@article{MonakhovaDiehl2022, author = {Monakhova, Yulia and Diehl, Bernd W.K.}, title = {Multinuclear NMR screening of pharmaceuticals using standardization by 2H integral of a deuterated solvent}, series = {Journal of Pharmaceutical and Biomedical Analysis}, volume = {209}, journal = {Journal of Pharmaceutical and Biomedical Analysis}, number = {Article number: 114530}, publisher = {Elsevier}, isbn = {0731-7085}, doi = {10.1016/j.jpba.2021.114530}, year = {2022}, abstract = {NMR standardization approach that uses the 2H integral of deuterated solvent for quantitative multinuclear analysis of pharmaceuticals is described. As a proof of principle, the existing NMR procedure for the analysis of heparin products according to US Pharmacopeia monograph is extended to the determination of Na+ and Cl- content in this matrix. Quantification is performed based on the ratio of a 23Na (35Cl) NMR integral and 2H NMR signal of deuterated solvent, D2O, acquired using the specific spectrometer hardware. As an alternative, the possibility of 133Cs standardization using the addition of Cs2CO3 stock solution is shown. Validation characteristics (linearity, repeatability, sensitivity) are evaluated. A holistic NMR profiling of heparin products can now also be used for the quantitative determination of inorganic compounds in a single analytical run using a single sample. In general, the new standardization methodology provides an appealing alternative for the NMR screening of inorganic and organic components in pharmaceutical products.}, language = {en} } @article{BurgerLindnerRumpfetal.2022, author = {Burger, Ren{\´e} and Lindner, Simon and Rumpf, Jessica and Do, Xuan Tung and Diehl, Bernd W.K. and Rehahn, Matthias and Monakhova, Yulia and Schulze, Margit}, title = {Benchtop versus high field NMR: Comparable performance found for the molecular weight determination of lignin}, series = {Journal of Pharmaceutical and Biomedical Analysis}, volume = {212}, journal = {Journal of Pharmaceutical and Biomedical Analysis}, number = {Article number: 114649}, publisher = {Elsevier}, address = {New York, NY}, isbn = {0731-7085}, doi = {10.1016/j.jpba.2022.114649}, year = {2022}, abstract = {Lignin is a promising renewable biopolymer being investigated worldwide as an environmentally benign substitute of fossil-based aromatic compounds, e.g. for the use as an excipient with antioxidant and antimicrobial properties in drug delivery or even as active compound. For its successful implementation into process streams, a quick, easy, and reliable method is needed for its molecular weight determination. Here we present a method using 1H spectra of benchtop as well as conventional NMR systems in combination with multivariate data analysis, to determine lignin's molecular weight (Mw and Mn) and polydispersity index (PDI). A set of 36 organosolv lignin samples (from Miscanthus x giganteus, Paulownia tomentosa and Silphium perfoliatum) was used for the calibration and cross validation, and 17 samples were used as external validation set. Validation errors between 5.6\% and 12.9\% were achieved for all parameters on all NMR devices (43, 60, 500 and 600 MHz). Surprisingly, no significant difference in the performance of the benchtop and high-field devices was found. This facilitates the application of this method for determining lignin's molecular weight in an industrial environment because of the low maintenance expenditure, small footprint, ruggedness, and low cost of permanent magnet benchtop NMR systems.}, language = {en} } @article{MonakhovaSobolevaFedotovaetal.2022, author = {Monakhova, Yulia and Soboleva, Polina M. and Fedotova, Elena S. and Musina, Kristina T. and Burmistrova, Natalia A.}, title = {Quantum chemical calculations of IR spectra of heparin disaccharide subunits}, series = {Computational and Theoretical Chemistry}, volume = {1217}, journal = {Computational and Theoretical Chemistry}, number = {Article number: 113891}, publisher = {Elsevier}, address = {New York, NY}, isbn = {2210-271X}, doi = {10.1016/j.comptc.2022.113891}, year = {2022}, abstract = {Heparin is a natural polysaccharide, which plays essential role in many biological processes. Alterations in building blocks can modify biological roles of commercial heparin products, due to significant changes in the conformation of the polymer chain. The variability structure of heparin leads to difficulty in quality control using different analytical methods, including infrared (IR) spectroscopy. In this paper molecular modelling of heparin disaccharide subunits was performed using quantum chemistry. The structural and spectral parameters of these disaccharides have been calculated using RHF/6-311G. In addition, over-sulphated chondroitin sulphate disaccharide was studied as one of the most widespread contaminants of heparin. Calculated IR spectra were analyzed with respect to specific structure parameters. IR spectroscopic fingerprint was found to be sensitive to substitution pattern of disaccharide subunits. Vibrational assignments of calculated spectra were correlated with experimental IR spectral bands of native heparin. Chemometrics was used to perform multivariate analysis of simulated spectral data.}, language = {en} } @article{PennerUsherovichNiedermeieretal.2022, author = {Penner, Crystal and Usherovich, Samuel and Niedermeier, Jana and B{\´e}langer-Champagne, Camille and Trinczek, Michael and Paulßen, Elisabeth and Hoehr, Cornelia}, title = {Organic Scintillator-Fibre Sensors for Proton Therapy Dosimetry: SCSF-3HF and EJ-260}, series = {electronics}, volume = {12}, journal = {electronics}, number = {1}, publisher = {MDPI}, address = {Basel}, issn = {2079-9292}, doi = {10.3390/electronics12010011}, pages = {12 Seiten}, year = {2022}, abstract = {In proton therapy, the dose from secondary neutrons to the patient can contribute to side effects and the creation of secondary cancer. A simple and fast detection system to distinguish between dose from protons and neutrons both in pretreatment verification as well as potentially in vivo monitoring is needed to minimize dose from secondary neutrons. Two 3 mm long, 1 mm diameter organic scintillators were tested for candidacy to be used in a proton-neutron discrimination detector. The SCSF-3HF (1500) scintillating fibre (Kuraray Co. Chiyoda-ku, Tokyo, Japan) and EJ-260 plastic scintillator (Eljen Technology, Sweetwater, TX, USA) were irradiated at the TRIUMF Neutron Facility and the Proton Therapy Research Centre. In the proton beam, we compared the raw Bragg peak and spread-out Bragg peak response to the industry standard Markus chamber detector. Both scintillator sensors exhibited quenching at high LET in the Bragg peak, presenting a peak-to-entrance ratio of 2.59 for the EJ-260 and 2.63 for the SCSF-3HF fibre, compared to 3.70 for the Markus chamber. The SCSF-3HF sensor demonstrated 1.3 times the sensitivity to protons and 3 times the sensitivity to neutrons as compared to the EJ-260 sensor. Combined with our equations relating neutron and proton contributions to dose during proton irradiations, and the application of Birks' quenching correction, these fibres provide valid candidates for inexpensive and replicable proton-neutron discrimination detectors}, language = {en} } @article{NiedermeierPennerUsherovichetal.2023, author = {Niedermeier, Jana and Penner, Crystal and Usherovich, Samuel and B{\´e}langer-Champagne, Camille and Paulßen, Elisabeth and Cornelia, Hoehr}, title = {Optical Fibers as Dosimeter Detectors for Mixed Proton/Neutron Fields - A Biological Dosimeter}, series = {electronics}, volume = {12}, journal = {electronics}, number = {2}, publisher = {MDPI}, address = {Basel}, issn = {2079-9292}, doi = {10.3390/electronics12020324}, pages = {11 Seiten}, year = {2023}, abstract = {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.}, language = {en} } @article{HaegerGrankinWagner2023, author = {Haeger, Gerrit and Grankin, Alina and Wagner, Michaela}, title = {Construction of an Aspergillus oryzae triple amylase deletion mutant as a chassis to evaluate industrially relevant amylases using multiplex CRISPR/Cas9 editing technology}, series = {Applied Research}, journal = {Applied Research}, number = {Early View}, publisher = {Wiley-VCH}, issn = {2702-4288}, doi = {10.1002/appl.202200106}, pages = {1 -- 15}, year = {2023}, abstract = {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.}, language = {en} } @article{BlockMayWetzeletal.2023, author = {Block, Franziska and May, Alexander and Wetzel, Katharina and Adels, Klaudia and Elbers, Gereon and Schulze, Margit and Monakhova, Yulia}, title = {What is the best spectroscopic method for simultaneous analysis of organic acids and (poly)saccharides in biological matrices: Example of Aloe vera extracts?}, series = {Talanta Open}, volume = {7}, journal = {Talanta Open}, number = {Art. No. 100220}, publisher = {Elsevier}, address = {Amsterdam}, issn = {2666-8319}, doi = {10.1016/j.talo.2023.100220}, pages = {1 -- 9}, year = {2023}, abstract = {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.}, language = {en} } @article{TrappLammersEngudaretal.2023, author = {Trapp, Svenja and Lammers, Tom and Engudar, Gokce and Hoehr, Cornelia and Denkova, Antonia G. and Paulßen, Elisabeth and de Kruijff, Robin M.}, title = {Membrane-based microfluidic solvent extraction of Ga-68 from aqueous Zn solutions: towards an automated cyclotron production loop}, series = {EJNMMI Radiopharmacy and Chemistry}, volume = {2023}, journal = {EJNMMI Radiopharmacy and Chemistry}, number = {8, Article number: 9}, publisher = {Springer Nature}, issn = {2365-421X}, doi = {10.1186/s41181-023-00195-2}, pages = {1 -- 14}, year = {2023}, language = {en} } @article{FalkenbergVossBottetal.2023, author = {Falkenberg, Fabian and Voß, Leonie and Bott, Michael and Bongaerts, Johannes and Siegert, Petra}, title = {New robust subtilisins from halotolerant and halophilic Bacillaceae}, series = {Applied Microbiology and Biotechnology}, volume = {107}, journal = {Applied Microbiology and Biotechnology}, publisher = {Springer Nature}, address = {Berlin}, issn = {1432-0614}, doi = {10.1007/s00253-023-12553-w}, pages = {3939 -- 3954}, year = {2023}, abstract = {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.}, language = {en} } @unpublished{GreinerJerominSitholeetal.2023, author = {Greiner, Lasse and Jeromin, G{\"u}nter Erich and Sithole, Patience and Petersen, Soenke}, title = {Preprint: Studies on the enzymatic reduction of levulinic acid using Chiralidon-R and Chiralidon-S}, series = {ChemRxiv}, journal = {ChemRxiv}, doi = {10.26434/chemrxiv-2023-jlvcv}, pages = {13 Seiten}, year = {2023}, abstract = {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.}, language = {en} }