oliver A1 - Geissel, H. A1 - Göbel, K. A1 - Griffin, Christopher J. A1 - Grisenti, R. A1 - Gumberidze, Alexandre A1 - Haettner, Emma A1 - Hagmann, Siegbert A1 - Heil, M. A1 - Heß, R. A1 - Hillenbrand, P.-M. A1 - Joseph, R. A1 - Jurado, B. A1 - Kozhuharov, Christophor A1 - Kulikov, I. A1 - Löher, Bastian A1 - Langer, Christoph A1 - Leckenby, Guy A1 - Lederer-Woods, C. A1 - Lestinsky, M. A1 - Litvinov, S. A. A1 - Lorenz, B. A. A1 - Lorenz, E. A1 - Marsh, J. A1 - Menz, Esther Babette A1 - Morgenroth, T. A1 - Petridis, N. A1 - Pibernat, Jerome A1 - Popp, U. A1 - Psaltis, Athanasios A1 - Sanjari, Shahab A1 - Scheidenberger, C. A1 - Sguazzin, M. A1 - Sidhu, Ragandeep Singh A1 - Spillmann, Uwe A1 - Steck, M. A1 - Stöhlker, T. A1 - Surzhykov, A. A1 - Swartz, J. A. A1 - Törnqvist, H. A1 - Varga, L. A1 - Vescovi, Diego A1 - Weick, H. A1 - Weigand, M. A1 - Woods, P. A1 - Xing, Y. A1 - Yamaguchi, Taiyo T1 - Proton capture on stored radioactive ¹¹⁸Te ions JF - EPJ Web of Conferences N2 - Experimental determination of the cross sections of proton capture on radioactive nuclei is extremely difficult. Therefore, it is of substantial interest for the understanding of the production of the p-nuclei. For the first time, a direct measurement of proton-capture cross sections on stored, radioactive ions became possible in an energy range of interest for nuclear astrophysics. The experiment was performed at the Experimental Storage Ring (ESR) at GSI by making use of a sensitive method to measure (p,γ) and (p,n) reactions in inverse kinematics. These reaction channels are of high relevance for the nucleosyn-thesis processes in supernovae, which are among the most violent explosions in the universe and are not yet well understood. The cross section of the ¹¹⁸Te(p,γ) reaction has been measured at energies of 6 MeV/u and 7 MeV/u. The heavy ions interacted with a hydrogen gas jet target. The radiative recombination process of the fully stripped ¹¹⁸Te ions and electrons from the hydrogen target was used as a luminosity monitor. An overview of the experimental method and preliminary results from the ongoing analysis will be presented. Y1 - 2023 U6 - http://dx.doi.org/10.1051/epjconf/202327911018 SN - 2100-014X N1 - Volume 279, 2023. Nuclear Physics in Astrophysics – X (NPA-X 2022). VL - 279 IS - Article Number: 11018 SP - 1 EP - 5 PB - EDP Sciences ER -