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In unserem Alltag sind Polymere weit verbreitet. In Form von funktionellen Polymeren werden sie u.a. als Wirk- oder Effektstoff eingesetzt. Sie bestehen aus einem Träger, an welchen über einen Spacer eine funktionelle Gruppe gebunden ist. Die Spacergruppen beeinflussen die chemischen, physikalischen und biologischen Eigenschaften der Polymere bzw. ermöglichen diese erst. Dadurch stellen sie in der pharmazeutischen Industrie und der medizinischen Chemie Schlüsselbausteine dar.
Auch Monoester von symmetrischen Dicarbonsäuren oder symmetrischen Diolen werden für die Einführung von Spacergruppen verwendet. Sie können durch die Hydrolyse von Diestern oder Dioldiestern chemisch synthetisiert werden. Da diese Reaktion nicht selektiv erfolgt, entstehen Nebenprodukte wie Disäuren oder Diole, die die Ausbeuten schmälern und eine aufwändige Aufarbeitung notwendig machen. Selektive enzymatische Verfahren stellen eine echte Alternative dar, denn eine Trennung des Produkts vom Nebenprodukt ist nicht notwendig. Bisher sind nur wenige Enzyme bekannt und verfügbar, die zur Synthese von Monoestern verwendet werden können.
Ziel dieser Arbeit ist die Entdeckung neuer Lipasen und Carboxylesterasen als Biokatalysatoren zur Synthese von Monoestern, die zudem in ausreichender Verfügbarkeit generiert werden sollen. Als Gendonor und Expressionssystem diente hierfür die Hefe Blastobotrys raffinosifermentans. Die nicht-konventionelle, nicht-pathogene und thermotolerante Hefe B. raffinosifermentans weist ein breites Kohlenstoff- und Stickstoff-Quellen Spektrum auf, was sie für industrielle Anwendungen interessant macht. Aufgrund einer bereits vielfach eingesetzten, effizienten Transformationsmethode wurde die Hefe bereits zur Produktion verschiedener Proteine wie humanem Serumalbumin, Interleukin-6, Phosphatasen mit Phytase-Aktivität, Tannasen und einer Lipase eingesetzt. Die exzellenten Wachstumsparameter garantieren hohe Enzymausbeuten.
Insgesamt wurden in dieser Arbeit 30 putative Lipase- und Carboxylesterase-Gene in ihrem Genom durch Annotationsanalysen identifiziert. Diese Gene wurden isoliert, amplifiziert und in der Hefe selbst überexprimiert. Die Proteinextrakte der erzeugten Stämme wurden anschließend auf Esteraseaktivität getestet, wovon sieben Kandidaten das Substrat p-Nitrophenylbutyrat (pNP-Butyrat) hydrolysierten. Anschließend wurde mittels eines Assays untersucht, ob die Enzyme die Hydrolyse der Substrate Adipinsäurediethylester (DEA), Dimethyl trans-1,4-cyclohexandicarboxylat (DMCH), Terephthalsäurediethylester (DETS) und Decandiol-dimethacrylsäureester (DDMAE) katalysieren. Vier Kandidaten hydrolysierten DEA und DMCH und ein Extrakt eignete sich zur Hydrolyse von DETS. Es folgten eine Testung auf Selektivität mittels Gaschromatographie mit gekoppeltem Flammenionisationsdetektor und eine affinitätschromatographische Reinigung der fünf Proteine. Dabei stellten sich die drei Kandidaten Alip2-6hp, 6h-Best1p und 6h-Best2p, eine putative Lipase und zwei putative Carboxylesterasen, als potenziell geeignete Kandidaten heraus.
Anschließend erfolgte die biochemische Charakterisierung der drei Proteine. Das Temperatur-Optimum der Enzyme lag zwischen 31 °C und 41 °C und das pH-Optimum zwischen 6,6 und 7,0. Die Metallionen Fe2+, Fe3+ und Cu2+ inhibierten alle drei Biokatalysatoren und auch die Zugabe verschiedener Lösungsmittel verringerte ihre Aktivität. Die Untersuchung des Substratspektrums mit p-Nitrophenylestern mit Kettenlängen von C2 bis C18 zeigte eine Präferenz von Alip2-6hp für mittelkettige pNP-Ester mit einem Maximum bei pNP-Caproat und von 6h-Best1p und 6h-Best2p für kurzkettige pNP-Ester mit einem Maximum bei pNP-Acetat. 6h-Best1p und 6h-Best2p zeigen damit das für Carboxylhydrolasen typische Substratspektrum. Da Lipasen üblicherweise langkettige Substrate bevorzugen, wurde die Klassifizierung für Alip2-6hp mittels Tween 20- und Olivenöl-Agarplattentest weiter untersucht. Das positive Ergebnis dieser Untersuchung lässt auf eine Lipase schließen.
Zur Bestimmung der Selektivität der Enzyme wurde die Hydrolyse von DEA und DMCH zeitlich per GC-FID verfolgt. Nach Derivatisierung der Carboxylgruppen war die quantitative Auswertung zum Gehalt an Monoester, Diester und Disäure möglich. Es ließ sich damit die Hydrolyse von DEA mit 6h-Best1p bestätigen. Bessere Ergebnisse wurden mit Alip2-6hp für das Substrat DMCH erzielt und mit Abstand die schnellste Hydrolyse wurde mit DEA als Substrat erreicht. In gereinigter Form hydrolysierte Alip2-6hp das Substrat DEA selektiv zu MEA, sodass bis zu 96 % Monoester synthetisiert werden konnten. Im Vergleich dazu wird MEA deutlich langsamer hydrolysiert.
Zusätzlich wurden fünf unterschiedliche Formulierungen des Enzyms Alip2-6hp mit dem Substrat DEA getestet: (1) Rohextrakt, (2) freies, gereinigtes Enzym, (3) immobilisiertes, gereinigtes Enzym (Beads) und als Ganzzellkatalysatoren (4) permeabilisierte (Triton-) Zellen und (5) permeabilisierte, immobilisierte (Triton-) Zellen. Die vielversprechendsten Ergebnisse wurden mit isoliertem gereinigtem Enzym erzielt. DEA wurde vollständig und spezifisch zu MEA umgesetzt.
Zur Gewährleistung einer ausreichenden Verfügbarkeit der Enzyme erfolgte die Kultivierung der Überexpressionsstämme im Fermenter im Fed-batch. Der Alip2-6hp produzierende Hefestamm erbrachte Aktivitäten von 674 U L-1, während der 6h-Best2p Überexpressionsstamm 2239 U L-1 produzierte.
Four aerobic bacteria with bacteriolytic capabilities were isolated from the brackish water site Strait Uzynaral of Lake Balkhash in Kazakhstan. The morphology and physiology of the bacterial isolates have subsequently been analyzed. Using matrix assisted laser desorption ionization-time of flight mass spectrum and partial 16S rRNA gene sequence analyses, three of the isolates have been identified as Pseudomonas veronii and one as Paenibacillus apiarius. We determined the capability of both species to lyse pre-grown cells of the Gram-negative strains Pseudomonas putida SBUG 24 and Escherichia coli SBUG 13 as well as the Gram-positive strains Micrococcus luteus SBUG 16 and Arthrobacter citreus SBUG 321 on solid media. The bacteriolysis process was analyzed by creating growth curves and electron micrographs of co-cultures with the bacteriolytic isolates and the lysis sensitive strain Arthrobacter citreus SBUG 321 in nutrient-poor liquid media. One metabolite of Paenibacillus apiarius was isolated and structurally characterized by various chemical structure determination methods. It is a novel antibiotic substance.
Proteasomes comprise a family of proteasomal complexes essential for maintaining protein homeostasis. Accordingly, proteasomes represent promising therapeutic targets in multiple human diseases. Several proteasome inhibitors are approved for treating hematological cancers. However, their side effects impede their efficacy and broader therapeutic applications. Therefore, understanding the biology of the different proteasome complexes present in the cell is crucial for developing tailor-made inhibitors against specific proteasome complexes. Here, we will discuss the structure, biology, and function of the alternative Proteasome Activator 200 (PA200), also known as PSME4, and summarize the current evidence for its dysregulation in different human diseases. We hereby aim to stimulate research on this enigmatic proteasome regulator that has the potential to serve as a therapeutic target in cancer.
Gallic acid, protocatechuic acid, catechol, and pyrogallol are only a few examples of industrially relevant aromatics. Today much attention is paid to the development of new microbial factories for the environmentally friendly biosynthesis of industrially relevant chemicals with renewable resources or organic pollutants as the starting material. The non–conventional yeast, Blastobotrys raffinosifermentans, possesses attractive properties for industrial bio-production processes such as thermo- and osmotolerance. An additional advantage is its broad substrate spectrum, with tannins at the forefront. The present study is dedicated to the characterization of catechol-1,2-dioxygenase (Acdo1p) and the analysis of its function in B. raffinosifermentans tannic acid catabolism. Acdo1p is a dimeric protein with higher affinity for catechol (KM = 0.004 ± 0.001 mM, kcat = 15.6 ± 0.4 s–1) than to pyrogallol (KM = 0.1 ± 0.02 mM, kcat = 10.6 ± 0.4 s–1). It is an intradiol dioxygenase and its reaction product with catechol as the substrate is cis,cis-muconic acid. B. raffinosifermentans G1212/YIC102-AYNI1-ACDO1-6H, which expresses the ACDO1 gene under the control of the strong nitrate-inducible AYNI1 promoter, achieved a maximum catechol-1,2-dioxygenase activity of 280.6 U/L and 26.9 U/g of dry cell weight in yeast grown in minimal medium with nitrate as the nitrogen source and 1.5% glucose as the carbon source. In the same medium with glucose as the carbon source, catechol-1,2-dioxygenase activity was not detected for the control strain G1212/YIC102 with ACDO1 expression under the regulation of its respective endogenous promoter. Gene expression analysis showed that ACDO1 is induced by gallic acid and protocatechuic acid. In contrast to the wild-type strain, the B. raffinosifermentans strain with a deletion of the ACDO1 gene was unable to grow on medium supplemented with gallic acid or protocatechuic acid as the sole carbon source. In summary, we propose that due to its substrate specificity, its thermal stability, and its ability to undergo long-term storage without significant loss of activity, B. raffinosifermentans catechol-1,2-dioxygenase (Acdo1p) is a promising enzyme candidate for industrial applications.
Feasible Cluster Model Method for Simulating the Redox Potentials of Laccase CueO and Its Variant
(2022)
Laccases are regarded as versatile green biocatalysts, and recent scientific research has focused on improving their redox potential for broader industrial and environmental applications. The density functional theory (DFT) quantum mechanics approach, sufficiently rigorous and efficient for the calculation of electronic structures, is conducted to better comprehend the connection between the redox potential and the atomic structural feature of laccases. According to the crystal structure of wild type laccase CueO and its variant, a truncated miniature cluster model method was established in this research. On the basic of thermodynamic cycle, the overall Gibbs free energy variations before and after the one-electron reduction were calculated. It turned out that the trends of redox potentials to increase after variant predicted by the theoretical calculations correlated well with those obtained by experiments, thereby validating the feasibility of this cluster model method for simulating the redox potentials of laccases.
Editorial: Streptococci in infectious diseases – pathogenic mechanisms and host immune responses
(2022)
Infective/bacterial endocarditis is a rare but life-threatening disease with a hospital mortality rate of 22.7% and a 1-year mortality rate of 40%. Therefore, continued research efforts to develop efficient anti-infective implant materials are of the utmost importance. Equally important is the development of test systems that allow the performance of new materials to be comprehensively evaluated. In this study, a novel antibacterial coating based on dalbavancin was tested in comparison to rifampicin/minocycline, and the suitability of a recently developed mouse tail vein model for testing the implant coatings was validated. Small polymeric stent grafts coated with a poly-L-lactic acid (PLLA) layer and incorporated antibiotics were colonized with Staphylococcus (S.) aureus before implantation into the tail vein of mice. The main assessment criteria were the hematogenous spread of the bacteria and the local tissue reaction to the contaminated implant. For this purpose, colony-forming units (CFU) in the blood, spleen and kidneys were determined. Tail cross sections were prepared for histological analysis, and plasma cytokine levels and expression values of inflammation-associated genes were examined. Both antibiotic coatings performed excellently, preventing the onset of infection. The present study expands the range of available methods for testing the anti-infectivity of cardiovascular implants, and the spectrum of agents for effective surface coating.
MicroRNAs (miRNA) are ubiquitous non-coding RNAs that have a prominent role in cellular regulation. The expression of many miRNAs is often found deregulated in prostate cancer (PCa) and castration-resistant prostate cancer (CRPC). Although their expression can be associated with PCa and CRPC, their functions and regulatory activity in cancer development are poorly understood. In this study, we used different proteomics tools to analyze the activity of hsa-miR-3687-3p (miR-3687) and hsa-miR-4417-3p (miR-4417), two miRNAs upregulated in CRPC. PCa and CRPC cell lines were transfected with miR-3687 or miR-4417 to overexpress the miRNAs. Cell lysates were analyzed using 2D gel electrophoresis and proteins were subsequently identified using mass spectrometry (Maldi-MS/MS). A whole cell lysate, without 2D-gel separation, was analyzed by ESI-MS/MS. The expression of deregulated proteins found across both methods was further investigated using Western blotting. Gene ontology and cellular process network analysis determined that miR-3687 and miR-4417 are involved in diverse regulatory mechanisms that support the CRPC phenotype, including metabolism and inflammation. Moreover, both miRNAs are associated with extracellular vesicles, which point toward a secretory mechanism. The tumor protein D52 isoform 1 (TD52-IF1), which regulates neuroendocrine trans-differentiation, was found to be substantially deregulated in androgen-insensitive cells by both miR-3687 and miR-4417. These findings show that these miRNAs potentially support the CRPC by truncating the TD52-IF1 expression after the onset of androgen resistance.
The main goal of this contribution was to determine the effect of predation of the often abundant to dominant doliolid Dolioletta gegenbauri (Tunicata, Thaliacea) on the abundance of co-occurring planktonic copepods by feeding on their eggs. Previous oceanographic investigations revealed that doliolids had ingested eggs of small calanoid copepods. The ecological significance of such feeding could not be quantified completely because the environmental abundance of such eggs was not known. In this study, the eggs and nauplii of the neritic calanoid Paracalanus quasimodo (Crustacea, Copepoda) were offered to gonozooids and phorozooids of D. gegenbauri with a 6–6.5 mm length together with three species of phytoplankton; i.e., simulating diet conditions on the shelf. We hypothesized that copepod eggs of a similar size as food particles would be readily ingested whereas small nauplii, which could escape, would hardly be eaten by the doliolids. Our results revealed that doliolids have the potential to control small calanoids by ingesting their eggs at high rates but not their nauplii or later stages. Late copepodid stages and adults of co-occurring calanoid species could cause less mortality because they prey less on such eggs than doliolids of a similar weight. However, certain abundant omnivorous calanoid species with pronounced perception and/or capture abilities can prey successfully on the nauplii of small calanoids.