Refine
Year of publication
Document Type
- Doctoral Thesis (151)
- Article (79)
Has Fulltext
- yes (230)
Is part of the Bibliography
- no (230)
Keywords
- - (78)
- Biokatalyse (24)
- Proteindesign (16)
- biocatalysis (15)
- Enzym (14)
- Biocatalysis (8)
- Biotechnologie (8)
- Biochemie (7)
- RNA (7)
- Ribozym (7)
- Protein-Engineering (6)
- protein engineering (6)
- Enzyme (5)
- Hydrolasen (5)
- Protein Engineering (5)
- Struktur (5)
- Elektrochemie (4)
- Gerichtete Evolution (4)
- Metabolomics (4)
- Molybdopterin (4)
- RNS (4)
- Thermodynamik (4)
- Transaminase (4)
- synthesis (4)
- Baeyer-Villiger monooxygenase (3)
- Baeyer-Villiger-Oxidation (3)
- Biokonversion (3)
- Biosensor (3)
- Chiral amines (3)
- DNA-Wirkstoff-Interaktion (3)
- DNA-drug interaction (3)
- DNS (3)
- Diabetes mellitus (3)
- Enzymkatalyse (3)
- Esterasen (3)
- Gold (3)
- Indolochinolin (3)
- Magnetische Kernresonanz (3)
- Massenspektrometrie (3)
- Monooxygenasen (3)
- Nucleinsäuren (3)
- Oxidativer Stress (3)
- Oxidoreduktase (3)
- Pankreatitis (3)
- Promiskuität (3)
- Protein engineering (3)
- RNS-Reparatur (3)
- RNS-Synthese (3)
- Rasterkraftmikroskopie (3)
- Reaktionsmechanismus (3)
- SELEX (3)
- Substratspezifität (3)
- Titandioxid (3)
- Transaminasen (3)
- Transaminases (3)
- antibacterial activity (3)
- asymmetric synthesis (3)
- catalytic promiscuity (3)
- electrochemistry (3)
- inhibition (3)
- metabolomics (3)
- regioselectivity (3)
- substituent (3)
- transaminases (3)
- transesterification (3)
- Acyltransferase (2)
- Amine (2)
- Arylesterase (2)
- Asymmetric synthesis (2)
- Baeyer-Villiger Monooxygenase (2)
- Bauchspeicheldrüsenentzündung (2)
- Benzazaphosphole (2)
- Chemie (2)
- Chirale Amine (2)
- Cyclovoltammetrie (2)
- Cytidindesaminierung (2)
- DNA Triplex (2)
- DNA triplex (2)
- Dithiolene (2)
- Enantioselektivität (2)
- Enoatreduktase (2)
- Flavonoide (2)
- Fluoreszenzmarkierung (2)
- G-quadruplexes (2)
- Haloalkan Dehalogenase (2)
- Heterologe Genexpression (2)
- High throughput screening (2)
- Hydrolases (2)
- Hydroxyl (2)
- Immobilisierung (2)
- Ketoreductase (2)
- Kristallographie (2)
- Kristallstruktur (2)
- Lipase (2)
- Metabolismus (2)
- Molekulardesign (2)
- Molekulardynamik (2)
- Molybdenum (2)
- NMR spectroscopy (2)
- Nukleoside (2)
- Phosphine (2)
- Proteine (2)
- Pterin (2)
- RNA recombination (2)
- SAM analogue (2)
- Schweineleberesterase (2)
- Selektionsassay (2)
- Simulation (2)
- Spektroskopie (2)
- Staphylococcus aureus (2)
- Strukturbiologie (2)
- Synthese (2)
- Tetracyclinrepressor (2)
- Transkriptionspriming (2)
- Twinribozym (2)
- Wasserstoffperoxid (2)
- acyl transfer (2)
- acyltransferase (2)
- acyltransferases (2)
- alpha-toxin (2)
- amine transaminase (2)
- atomic force microscopy (2)
- biomarker (2)
- biosensor (2)
- chemical ecology (2)
- chiral amines (2)
- dehalogenase (2)
- directed evolution (2)
- enzyme cascade (2)
- enzyme catalysis (2)
- epoxide hydrolase (2)
- flavonoid (2)
- high-throughput screening (2)
- hydrolases (2)
- indoloquinoline (2)
- katalytische Promiskuität (2)
- methylation (2)
- natural products (2)
- oligonucleotides (2)
- pancreatitis (2)
- plastic degradation (2)
- quadruplex (2)
- rational design (2)
- rational protein design (2)
- rationales Proteindesign (2)
- redox chemistry (2)
- ribozyme (2)
- structure-activity (2)
- thermodynamics (2)
- transcription priming (2)
- twin ribozyme (2)
- -Enzym (1)
- -amino acid oxidase (LAAO) (1)
- 1,4-naphthoquinones (1)
- 1-deoxy sphingolipids (1)
- 3-Bis(Silyl Enol Ethers) (1)
- 31P-NMR (1)
- 3DM (1)
- 4070512-2 (1)
- 4165547-3 (1)
- 4193016-2 (1)
- 4248339-6 (1)
- 4323543-8 (1)
- 4441960-0 (1)
- 4796824-2 (1)
- 7 beta Hydroxylierung (1)
- 7β-Hydroxylierung (1)
- <i>Bacillus subtilis</i> (1)
- <i>S. aureus</i> (1)
- <i>S. pneumoniae</i> (1)
- <i>Staphylococcus aureus</i> (1)
- API Drug synthesis (1)
- Abbau (1)
- Acyltransfer (1)
- Acyltransferasen (1)
- Adenosindesaminierung (1)
- Adhesion (1)
- Adsorption (1)
- Aktive Zentren (1)
- Akute Bauchspeicheldrüsenentzündung (1)
- Akute Pankreatitis (1)
- Alcohol dehydrogenase (1)
- Alkanolamine (1)
- Alkohol (1)
- Alkohole (1)
- Alphaherpesviren (1)
- Alphaherpesvirus (1)
- Amin-Transaminasen (1)
- Amine transaminase, ketoreductase (1)
- Amino Alcohol (1)
- Aminoacylierung (1)
- Aminoalkohol (1)
- Aminosäuren (1)
- Amperometric detection; EstraMonitor (1)
- Amperometrische Detektion; EstraMonitor (1)
- Analytische Chemie (1)
- Annotation (1)
- Anti-cancer and anti-microbials (1)
- Antibiotikaresistenz (1)
- Antibiotikum (1)
- Antimicrobial (1)
- Antimicrobial Resistance (1)
- Antioxidans (1)
- Antiphospholipidsyndrom (1)
- Antiterminator-Proteine (1)
- Aptamer (1)
- Aptamers (1)
- Aptazym (1)
- Aquaporins (1)
- Aquatic Interfaces (1)
- Aryl fluorides (1)
- Arzneimitteldesign (1)
- Aspzinkin (1)
- Assemblin (1)
- Atmosphärendruckplasma (1)
- Autoantikörper (1)
- Azaphospholderivat (1)
- Azides (1)
- BCL11B (1)
- BVMO (1)
- Bacillus subtilis (1)
- Baclofen (1)
- Baeyer-Villiger monooxygenases (1)
- Benzazaphospholes (1)
- Bgl (1)
- Biaryle (1)
- Bidirectional electron transfer (1)
- Biocatalytic alkylation (1)
- Biogeochemie (1)
- Biokatalyse , Enzym , Alkohol , Amine , Enzymkatalyse , Asymmetrische Katalyse (1)
- Biokatalyse , Organische Synthese , Enzym , Prozessoptimierung (1)
- Biokompatibilität (1)
- Biomimetic membrane; cold physical plasma;membrane oxidation; lipid bilayer; electrochemistry; mass spectrometry; atomic force microscopy (1)
- Biophysikalische Chemie (1)
- Bioreaktor (1)
- Biosynthesis of bile acids (1)
- Biotechnologie; Biokatalyse; Metagenom; Enzym (1)
- Butyrolactonderivate (1)
- C-F activation (1)
- CAL-A (1)
- CANDLE/PRAAS (1)
- CAZymes (1)
- CODEHOP (1)
- COVID-19 (1)
- Calcium (1)
- Campher (1)
- Caprolacton <epsilon-> (1)
- Carbamoylase (1)
- Carboxylester-Hydrolasen (1)
- Carboxylesterase (1)
- Cascade reaction (1)
- Cathepsin B (1)
- Cathepsine (1)
- Ceramide (1)
- Chalconisomerase (1)
- Chemical Ecology (1)
- Chemical Stability (1)
- Chemische Stabilität (1)
- Chemische Synthese (1)
- Chemo-enzymatic synthesis (1)
- Chinhydron (1)
- Chiral amine (1)
- Chondroitinsulfate (1)
- Chronische Bauchspeicheldrüsenentzündung (1)
- Chronische Pankreatitis (1)
- Click chemistry (1)
- Cofaktor (1)
- Cold atmospheric plasma (1)
- Collagen (1)
- Corrosion-electrochemical behaviour (1)
- Cross-coupling (1)
- Cyanoethyl (1)
- Cyclisation (1)
- Cycloalkanonmonooxygenase (1)
- Cyclohexanon-Monooxygenase (1)
- Cyclohexanone monooxygenase (1)
- Cylindrocarpon radicicola (1)
- Cystatin C (1)
- Cytochrom P450 Monooxygenase (1)
- Cytochrome P-450 (1)
- Cytotoxizität (1)
- C–C bond cleavage (1)
- DNA (1)
- DNA-Wirkstoff-Struktur (1)
- DNA-drug structure (1)
- DNA-microarray (1)
- DNAzym (1)
- DNAzyme (1)
- Darm (1)
- Dehydrocyclisation (1)
- Desorption (1)
- Diastereomere (1)
- Dihydropyrimidinase (1)
- Directed evolution (1)
- Dithiol (1)
- Dithiolen (1)
- Durchflusscytometrie (1)
- ESKAPE pathogens (1)
- Eicosanoide (1)
- Eintopfreaktion (1)
- Electrochemical Stability (1)
- Electrochemically active bacteria (1)
- Electrochemie (1)
- Elektrochemische Stabilität (1)
- Elektrochemischer Sensor (1)
- Elektrokatalyse (1)
- Enantiopreference (1)
- Enzymatic Degradation (1)
- Enzyme Discovery (1)
- Enzyme Engineering (1)
- Enzyme discovery (1)
- Enzyme identification (1)
- Enzymidentifizierung (1)
- Enzymkaskade (1)
- Enzymkinetik (1)
- Epoxid-Hydrolase (1)
- Epoxidhydrolase (1)
- Error-prone PCR (1)
- Erucic acid (1)
- Ethylen Oligomerisation (1)
- Ethylene oligomerisation (1)
- Ethylene oligomerization (1)
- Ethylenoligomerisation (1)
- Eubacterium ramulus (1)
- FAIR data (1)
- FRET (1)
- Familie-VIII-Carboxylesterase (1)
- Fatty acid enrichment (1)
- Fermentation (1)
- Festphase (1)
- Festphasensynthese (1)
- Fibronectin (1)
- Fibrose (1)
- Flavine (1)
- Flavinemononucleotide (1)
- Flavinmononukleotid (1)
- Flavivirus (1)
- Fließinjektionsanalyse (1)
- Flow catalysis (1)
- Function prediction (1)
- Funktionalisierung <Chemie> (1)
- Fusionsprotein (1)
- G-Quadruplex (1)
- GBM (1)
- GC-MS (1)
- Genbibliothek (1)
- Gendrift (1)
- Genregulation (1)
- Gentherapie (1)
- Gerüstprotein (1)
- Gleichgewicht (1)
- Glycolysis (1)
- Glykosaminoglykane (1)
- Gold-Nanopartikel (1)
- Gondoic acid (1)
- Graphische Darstellung (1)
- HEV (1)
- Hairpin-Ribozym (1)
- Hairpinribozym (1)
- Haloalkan-Dehalogenase (1)
- Hautkrebs (1)
- Hefeartige Pilze (1)
- Hepatitis-E-Virus , Polymerase-Kettenreaktion , Microarray , West-Nil-Virus , Flaviviren , RNS-Viren , Genotypisierung (1)
- Herpesviren (1)
- Herpesviridae (1)
- Heterocycle synthesis (1)
- Heterocyclische Verbindungen (1)
- High-throughput assay (1)
- Hirudin (1)
- Hirudin‐like factors (1)
- Histidin (1)
- Histidinphosphorylierung (1)
- Hyaluronsäure (1)
- Hybrid Liganden (1)
- Hybrid ligand (1)
- Hydantoinase-Prozess (1)
- Hydrogenphosphate (1)
- Hydroxytyrosol (1)
- Identifikation (1)
- Imin-Reduktase (1)
- Imine (1)
- Iminreduktase (1)
- Immobilization (1)
- Immunantwort (1)
- Immunsystem (1)
- Impedance Spectroscopy (1)
- Impedanzspektroskopie (1)
- Implant (1)
- Implantat (1)
- In situ product recovery (1)
- Industrie (1)
- Infektionen (1)
- Integrin (1)
- Integrin αIIbβ3 (1)
- Integrin αiibβ3 (1)
- Interleukin 33 (1)
- Isopropylamine (1)
- Isothermale Titrationskalorimetrie (1)
- KHV (1)
- KHVD (1)
- Kaltes Plasma (1)
- Karzinom (1)
- Kaskade (1)
- Kaskadenreaktion (1)
- Katalyse (1)
- Kazal inhibitor (1)
- Ketene (1)
- Ketoreduktase (1)
- Kip1 (1)
- Klarzelliges Nierenzellkarzinom (1)
- Kleinmolekülaktivierung (1)
- Klinische Pathologie (1)
- Knoevenagel ligation (1)
- Kohlendioxid (1)
- Komplexe (1)
- Kontinuierliche gerichtete Evolution (1)
- Korrosion (1)
- Korrosionselektrochemische Verhalten (1)
- Kraftfeld-Rechnung (1)
- Kristallfläche (1)
- Künstliche Evolution (1)
- LC-MS (1)
- LCA (1)
- Landsat (1)
- Ligand (1)
- Ligand-DNA-Interaktion (1)
- Ligation (1)
- Limnologie (1)
- Linker (1)
- Lipasen (1)
- Lipases (1)
- Lipid Modification (1)
- Liquid chromatography (1)
- Lithocholsäure (1)
- Lysosomen (1)
- Makrophagen (1)
- Mass spectrometry (1)
- Maus (1)
- Meerespilze (1)
- Membrane (1)
- Metabolism (1)
- Metabolom (1)
- Metadata standard (1)
- Methylation (1)
- Methyltransferase (1)
- Microbial electrochemistry (1)
- MoCo (1)
- Modelle des Molybdän-Cofaktors (1)
- Modellverbindungen (1)
- Molecular Modeling (1)
- Molecular Modelling (1)
- Molecular modeling (1)
- Molecular modelling (1)
- Molybdenum Cofactor (1)
- Molybdenum cofactor (moco) (1)
- Molybdenum cofactor (moco) models (1)
- Molybdenum mediated pentathiepin synthesis (1)
- Molybdän-Cofaktor (1)
- Molybdän-Cofaktor-Defizienz (1)
- Monodithiolenkomplex (1)
- Monodithiolenkomplexe (1)
- Monooxygenase (1)
- Muskelatrophie (1)
- Mutagenese (1)
- N-Acyl-L-Homoserinlakton (1)
- N-Aryl Phosphinoglycines (1)
- N-heterocyclic olefins (1)
- N-substituierter Phosphanylglycine (1)
- N34S (1)
- NNMT (1)
- Naringenin (1)
- Naturstoff (1)
- Nickel (1)
- Nickel catalysts (1)
- Nickelkatalysator (1)
- Niere (1)
- Nitric oxide (1)
- Nnucleophilic substitution (1)
- Nucleoside modification and labeling (1)
- Nukleinsäuren (1)
- Nukleosidanaloga (1)
- OAT Reaction (1)
- ONX-0914 (1)
- Oberflächenbehandlung (1)
- Oberflächenmodifizierung (1)
- Oberflächenveränderung (1)
- OleP (1)
- Oligomerisation (1)
- Oligonucleotides (1)
- Oligonukleotid Konjugate (1)
- Olive mill wastewaters valorization (1)
- One‐pot reaction (1)
- Ontology (1)
- Optisch active Dithiolene (1)
- Organic Matter (1)
- Organischer Stoff (1)
- Organschaden (1)
- Ovalbumin (1)
- Oxidoreductase (1)
- Oxidoreductasen (1)
- Oxidoreductases (1)
- Oxidoreduktasen (1)
- Oxidschi (1)
- Oxocarbonsäureester (1)
- P-Arylation (1)
- P450 (1)
- P=C Verbindungen (1)
- P=C-N-Heterocyclen (1)
- P=C-N-Heterocycles (1)
- PET (1)
- PET Hydrolases (1)
- PETase activity (1)
- PETase-like enzymes (1)
- Pancreatitis (1)
- Passivierung (1)
- Pb-UPD (1)
- Pd/PTABS catalyst (1)
- Pentathiepins (1)
- Peptide (1)
- PestE (1)
- Phase Equilibrium (1)
- Phasengleichgewicht (1)
- Phenylalanin-Ammoniumlyase (1)
- Phloretin (1)
- Phosphane (1)
- Phosphanyl- (1)
- Phosphanylaminosäure (1)
- Phosphanylaniline (1)
- Phosphanylglycine (1)
- Phosphaproline (1)
- Phosphaprolines (1)
- Phosphate Substituted Dithiolene (1)
- Phosphino amino acids (1)
- Phosphinoaminosäuren (1)
- Phosphinoaniline (1)
- Phosphinoanilines (1)
- Phosphinoglycines (1)
- Phospholipase A2 (1)
- Phosphonium glycolates (1)
- Phosphoniumglykolate (1)
- Phosphoniumsalze (1)
- Phosphor-31-NMR-Spektroskopie (1)
- Phosphoramidite (1)
- Phosphorylierung (1)
- Phosphotransferasesystem (1)
- Physikalische Chemie (1)
- Plasma chemistry (1)
- Plasmachemie (1)
- Plasmamedizin (1)
- Platelet Factor 4 (1)
- Platin (1)
- Platinabscheidung auf Gold (1)
- Plesiocystis pacifica (1)
- Plättchenfaktor 4 (1)
- Polycaprolactone (1)
- Polyethylenterephthalate (1)
- Polykristall (1)
- Polyphenole (1)
- Polyvinylalkohol (1)
- Post-translational modification (1)
- Potential – pH Diagram (1)
- Potentiometrie (1)
- Potenzial – pH-Diagramm (1)
- Pr (1)
- Process engineering (1)
- Promiscuous acyltransferase (1)
- Protease (1)
- Proteases (1)
- Proteinfaltung (1)
- Proteingerüst (1)
- Proteinogene Aminosäuren (1)
- Proteinreinigung (1)
- Proteinsequenz (1)
- Protonen-NMR-Spektroskopie (1)
- Prozesstechnik (1)
- Pseudomonas putida (1)
- Pseudorabies-Virus (1)
- Push–pull imines (1)
- Pyruvatdecarboxylase (1)
- QCM (1)
- QM/MM (1)
- Quadruplex (1)
- Quantenchemie (1)
- Quorum quenching (1)
- Quorum sensing (1)
- RIDD (1)
- RNA engineering (1)
- RNA repair (1)
- RNA self-splicing (1)
- RNA, modifizierte Nukleoside, Chemische Synthese (1)
- RNA-Reparatur (1)
- RNA-World (1)
- RNS-Edierung (1)
- RPE (1)
- RT-qPCR (1)
- Radikalfänger (1)
- Rasterelektronenmikroskop (1)
- Reactive species (1)
- Reaktionskaskade (1)
- Reaktive Sauerstoffspezies (1)
- Redox Biogeochemie (1)
- Redox Biogeochemistry (1)
- Redoxchemie (1)
- Reduktive Aminierung (1)
- Regioselektivität (1)
- Regulatorische T-Zelle (1)
- Rekombination (1)
- Renilla Luciferase (1)
- Repressorproteine (1)
- Resistenz (1)
- Rhenium (1)
- Rhodococcus rhodochrous (1)
- Riboswitch (1)
- Ribozyme (1)
- Ring-opening polymerization (1)
- Ringöffnungspolymerisation (1)
- Röntgenkristallographie (1)
- Röntgenstrukturanalyse (1)
- SARS-CoV-2 (1)
- SAXS (1)
- SNPs (1)
- SPINK1 (1)
- Sarkopenie (1)
- Schweineleber-Esterase (1)
- Schwermetalle (1)
- Scilab (1)
- Screening (1)
- Sekundärstruktur (1)
- Semantics (1)
- Sensor (1)
- Sensoren (1)
- Serinproteasen (1)
- SiMoA (1)
- Small RNA (1)
- Social Spiders (1)
- Spektroelektrochemie (1)
- Sphingolipide (1)
- Spinmarkierung (1)
- Stabilität (1)
- Steroidmonooxygenase (1)
- Stickstoffmonoxid (1)
- Stoffwechsel (1)
- Streptococcus pneumoniae (1)
- Streptomyces (1)
- Strukturanalyse (1)
- Strukturaufklärung (1)
- Strukturverfeinerung (1)
- Study of Health in Pomerania (1)
- Substrat <Chemie> (1)
- Suides Herpesvirus (1)
- Sulfoxidation (1)
- Synthetic Biology (1)
- Synthetic design (1)
- Synthetic methods (1)
- Synthetische Biologie (1)
- TME (1)
- Targeted Proteomics (1)
- Temperaturbeständigkeit (1)
- TetR‐family repressor (1)
- Tetracyclin (1)
- Tetracycline (1)
- Thermal Desorption Spectrometry (1)
- Thermische Desorptionsspektroskopie (1)
- Tierphysiologie (1)
- Titan (1)
- Titration (1)
- Transition Metal Complexes (1)
- Transitional metal catalysis (1)
- Trinukleotid (1)
- Tris inhibition (1)
- Trypsin (1)
- Tyrosin-Ammonium-Lyase (1)
- UDCA (1)
- UPR (1)
- Umesterung (1)
- Uncoupling (1)
- Ursodeoxycholsäure (1)
- V-loop (1)
- VEEV (1)
- VHL (1)
- Vanadiumkomplexe (1)
- Veresterung (1)
- Volatiles (1)
- Voltammetrie (1)
- Vorhersage (1)
- WNV (1)
- Warburg effect (1)
- Wasserhärte (1)
- Water-soluble catalysis (1)
- Wein (1)
- Whole-cell enzyme cascade (1)
- Wirtsorganismus (1)
- Wnt signalling (1)
- Wnt-Signalweg (1)
- X-ray structure (1)
- X‐ray crystallography (1)
- Z-score standardization (1)
- Zellen (1)
- active centers (1)
- acylation (1)
- adaptation (1)
- adenosine deamination (1)
- advanced training (1)
- aging (1)
- agreement (1)
- airway epithelial cells (1)
- alanine scanning (1)
- alcohol dehydrogenase (1)
- alcohol dehydrogenases (1)
- aldehyde production (1)
- aldehydes (1)
- alkenes (1)
- alkyl iodide (1)
- alkylation (1)
- alpha- Phosphino Amino Acids (1)
- alphaherpesvirus (1)
- amination (1)
- amino alcohols (1)
- analytical measurements (1)
- antibacterial (1)
- antibacterial drug resistance (1)
- antibiotic resistance (1)
- antibiotics (1)
- antifungal (1)
- antimicrobial (1)
- antimicrobial resistance (1)
- antimicrobial resistance crisis (1)
- antimicrobial substance (1)
- antimicrobial surface (1)
- antiterminator protein (1)
- aptazyme (1)
- aptazymes (1)
- aquaculture (1)
- aquatische Grenzzonen (1)
- assemblin (1)
- assembly protein (1)
- asymmetric catalysis (1)
- atmospheric-pressure plasma jets (1)
- autoligation (1)
- batteries (1)
- beta coefficient (1)
- beta-Catenin (1)
- beta-catenin (1)
- bias (1)
- bioactive compounds (1)
- biobank education (1)
- biobank technician (1)
- biocatalytic cascades (1)
- biochemical-clinical traits (1)
- biodegradable plastics (1)
- bioinformatic (1)
- biological membranes (1)
- bioluminescence (1)
- blood coagulation (1)
- bone erosion (1)
- bone remodelling (1)
- bone turnover marker (1)
- boronate linkage (1)
- boronic acid (1)
- boronic acids (1)
- bortezomib (1)
- bulk chemical production (1)
- capsid assembly (1)
- carbamoylase (1)
- carbocations (1)
- carbon catabolite repression (1)
- carrageenan (1)
- cascade reaction (1)
- castration-resistant prostate cancer (1)
- catalytic activity (1)
- catalytic triad (1)
- ccRCC (1)
- cell cycle regulator (1)
- cell proliferation (1)
- cellular sensitivity (1)
- cellulose degradation (1)
- chalcone isomerase (1)
- chemical identification (1)
- chemische Identifizierung (1)
- chitin-binding domain (1)
- chondroitin sulfate (1)
- circular (1)
- cloud removal (1)
- co-infection (1)
- co-substrate recycling (1)
- cold physical plasma (1)
- cold physical plasmas (1)
- collagen-induced arthritis (1)
- compensatory growth (1)
- corrosion (1)
- crystal structure (1)
- cyclin-dependent kinase inhibitor (1)
- cycloalkanone monooxygenase (1)
- cytidine deamination (1)
- deep tracheal aspirate (1)
- degradation (1)
- dehalogenasen (1)
- derivatives (1)
- detergents (1)
- deubiquitination (1)
- differentiation (1)
- dihedral principal component analysis (1)
- dihydrogen phosphate (1)
- diketocamphane monooxygenase (1)
- diseases (1)
- dissolution mechanism (1)
- dithiolene ligand (1)
- drug discovery (1)
- duale Substraterkennung (1)
- eQTL (1)
- early-stage functionalisation (1)
- eicosanoids (1)
- enantiopure ( (1)
- enantioselectivity (1)
- endosymbionts (1)
- enzyme (1)
- enzyme evolution (1)
- enzyme kits (1)
- enzyme promiscuity (1)
- enzymes (1)
- epoxid hydrolasen (1)
- esterase (1)
- esterases (1)
- extracellular matrix (1)
- eye lens cell membrane (1)
- family VIII carboxylesterase (1)
- fish (live) (1)
- flavin reductase (1)
- fluorescence (1)
- fluorine (1)
- formylglycine-generating enzyme (1)
- free radicals (1)
- fusion protein (1)
- gamma-Lactamase (1)
- gamma-lactamase (1)
- genetic (1)
- genetic code expansion (1)
- genetically encoded sensors (1)
- genotyping (1)
- gerichtete Evolution (1)
- glia (1)
- glucose tolerance (1)
- glycosaminoglycans (1)
- glycosidases (1)
- glycoside hydrolase (1)
- glycosidic torsion angles (1)
- gold dissolution (1)
- gold nanoparticles (1)
- group A streptococcus (1)
- guanosine analogs (1)
- hairpin-ribozyme (1)
- halide methyltransferase (1)
- halides (1)
- haloalkane (1)
- haloalkane dehalogenases (1)
- haloperoxidase (1)
- heavy metal (1)
- heterologous gene expression (1)
- histidine (1)
- homodimerization (1)
- host pathogen interactions (1)
- hyaluronic acid (1)
- hydantoinase (1)
- hydantoinase-process (1)
- hydrogen bond (1)
- hydrogen peroxide (1)
- hydrogen phosphate (1)
- hydrolysis (1)
- hydrophobicity (1)
- hydroxytyrosol (1)
- hydroxytyrosol acetate (1)
- hypoxia-ischemia (1)
- image reconstruction (1)
- immobilization (1)
- immunology (1)
- imprecision (1)
- in vivo selection (1)
- industrial catalysis (1)
- industrielle Biokatalyse (1)
- infection (1)
- influenza A virus (1)
- inorganic materials (1)
- insulin-producing cells (1)
- integrin αIIbβ3 (1)
- intestinal (1)
- in situ-ICP-MS (1)
- ion insertion (1)
- isoflavonoid (1)
- isolated sulfite oxidase deficiency (iSOD) (1)
- isothermal titration calorimetry (ITC) (1)
- kINPen (1)
- ketoreductase (1)
- kinases (1)
- kinetic resolution (1)
- kinetics (1)
- laminarin (1)
- late-stage functionalisation (1)
- lead compounds (1)
- lead diversification (1)
- lead structure (1)
- leaf-branch compost (1)
- learning objectives (1)
- lipase (1)
- lipid mediators (1)
- lipid monolayers (1)
- lipids (1)
- liposomes (1)
- liquid-liquid extraction (1)
- liver regeneration (1)
- luciferase (1)
- lysine acetylation (1)
- lysine acetyltransferases (1)
- lysine deacetylases (1)
- m6A (1)
- mQTL (1)
- magnetic moment (1)
- marine biodegradation (1)
- marine fungi (1)
- marine polysaccharides (1)
- mass spectrometry (1)
- medicinal leeches (1)
- membrane protein (1)
- menaquinones (1)
- metabolite (1)
- metabolites (1)
- metadynamics (1)
- metal oxides (1)
- methyltransferases (1)
- metrological controls (1)
- microglia (1)
- microorganisms (1)
- mitochondria (1)
- mixed-valence complex (1)
- molecular dynamics simulations (MDS) (1)
- molybdenum cofactor deficiency (MoCoD) (1)
- molybdopterin (1)
- monooxygenase (1)
- monoterpene acylation (1)
- morphology (1)
- n/a (1)
- neurofilament (1)
- neurons (1)
- neutral genetic drift (1)
- nitrosative stress (1)
- non-identical reversible reaction (1)
- novel immunotherapy (1)
- nucleophilic substitution (1)
- nucleoredoxin (1)
- oil (1)
- oligonucleotide conjugate (1)
- olive mill wastewaters valorization (1)
- optically active dithiolene (1)
- organic synthesis (1)
- overflow metabolites (1)
- oxidation (1)
- oxidative and nitrosative stress (1)
- oxidative post-translational modifications (1)
- oxidative stress (1)
- oxidized lipids (1)
- oxylipins (1)
- pH electrode (1)
- pH-Elektrode (1)
- pH-assay (1)
- pan-cancer (1)
- pancreas (1)
- papain-like protease (1)
- periodontitis (1)
- phosphorylation (1)
- phosphotransferase system (1)
- pig liver esterase (1)
- pig model (1)
- plasma liquid chemistry (1)
- plasma medicine (1)
- plastic (1)
- plastic pollution (1)
- platinum deposition on gold (1)
- polycrystalline gold (1)
- polyethylene terephthalate (1)
- porphyran (1)
- proteasome (1)
- protein expression and purification (1)
- protein interaction (1)
- protein-engineering (1)
- proteindesign (1)
- protein–protein interaction (1)
- proteomics (1)
- pseudorabies virus (1)
- pyrrolobenzodiazepine (1)
- pyruvate (1)
- pyruvate kinase (1)
- quality control (1)
- quinhydrone (1)
- radical polishing (1)
- radical reactions (1)
- radiometric interpolation (1)
- rat hepatocytes (1)
- rationales Design (1)
- reactive nitrogen species (1)
- reactive species (1)
- recombinant enzyme (1)
- recombinant expression of proteins (1)
- recombination (1)
- reconstitution (1)
- recycling (1)
- redox regulation (1)
- redox signaling (1)
- rekombinante Proteinexpression (1)
- reoxygenation (1)
- respiratory tract infection (1)
- retina (1)
- rheumatoid arthritis (1)
- riboswitch (1)
- ribozymes (1)
- rice husk (1)
- scaffold (1)
- scale up for bulk chemical production (1)
- schwere akute Pankreatitis (1)
- secondary plantmetabolites (1)
- secondary structure (1)
- selection assay (1)
- selection-assay (1)
- self assembled monolayer (1)
- sensors (1)
- sepsis (1)
- sepsis patients (1)
- septic arthritis (1)
- serine protease (1)
- serum markers (1)
- silver (1)
- sirtuin (1)
- social arthropods (1)
- spectral matching (1)
- sphingomyelin (1)
- spinlabel (1)
- split hairpin ribozyme (1)
- standard mechanism (1)
- steered molecular dynamics (1)
- stereoselectivity (1)
- structure activity (1)
- structure–activity (1)
- substrate specificity (1)
- sugar conformation (1)
- sugar pucker (1)
- suid herpesvirus (1)
- sulfoxidation (1)
- supercapacitors (1)
- surface change (1)
- surface modification (1)
- surface plasmon resonance (SPR) (1)
- surface state (1)
- surface treatment (1)
- talin (1)
- tandem PETases (1)
- temporal fitting (1)
- tertiary alcohol (1)
- tertiärer Alkohole (1)
- tetra-nuclear nickel complex (1)
- tetracycline (1)
- three-phase electrochemistry (1)
- titanium dioxide (1)
- transaminase (1)
- transbilayer lipid (flip-flop) motion (1)
- transcript (1)
- transmembrane pores (1)
- type I IFN response (1)
- ulvan (1)
- uncertainty of measurement (1)
- upcycling (1)
- ustilagic acid (1)
- viral diagnosis (1)
- virology (1)
- virulence factors (1)
- virus (1)
- virus host interaction (1)
- viruses (1)
- vitamin K2 (1)
- volatile organic compound (1)
- volatile organic compounds (1)
- volatiles (1)
- white spot syndrome virus (1)
- whole-cell biocatalysis (1)
- x-ray crystallography (1)
- zinc finger (1)
- zirkular (1)
- Östrogen-Rezeptor-Modulator (1)
- Östrogene (1)
- Übergangsmetallkomplexe (1)
- β-amino acid (1)
- β-glucosidase (1)
- β-phenylalanine ethyl ester (1)
- ω-hydroxy fatty acid (1)
- ω-transaminase (1)
Institute
- Institut für Chemie und Biochemie (230) (remove)
Publisher
- MDPI (27)
- Frontiers Media S.A. (13)
- John Wiley & Sons, Ltd (12)
- Wiley (11)
- John Wiley & Sons, Inc. (3)
- Blackwell Publishing Ltd (1)
- SAGE Publications (1)
- Wiley-VCH Verlag GmbH & Co. KGaA (1)
The relevance of cold atmospheric plasmas (CAPs) in biomedicine has recently grown. The potential of CAPs has been discussed in multiple scientific works, highlighting its effectiveness in promoting wound healing, limiting cancer progression, and for sterilization of surfaces. Main bioactive molecules, such as reactive oxygen and nitrogen species (RONS), are proposed as key candidates in these processes. Indeed, the generation of cold plasma induces noble gas ionization which, reacting with atmospheric air molecules, generates species such as singlet oxygen, atomic oxygen radicals, nitric oxide radicals. Although molecular simulations have been conducted, the mechanism of action on biological molecules, as well as the possibility to tune plasmas to produce specific species cocktails (e.g., with different degree of oxidation power) has been not fully unleashed. In this dissertation, presented in form of 5 published scientific articles, focus has been placed on the interaction of plasmas with peptides and proteins, which are main biological effectors in cellular compartments. Precisely, through the development of liquid chromatography coupled mass spectrometry (LC-MS) methods, the effects of plasmas on peptides and proteins in form of oxidative post-translational modifications (oxPTMs) has been investigated. The characterization of these oxPTMs has been performed by treating peptide or protein aqueous solutions and on porcine skin tissues. It has been found that, introducing small amounts of different gases (oxygen, nitrogen, or both) or even water molecules, can made CAPs tunable tools to produce oxygen-species dominating effects versus nitrogen-species dominating effects. In addition to this, it was found that the amino acid position in a peptide or protein influences the quality and quantity of the resulting oxPTMs. Besides this, other important parameters like driven gases, admixture gases or treatment duration were identified as relevant factors for the modification of amino acids in the peptide structure. By comparing the effects between peptide solutions and complex matrices such as porcine skin, water has been identified as a valid vehicle to transport and amplify the plasma chemistry. In an experimental study, the inactivation of a protein (PLA2) was observed after CAP treatment and together with simulation studies, the specific dioxidation of tryptophane W128 was detected as a potential explanation for this inactivation, indicating the strong impact of plasma on biological targets. In summary, oxidative modifications found in peptide solutions were observed also in complex protein structures and sample matrices. In conclusion, this work provides a starting point for future studies of oxidative modifications in complex models and may thus be helpful for further investigations in the fields of plasma medicine and redox chemistry.
This work investigated the enzymatic degradation of polyethylene terephthalate (PET) (ArticlesI and II) and polyvinyl alcohol (PVA) (Article III). Physical or chemical degradation of plastic polymers is often performed under extreme conditions like high temperatures or pressure. In comparison to that, recycling of plastics with enzymes can be carried out at ambient temperatures and neutral pH. Enzymes themselves are non- toxic, environmentally friendly, and have been used successfully in a variety of industrial processes.
Enzymatic degradation of polyesters is well studied. Their heteroatomic backbone, which is connecting monomers via ester bonds offers a target for an enzymatic attack. Especially PET, one of the most common polyesters, has been in the focus of research. The first enzyme capable of degrading the polymer was found in 2005. Since then, researchers discovered several enzymes with similar functions and subjected them to enzyme engineering. Improving the enzyme's substrate affinity, activity, and stability aims at making PET recycling more efficient. Article I provides an overview of limitations that enzymatic PET recycling is still facing and the research carried out to overcome them. More precisely, enzyme−substrate interactions, thermostability, catalytic efficiency, and inhibition caused by oligomeric degradation intermediates are summarized and discussed in detail.
Article II further addresses one of the above-mentioned limitations, namely product inhibition of PET hydrolyzing enzymes. We elucidated the crystal structure of TfCa, a carboxylesterase from Thermobifida fusca (T. fusca), and applied semi-rational enzyme engineering. The article discusses the structure-function relationship of TfCa based on the apo-structure as well as ligand-soaked structures. Furthermore, it compares the structures of TfCa and MHETase, another PET hydrolase helper enzyme. Lastly, we determined the substrate profile of the carboxylesterase based on terephthalate-based oligo-esters of various lengths and one ortho-phthalate ester. In a dual enzyme system, TfCa degraded intermediate products derived from the PET hydrolysis of a variant of PETase hydrolase from Ideonella sakaiensis (I. sakaiensis). The dual enzyme system utilized PET more efficiently in comparison to solely PETase due to relieved product inhibition. Since TfCa successfully degraded oligomeric intermediates, the reaction not only released terephthalic acid as the sole product but also increased the overall product yield.
While PET contains an ester bond that can be attacked and hydrolyzed by esterases or lipases, PVA consists of a homoatomic C-C-backbone with repeating 1,3-diol units. The polymer is water soluble with remarkable physical properties such as thermostability and viscosity. PVA is often described as biodegradable, but microbial degradation is slow and frequently involves cost-intensive cofactors. In this study, we present an improved PVA polymer with derivatized side chains and an enzyme cascade that can degrade not only modified but also unmodified PVA in a one-pot reaction. The enzyme cascade consists of a lipase, an alcohol dehydrogenase (ADH), and a Baeyer-Villiger monooxygenase (BVMO). In comparison to the scarcely published research on PVA degradation with free enzyme, this cascade is not only independent from the frequently required cofactor pyrroloquinoline quinone (PQQ) but, in principle, contains an in vitro cofactor recycling mechanism.
This thesis deals with the characterisation and engineering of new thermophilic PET hydrolases as potential candidates for an eco-friendly biocatalytic recycling approach for the upcycling or downcycling of polyethylene terephthalate (PET) on industrial scale. Furthermore, high-throughput screening methods are described that detect the products of PET hydrolysis. The high demand of PET in the packaging and textile industries with a global production of 82 million metric tons per year has significantly contributed to the global solid waste stream and environmental plastic pollution after its end-of-life. Although PET hydrolases have been identified in various microorganisms, only a handful of benchmark enzymes have been engineered for industrial applications. Therefore, the identification of new PET hydrolases from metagenomes or via protein engineering approaches, especially thermophilic PET hydrolases with optimal operating temperatures (i.e., increased thermostability and activity) near the glass transition temperature of the polymer PET, is a crucial step towards a bio-based circular plastic economy. Article I demonstrates that metagenome-derived thermophilic PET hydrolases can be significantly improved using different engineering approaches to achieve a similar activity level as the well-established leaf-branch-compost cutinase (LCC) F243I/D238C/S283C/Y127G variant (LCC ICCG). In Article II, thermostable variants of a mesophilic enzyme (PETase from Ideonella sakaiensis) were identified from a mutant library and characterised against PET substrates in various forms. Articles III and IV describe the application of high-throughput methods for the identification of novel PET hydrolases by directly assaying terephthalic acid (TPA), one of the monomeric building blocks of PET. Furthermore, Article IV describes the possibility of a one-pot conversion of the TPA-based aldehydes produced to their diamines as example for an open-loop upcycling method.
Ziel der Arbeit war es, Mono-Dithiolen-Vanadiumkomplexe zu synthetisieren, die als Katalysatoren in Oxidationsreaktion von prochiralen Sulfiden zu chiralen Sulfoxiden getestet werden sollten.
Es konnten verschiedene Ansätze entwickelt werden, die vielversprechend waren, um durch weitere Forschung Mono-Dithiolen-Vanadiumkomplexen erhalten zu können.
Insbesondere konnte eine universell anwendbare Syntheseroute für die Verwendung von aliphatischen Dithiolenen in der Komplexsynthese erfolgreich gezeigt werden. Außerdem wurden neue Kristallstrukturen verschiedener Dithiolen-Vanadiumkomplexe erhalten.
Die Dissertation beschreibt die Synthese verschiedener Nukleosidanaloga mit den notwendigen Modifizierungen und Funktionalitäten für einen Einsatz in der Phosphoramidit-basierten chemischen Oligonukleotidsynthese an fester Phase. Im Rahmen der Arbeit wurde ein nicht-kanonisches Desoxyadenosinderivat ausgehend von Allopurinol hergestellt. Außerdem wurden verschiedene Azid-modifizierte Nukleoside synthetisiert und Untersuchungen zur Herstellung eines Borono-modifizierten Adenosinderivats durchgeführt. Des Weiteren wurde ein Verfahren zur Bestimmung der Stabilität der Azidogruppe unter Standardbedingungen der Phosphoramidit-basierten chemischen Oligonukleotidsynthese demonstriert.
In dieser Doktorarbeit konnte in zwei verschiedenen experimentellen Modellen der chronischen Pankreatitis in C57BL/6 Mäusen gezeigt werden, dass die chronische Pankreatitis mit einem Gewichtsverlust und einer Verminderung der muskuloskelettalen Kraft assoziiert sind. Untersuchungen im Kleintier-MRT belegten eine signifikante Verminderung des Durchmessers des Quadrizepsmuskels in beiden Modellen. Auf Proteinebene fanden sich im Skelettmuskel von Mäusen mit chronischer Pankreatitis Expressionssteigerungen von growth differentiation factor 8 (GDF8) und Muscle RING-finger protein-1 (MuRF1). Auf mRNA Ebene konnten wir zeigen, dass Activin A und das transforming growth factor β (TGFβ) in beiden Modellen erhöht waren, wohingegen Follistatin und teilweise auch Inhibin A vermindert waren. Die Anzahl apoptotischer Zellen stieg im Quadrizepsmuskel in beiden Modellen signifikant an, was darauf schließen lässt, dass die Apoptose beim Muskelabbau eine Rolle spielt. Des Weiteren fanden sich in Mäusen mit chronischer Pankreatitis und Sarkopenie Veränderungen des Serummetaboloms und des Stuhlmikrobioms, die jedoch in Abhängigkeit des verwendeten Modells stark variierten. Modellübergreifend war eine Vermehrung von Akkermansia spp. in der chronischen Pankreatitis nachweisbar.
Pancreatitis is an inflammatory disorder of the pancreas with a mortality rate of 5% and severe negative effects on the quality of life. Of all non-malignant gastrointestinal diseases, it is the most common reason for hospitalization. Pancreatitis is a disease of multiple etiologies with different underlying pathomechanisms. Due to the diversity of mechanisms by which homeostasis within the exocrine pancreas can be disrupted, finding appropriate therapeutic approaches is challenging. Current treatment options are inadequate and are mostly limited to supportive treatment like fluid administration, bowel rest, antibiotics and pain control. Although significant advancements have been achieved in recent decades, the mortality rate for pancreatitis has not decreased. Furthermore, progress is slow due to limited patient sample availability and lack of an appropriate cell model. Taking samples from a human pancreas is typically avoided, because damaging the pancreatic tissue can itself induce pancreatitis. Additionally, while it is possible to keep individual acini in culture, it is not possible to grow pancreatic acinar cells. Thus, less appropriate cell models, often derived from pancreatic cancer samples, have to be used. The most common animal model for pancreatitis is mice, with caerulein administration being the most common method of inducing pancreatitis. However, the use of animal models has significant drawbacks, as they are time-consuming, costly, and pose ethical questions. Furthermore, exposing the pancreas to appropriate stimuli in animal models is difficult. For example, alcohol is the leading cause of pancreatitis in humans, but is typically avoided by animals. Thus, alcohol feeding methods had to be developed to overcome the natural aversion of rodents to alcohol. Results obtained from animal models are also often not transferable into clinical trials and outcomes in humans remain largely unpredictable. Due to the lack of experimental models, our understanding of this highly complex disease is still limited and significant progress is required for the development of effective therapy options.
In this dissertation recombinantly expressed trypsin isoforms and variants of the serine protease inhibitor Kazal-type 1 (SPINK1) inhibitor are used to investigate mechanisms, by which tryptic activity is regulated in pancreatic acinar cells. With premature tryptic activity in the exocrine pancreas being the common focal point of most etiologies connected to pancreatitis, trypsin represents by far the most promising target for treating pancreatitis. Understanding the mechanisms by which the pancreas protects itself and rationalizing mutations that can undermine these protective mechanisms, are important steps towards developing effective therapies.
Enzymes are well-known for being remarkably selective catalysts. They are often able to catalyse reactions for certain molecules while leaving other similar molecules completely unchanged. Nevertheless, many enzymes are capable of catalysing other reactions and/or transforming other substrates than their physiologically relevant activities. This phenomenon is referred to as enzyme promiscuity and it is thought to play an important role in the emergence of novel functions by providing a starting point for divergent evolution towards different enzymatic activities. It is important for enzymes to be selective to avoid harmful side-products and increase reaction efficiency, but often catalysts are not optimised beyond what is required for their function. Life profits from the cross-reactivity and enzyme promiscuity through accidental discovery of new helpful molecules and pathways, while using regulation to quickly adapt to changing circumstances.
Enzymes are grouped together with other similar proteins into structural families and superfamilies. Members of a structural family share significant structural elements and often have similar catalytic mechanisms. However, they often catalyse very different chemical reactions and accept a variety of different substrates. Promiscuous activities are common within superfamilies, where the primary function of one family member is often found as promiscuous activity in other family members. Together with the structural similarities, this prevalent cross-reactivity suggests a common evolutionary origin. One of the largest structural superfamilies is the α/β-hydrolase-fold family. Despite sharing a highly conserved core structure, this superfamily is catalytically diverse and spans several distinct enzyme classes including hydrolases, acyltransferases, oxidoreductases, lyases, and isomerases. Epoxide hydrolases and dehalogenases of the α/β-hydrolase-fold family even share the same Asp/Glu-His-Asp catalytic triad and form similar covalent alkyl-enzyme reaction intermediates, yet they are known for attacking either epoxides or C-X bonds with perfect chemoselectivity. Although promiscuity is often observed within the α/β-hydrolase fold family and despite their mechanistic similarities, no α/β-hydrolases were known that exhibit both epoxide hydrolase and dehalogenase activity simultaneously.
The versatility of the catalytic triads used by α/β-hydrolases makes these enzymes attractive targets for the conversion of catalytic activity through protein engineering. Several attempts were made to introduce dehalogenase activity in an epoxide hydrolase, and after several rounds of designing and screening different variants of the epoxide hydrolase PaeCIF from Pseudomonas aeruginosa, minor dehalogenase activity was detected for some of the variants. However, despite promising first results it proved extremely difficult to reliably reproduce the results, primarily due to expression problems and low sensitivity of the halide detection assays that were available at the time. Since the conversion proved to be more difficult than expected (unpublished data), it was decided to investigate other potential protein scaffolds.
Considering the prevalence of catalytic promiscuity among members of the α/β-hydrolase-fold superfamily, and the close relationship and catalytic similarities between epoxide hydrolases and dehalogenases, it seemed odd that no enzyme is known to have both epoxide hydrolase and dehalogenase activity. We argued that it is highly probable that a promiscuous epoxide hydrolase-dehalogenase enzyme exists, but it simply has not been found yet due to the absence of sensitive high-throughput halide assays and not screening the right set of enzymes. Although several established assays were available for the determination of dehalogenase activity, these assays suffer major drawbacks. For example, one of the most popular assays, the Iwasaki assay, is not very sensitive and uses extremely toxic chemicals, while pH assays like the phenol red assay are inherently unreliable and insensitive due to the low buffer concentrations employed107,114. Thus, a new assay for the screening of dehalogenase activity through the selective detection of halides was developed115. The halide oxidation assay provides a safer, more reliable, and most importantly, much more sensitive method to detect dehalogenase activity.
Using molecular phylogenetics, we studied the evolutionary relationship between epoxide hydrolases and dehalogenases to identify interesting extant epoxide hydrolases. Molecular phylogenetics uses a multiple sequence alignment of the amino acid or nucleotide sequences of extant enzymes to construct a phylogenetic tree. At first, we tried using a large dataset with almost 3,500 putative epoxide hydrolase and dehalogenase sequences, but we quickly realised the resulting phylogenetic tree was impractical. Most of the sequences in this large dataset were not characterised experimentally but annotated automatically based on their sequence similarity to a rather limited number of characterised sequences. Although automated annotations can be used as predictions for catalytic activity, they are often wrong. As we were particularly interested in the interface of both epoxide hydrolase and dehalogenase activities, we needed more certainty and a change in direction was necessary.
Instead of trying to filter the α/β-hydrolase fold database, experimentally characterised sequences were collected through literature research. This smaller dataset consisting of characterised sequences resulted in a phylogenetic tree containing 45 epoxide hydrolases, 30 haloalkane dehalogenases and 7 haloacetate dehalogenases from a variety of different organisms. Ancestral sequence reconstruction was attempted for several interesting nodes in this phylogenetic tree. By combining the multiple sequence alignment, the evolutionary relationships from the phylogenetic tree, and evolutionary models, a hypothetical sequence of the theoretical ancestor can be determined. Unfortunately, it was difficult to get good soluble protein expression with the ancestral sequences and despite our best efforts it was not possible to obtain reliable and reproducible screening results. Instead of trying to improve protein expression and purification protocols for the ancestral sequences, we decided to focus on screening extant sequences with the newly developed halide oxidation assay to find a promiscuous epoxide hydrolase-dehalogenase.
In addition to reconstructing ancestral sequences, eight extant epoxide hydrolases could be selected for screening towards dehalogenase activity and as promising potential engineering scaffolds from this phylogenetic tree. The eight selected epoxide hydrolases were screened for dehalogenase activity with several haloalkane substrates and the epoxide hydrolase CorEH from Corynebacterium sp. C12 was found to exhibit promiscuous dehalogenase activity. Interestingly, the measured concentrations of bromide for the initial hit with CorEH were only 150-250 nM, well below the lowest detection limit of 20 µM achievable in microtiter plate format with the Iwasaki assay. This means that the dehalogenase activity of CorEH would probably not have been detected were it not for the development of the sensitive halide oxidation assay.
CorEH is an epoxide hydrolase that can also catalyse the dehalogenation of haloalkanes, particularly bromoalkanes such as 1-bromobutane and 1-bromohexane. The dehalogenase activity of wild-type CorEH with 1-bromobutane (0.25 nmol·min-1·mg-1) is about 4,000-fold lower than the average activity of several natural dehalogenases with two halide-stabilising residues (1 μmol·min-1·mg-1) and approximately 400-fold lower compared to the dehalogenases with a single halide-stabilising residue. The crystal structure of CorEH was determined to 2.2 Å. Our structure-function studies suggest that the dehalogenase activity of CorEH probably stems from the presence of at least one halide-stabilising residue. Unfortunately, this could not be confirmed experimentally via mutagenesis as the W100A variant lost both the dehalogenase and epoxide hydrolase activity in equal measure, making it difficult to demonstrate that W100 is involved in halide stabilisation. The loss of both activities for variant W100A can possibly be explained by the secondary function of the tryptophan; removal of W100 might lead to the incorrect positioning of the catalytic nucleophile for the nucleophilic attack involved in both epoxide hydrolysis and dehalogenation. Nevertheless, computational modelling of Michaelis-Menten complexes, utilising the crystal structure of CorEH, supports the hypothesis that the tryptophan W100 is involved in halide stabilisation in CorEH. Based on docking studies, the epoxide ring-opening tyrosine is also close enough to form hydrogen bonds to stabilise the substrate. However, it is also possible that like several characterised haloalkane dehalogenases, CorEH only uses a single residue to stabilise the halide. Removal of the tryptophan at the primary halide-stabilising position resulted in the loss of both activities, likely due to the loss of its secondary function to properly position the catalytic nucleophile. Substitution of the uncommon tryptophan in the HGxP-motif with phenylalanine does not completely remove the dehalogenase activity. Nevertheless, it causes a significant drop in both haloalkane dehalogenase and epoxide hydrolase activities, indicating that this residue is important for catalysis or the structural integrity of CorEH.
Enzyme promiscuity plays an important role in enzyme evolution and the diversification of enzymes. Several researchers have attempted to interconvert epoxide hydrolase and dehalogenase activity, or to find an enzyme with both activities, without success. It would be hard to maintain the view that promiscuity is a fundamental property crucial to enzyme evolution if we could not observe promiscuity between two enzyme classes with such similar reaction mechanisms. Our findings show that dual epoxide hydrolase and dehalogenase activity can occur in one natural protein scaffold. We believe that we succeeded because we used a phylogenetic analysis of characterised sequences to select the right subset of epoxide hydrolases to investigate and due to the much more sensitive halide assays not available to those before us. The versatility of the catalytic triad in α/β-hydrolases combined with the variety of possible supporting residues found in both epoxide hydrolases and dehalogenases shows that catalytic mechanisms can be flexible. This flexibility allows space for diversification of catalytic residues without loss of function, giving rise to novel (promiscuous) functions and new cross-reactivities.
Abstract
Halide methyltransferases (HMTs) enable the enzymatic synthesis of S‐adenosyl‐l‐methionine (SAM) from S‐adenosyl‐l‐homocysteine (SAH) and methyl iodide. Characterisation of a range of naturally occurring HMTs and subsequent protein engineering led to HMT variants capable of synthesising ethyl, propyl, and allyl analogues of SAM. Notably, HMTs do not depend on chemical synthesis of methionine analogues, as required by methionine adenosyltransferases (MATs). However, at the moment MATs have a much broader substrate scope than the HMTs. Herein we provide an overview of the discovery and engineering of promiscuous HMTs and how these strategies will pave the way towards a toolbox of HMT variants for versatile chemo‐ and regioselective biocatalytic alkylations.
Abstract
This work presents the reactivity and dissolution of an as‐polished and electrochemically pre‐treated polycrystalline Au electrode, which is used as a model system. The effect of the electrochemical pre‐treatment in corrosive 0.37 M HCl solutions on the Au surface roughness and dissolution is investigated by varying the number of pre‐treatment steps at 1.16 V against the reversible hydrogen electrode. It is shown that the first 10 s pre‐treatment of the as‐polished Au results in a higher surface roughness and thus higher electrochemically active surface area (ECSA) than that of the as‐polished Au. With the subsequent pre‐treatments, however, the ECSA is gradually decreasing reaching a steady value. The dissolution rate of the pre‐treated Au electrodes upon potential cycling in 0.1 M H2SO4 is determined by in situ inductively coupled plasma mass spectrometry. A non‐linear dependence of Au dissolution amount is found with respect to the number of pre‐treatments. The overall total Au dissolution rate follows a similar trend as ECSA/roughness. However, an important difference in the dissolution behavior is identified with respect to dissolution processes during Au oxidation (anodic dissolution) and Au reduction (cathodic dissolution): the former is more sensitive to the surface roughness. Thus, the ratio between Au anodic and cathodic dissolution amounts decreases substantially with decrease in surface roughness. This finding is explained by the slow and fast dissolution kinetics for anodic and cathodic processes, respectively. Current work further advances our understanding of the complex Au dissolution mechanism.