Thermomonospora curvata is a thermophilic actinomycete hylogenetically related to Thermobifida fusca that produces extracellular hydrolases capable of degrading synthetic polyesters. Analysis of the genome of T. curvata DSM43183 revealed two genes coding for putative polyester hydrolases Tcur1278 and Tcur0390 sharing 61% sequence identity with the T. fusca enzymes. Mature proteins of Tcur1278 and Tcur0390 were cloned and expressed in Escherichia coli TOP10. Tcur1278 and Tcur0390 exhibited an optimal reaction temperature against p-nitrophenyl butyrate at 60°C and 55°C, respectively. The optimal pH for both enzymes was determined at pH 8.5. Tcur1278 retained more than 80% and Tcur0390 less than 10% of their initial activity following incubation for 60 min at 55°C. Tcur0390 showed a higher hydrolytic activity against poly(ε-caprolactone) and polyethylene terephthalate (PET) nanoparticles compared to Tcur1278 at reaction temperatures up to 50°C. At 55°C and 60°C, hydrolytic activity against PET nanoparticles was only detected with Tcur1278. In silico modeling of the polyester hydrolases and docking with a model substrate composed of two repeating units of PET revealed the typical fold of α/β serine hydrolases with an exposed catalytic triad. Molecular dynamics simulations confirmed the superior thermal stability of Tcur1278 considered as the main reason for its higher hydrolytic activity on PET.

Elevated reaction temperatures are crucial for the efficient enzymatic degradation of polyethylene terephthalate (PET). A disulfide bridge was introduced to the polyester hydrolase TfCut2 to substitute its calcium binding site. The melting point of the resulting variant increased to 94.7°C (wild-type TfCut2: 69.8 °C) and its half-inactivation temperature to 84.6 °C (TfCut2: 67.3 °C). The variant D204C-E253C-D174R obtained by introducing further mutations at vicinal residues showed a temperature optimum between 75 and 80 °C compared to 65 and 70 °C of the wild-type enzyme. The variant caused a weight loss of PET films of 25.0 +/- 0.8% (TfCut2: 0.3 +/-0.1%) at 70 °C after a reaction time of 48 h. The results demonstrate that a highly efficient and calcium-independent thermostable polyester hydrolase can be obtained by replacing its calcium binding site with a disulfide bridge.

The enzymatic degradation of polyethylene terephthalate (PET) occurs at mild reaction conditions and may find applications in environmentally friendly plastic waste recycling processes. The hydrolytic activity of the homologous polyester hydrolases LC cutinase (LCC) from a compost metagenome and TfCut2 from Thermobifida fusca KW3 against PET films was strongly influenced by the reaction medium buffers tris(hydroxymethyl)aminomethane (Tris), 3-(N-morpholino)propanesulfonic acid (MOPS), and sodium phosphate. LCC showed the highest initial hydrolysis rate of PET films in 0.2 M Tris, while the rate of TfCut2 was 2.1-fold lower at this buffer concentration. At a Tris concentration of 1 M, the hydrolysis rate of LCC decreased by more than 90% and of TfCut2 by about 80%. In 0.2 M MOPS or sodium phosphate buffer, no significant differences in the maximum initial hydrolysis rates of PET films by both enzymes were detected. When the concentration of MOPS was increased to 1 M, the hydrolysis rate of LCC decreased by about 90%. The activity of TfCut2 remained low compared to the increasing hydrolysis rates observed at higher concentrations of sodium phosphate buffer. In contrast, the activity of LCC did not change at different concentrations of this buffer. An inhibition study suggested a competitive inhibition of TfCut2 and LCC by Tris and MOPS. Molecular docking showed that Tris and MOPS interfered with the binding of the polymeric substrate in a groove located at the protein surface. A comparison of the Ki values and the average binding energies indicated MOPS as the stronger inhibitor of the both enzymes.

Thermomonospora curvata is a thermophilic actinomycete hylogenetically related to Thermobifida fusca that produces extracellular hydrolases capable of degrading synthetic polyesters. Analysis of the genome of T. curvata DSM43183 revealed two genes coding for putative polyester hydrolases Tcur1278 and Tcur0390 sharing 61% sequence identity with the T. fusca enzymes. Mature proteins of Tcur1278 and Tcur0390 were cloned and expressed in Escherichia coli TOP10. Tcur1278 and Tcur0390 exhibited an optimal reaction temperature against p-nitrophenyl butyrate at 60°C and 55°C, respectively. The optimal pH for both enzymes was determined at pH 8.5. Tcur1278 retained more than 80% and Tcur0390 less than 10% of their initial activity following incubation for 60 min at 55°C. Tcur0390 showed a higher hydrolytic activity against poly(ε-caprolactone) and polyethylene terephthalate (PET) nanoparticles compared to Tcur1278 at reaction temperatures up to 50°C. At 55°C and 60°C, hydrolytic activity against PET nanoparticles was only detected with Tcur1278. In silico modeling of the polyester hydrolases and docking with a model substrate composed of two repeating units of PET revealed the typical fold of α/β serine hydrolases with an exposed catalytic triad. Molecular dynamics simulations confirmed the superior thermal stability of Tcur1278 considered as the main reason for its higher hydrolytic activity on PET.:Introduction; Materials and methods; Results; Discussion

Der Actinomycet T. fusca KW3, isoliert aus Kompost, bildete während der Kultivierung im Mineralsalz-Spurenelement-Vitamin-Minimalmedium nach Zusatz von PET-Fasern eine 52 kDa Carboxylesterase (TfCa), welche effizient zyklische PET Trimere (CTR) hydrolysiert. Die TfCa besitzt einen pI von 4,8, eine Substratspezifität gegenüber kurzkettigen p-Nitrophenyl-Estern und wird durch Phenylmethylsulfonylfluorid (PMSF) und Tosyl-L-Phenylalanin-Chloromethylketon (TPCK) in der Aktivität gehemmt. Die Carboxylesterase hydrolysiert kein Cutin oder Poly-ε-caprolacton (PCL). CTR hingegen wurden durch die TfCa mit einem Km von 0,5 mM und einer Vmax von 9,3 μmol/min/mg bei optimalen Bedingungen (60°C, pH 6) hydrolysiert. Das aktive Zentrum der Carboxylesterase besteht aus den Aminosäuren Ser185, Glu319 und His415, wobei das Serin in das katalytische Motiv G-E-S-A-G eingebettet ist. Während der Reaktion setzte die TfCa auch Hydrolyseprodukte aus PET-Fasern und -Filmen frei. Der Nachweis der Hydrolyse erfolgte durch Umkehrphasen-Hochleistungsflüssigkeitschromatographie der Abbauprodukte und bei den PET-Filmen zusätzlich mittels Rasterelektronenmikroskopie. Dabei zeigte die Carboxylesterase verglichen mit anderen PET-Hydrolasen eine geringere Effizienz, was durch die Lage des aktiven Zentrums in einer Bindungstasche und der daraus folgenden schlechten Zugänglichkeit für polymere Substrate begründet werden kann. Bei der Hydrolyse der viel kleineren CTR war die TfCa deutlich effektiver, was auf eine höhere Spezifität gegenüber kurzkettigen PET Substraten hinweist.

Elevated reaction temperatures are crucial for the efficient enzymatic degradation of polyethylene terephthalate (PET). A disulfide bridge was introduced to the polyester hydrolase TfCut2 to substitute its calcium binding site. The melting point of the resulting variant increased to 94.7°C (wild-type TfCut2: 69.8 °C) and its half-inactivation temperature to 84.6 °C (TfCut2: 67.3 °C). The variant D204C-E253C-D174R obtained by introducing further mutations at vicinal residues showed a temperature optimum between 75 and 80 °C compared to 65 and 70 °C of the wild-type enzyme. The variant caused a weight loss of PET films of 25.0 +/- 0.8% (TfCut2: 0.3 +/-0.1%) at 70 °C after a reaction time of 48 h. The results demonstrate that a highly efficient and calcium-independent thermostable polyester hydrolase can be obtained by replacing its calcium binding site with a disulfide bridge.

The enzymatic degradation of polyethylene terephthalate (PET) occurs at mild reaction conditions and may find applications in environmentally friendly plastic waste recycling processes. The hydrolytic activity of the homologous polyester hydrolases LC cutinase (LCC) from a compost metagenome and TfCut2 from Thermobifida fusca KW3 against PET films was strongly influenced by the reaction medium buffers tris(hydroxymethyl)aminomethane (Tris), 3-(N-morpholino)propanesulfonic acid (MOPS), and sodium phosphate. LCC showed the highest initial hydrolysis rate of PET films in 0.2 M Tris, while the rate of TfCut2 was 2.1-fold lower at this buffer concentration. At a Tris concentration of 1 M, the hydrolysis rate of LCC decreased by more than 90% and of TfCut2 by about 80%. In 0.2 M MOPS or sodium phosphate buffer, no significant differences in the maximum initial hydrolysis rates of PET films by both enzymes were detected. When the concentration of MOPS was increased to 1 M, the hydrolysis rate of LCC decreased by about 90%. The activity of TfCut2 remained low compared to the increasing hydrolysis rates observed at higher concentrations of sodium phosphate buffer. In contrast, the activity of LCC did not change at different concentrations of this buffer. An inhibition study suggested a competitive inhibition of TfCut2 and LCC by Tris and MOPS. Molecular docking showed that Tris and MOPS interfered with the binding of the polymeric substrate in a groove located at the protein surface. A comparison of the Ki values and the average binding energies indicated MOPS as the stronger inhibitor of the both enzymes.

Der Actinomycet T. fusca KW3, isoliert aus Kompost, bildete während der Kultivierung im Mineralsalz-Spurenelement-Vitamin-Minimalmedium nach Zusatz von PET-Fasern eine 52 kDa Carboxylesterase (TfCa), welche effizient zyklische PET Trimere (CTR) hydrolysiert. Die TfCa besitzt einen pI von 4,8, eine Substratspezifität gegenüber kurzkettigen p-Nitrophenyl-Estern und wird durch Phenylmethylsulfonylfluorid (PMSF) und Tosyl-L-Phenylalanin-Chloromethylketon (TPCK) in der Aktivität gehemmt. Die Carboxylesterase hydrolysiert kein Cutin oder Poly-ε-caprolacton (PCL). CTR hingegen wurden durch die TfCa mit einem Km von 0,5 mM und einer Vmax von 9,3 μmol/min/mg bei optimalen Bedingungen (60°C, pH 6) hydrolysiert. Das aktive Zentrum der Carboxylesterase besteht aus den Aminosäuren Ser185, Glu319 und His415, wobei das Serin in das katalytische Motiv G-E-S-A-G eingebettet ist. Während der Reaktion setzte die TfCa auch Hydrolyseprodukte aus PET-Fasern und -Filmen frei. Der Nachweis der Hydrolyse erfolgte durch Umkehrphasen-Hochleistungsflüssigkeitschromatographie der Abbauprodukte und bei den PET-Filmen zusätzlich mittels Rasterelektronenmikroskopie. Dabei zeigte die Carboxylesterase verglichen mit anderen PET-Hydrolasen eine geringere Effizienz, was durch die Lage des aktiven Zentrums in einer Bindungstasche und der daraus folgenden schlechten Zugänglichkeit für polymere Substrate begründet werden kann. Bei der Hydrolyse der viel kleineren CTR war die TfCa deutlich effektiver, was auf eine höhere Spezifität gegenüber kurzkettigen PET Substraten hinweist.:Inhaltsverzeichnis 1 Einführung 1 1.1 Actinomyceten 1 1.1.1 Klassifizierung 1 1.1.2 Thermobifida (Thermomonospora) fusca 2 1.2 Biopolyester Cutin und Suberin 4 1.2.1 Bestandteile des Cutins 4 1.2.2 Bestandteile des Suberins 5 1.2.3 Struktur der Biopolymere 7 1.3 Hydrolasen 12 1.3.1 Struktur und katalytischer Mechanismus 12 1.3.2 Carboxylesterasen 16 1.3.3 Lipasen 19 1.3.4 Cutinasen 22 1.4 Polyethylenterephthalat 23 1.4.1 Konventionelle PET-Faserbehandlung 24 1.4.2 Charakterisierung von PET 26 1.4.3 Biofunktionalisierung von PET 30 1.4.4 PET-Hydrolasen 38 1.5 Zielsetzung 49 2 Material und Methoden 50 2.1 Materialien 50 2.1.1 Verwendete Mikroorganismen 50 2.1.2 Verwendete Enzyme 50 2.1.3 Größenstandards 51 2.1.3.1 Low Molecular Weight Marker (GE Healthcare) 51 2.1.3.2 Roti®-Mark Standard (Fa. Carl Roth GmbH) 51 2.1.3.3 SpectraTM Multicolor Broad range Protein Ladder (Fermentas) 51 2.1.3.4 PageRulerTM plus prestained protein ladder (Fermentas) 51 2.1.3.5 Kalibrierungskit für pI Bestimmung (pH 3-10, GE Healthcare) 52 2.1.3.6 Kalibrierungskit für die Größenausschlusschromatographie (LMW, GE Healthcare) 52 2.1.4 Chemikalien 53 2.1.5 Geräte und Materialien 55 VI 2.1.6 Software 58 2.1.7 Nährmedien 58 2.1.8 Suberin- und Cutin-Präparationen 60 2.1.9 PET-Substrate für die Abbauuntersuchungen 60 2.1.10 Puffer und Lösungen 60 2.2 Mikrobiologische Methoden 65 2.2.1 Stammhaltung und Kultivierung 65 2.2.2 Mikroskopische Untersuchungen 65 2.2.3 Trockengewichtsbestimmung 65 2.3 Proteinchemische Methoden 66 2.3.1 Proteinaufreinigung der Wildtyp TfCa 66 2.3.2 Proteinaufreinigung der rekombinanten TfCa, TfCut1 und TfCut2 67 2.4 Analytische Methoden 67 2.4.1 Esterase-Aktivitätsbestimmung 67 2.4.1.1 Bestimmung mittels Spektrophotometer 68 2.4.1.2 Bestimmung mittels Plattenleser 68 2.4.2 Cutinase-Aktivitätsbestimmung 68 2.4.3 PCL-Abbauuntersuchung 69 2.4.3.1 Bestimmung mittels Hofbildung 69 2.4.3.2 Bestimmung mittels Plattenleser 69 2.4.4 Quantitative Protein-Bestimmung nach Bradford (1976) 69 2.4.5 SDS Polyacrylamidgelelektrophorese (SDS-PAGE) 70 2.4.5.1 Esteraseaktivitäts-Färbung 70 2.4.5.2 Coomassie-Färbung 71 2.4.5.3 Silberfärbung der Proteine 71 2.4.6 Bestimmung des pI 71 2.4.7 Bestimmung der Molaren Masse 72 2.4.8 Bestimmung der Temperatur und pH-Wert Stabilität 72 2.4.9 Bestimmung der Stabilität gegenüber Inhibitoren 72 2.4.10 Bestimmung der kinetischen Konstanten für die Hydrolyse von p-NP Ester 72 2.4.11 Bestimmung der kinetische Konstanten für die Hydrolyse von CTR 73 2.4.12 Bestimmung optimaler Temperatur und pH-Wert für die Hydrolyse von CTR 73 2.4.13 N-terminale Sequenzierung 73 2.4.14 MALDI-TOF Sequenzierung 74 2.4.15 Abbaustudien 74 VII 2.4.16 Analytik der PET Abbauprodukte 75 2.4.17 Konzentrationabhängige CTR-Abbaustudien 75 2.5 Homologie-Modelling der TfCa und weitere PET-Hydrolasen 76 3 Ergebnisse und Diskussion 77 3.1 Screening nach PET-Hydrolasen aus T. fusca 77 3.1.1 Wachstum von T. fusca KW3 im Czapek-Medium 77 3.1.2 Wachstum von T. fusca KW3 im MSV-Medium 78 3.1.2.1 Esterasebildung mit verschiedenen synthetischen und natürlichen Polyestern 78 3.1.2.2 Esterasebildung mit einer Suberinpräparation 81 3.1.2.3 Esterasebildung mit PET-Fasern 83 3.1.3 Esterasebildung bei T. fusca KW3 DSM 6013, T. fusca DSM 43792 und DSM 43793 mit PET-Fasern und Diethylterephthalat 85 3.2 Charakterisierung der PET-Hydrolasen aus T. fusca KW3 95 3.2.1 Aufreinigung 95 3.2.1.1 Aufreinigung der TfCa 95 3.2.1.2 Aufreinigung der rekombinanten PET-Hydrolasen 105 3.2.2 pI der TfCa 113 3.2.3 Molare Masse der TfCa 113 3.2.4 Temperatur- und pH-Stabilität der TfCa 114 3.2.5 Wirkung von Inhibitoren auf die TfCa 117 3.2.6 Kinetik der Hydrolyse von verschiedenen Esterasesubstraten durch die TfCa 118 3.2.7 Optimale Temperatur und optimaler pH-Wert der CTR-Hydrolyse durch die TfCa 119 3.2.8 Cutinaseaktivität der TfCa 120 3.2.9 PCL-Abbau durch die TfCa 121 3.2.10 N-terminale Sequenz der TfCa 122 3.2.11 MALDI-TOF Sequenzierung der TfCa 123 3.2.12 Homologie-Modeling der TfCa und Vergleich der Struktur mit anderen Hydrolasen 126 3.2.13 Vergleich der TfCa mit bekannten PET-Hydrolasen 128 3.3 Hydrolyse von PET Substraten durch prokaryotische und eukaryotische Hydrolasen 138 3.3.1 Partielle Hydrolyse von APET-Filmen durch die PET-Hydrolasen 140 3.3.2 Partielle Hydrolyse von PET-Fasern durch die PET-Hydrolasen 148 3.3.3 Hydrolyse von PET-Trimeren durch die PET-Hydrolasen 151 4 Zusammenfassung 165 VIII 5 Literaturverzeichnis 166 6 Publikationen 181 7 Poster und Vorträge 182 8 Lebenslauf 183

The aim of this research is to develop a wet-chemical silvering method for a three-dimensional (3D) textile material made of polyethylene terephthalate (PET) to prevent and eliminate biological contaminants in drinking water and other liquid-containing systems. Three-dimensional textile fabrics are particularly well-suited as silvered disinfection materials in water systems, because they have 3D structures, pressure-elastic textile design, and provide large contact areas. Furthermore, water can easily be passed through the structure. The developed wet-chemical procedures are based on aminosilane, which consists of at least two amine groups and is well-suited to form a silver diamine complex. The silvered textile material was coated with cationic silver. After the chemical reduction, the cationic silver turns into metallic silver on the surface of PET spacer fabrics. The surface morphology of silver-coated spacer fabrics was analyzed and the uniform silver layer on the PET fiber surface was found. X-ray diffraction and energy-dispersive X-ray spectroscopy analysis spectrums showed that the silver was immobilized on the PET fiber surface. The layer thickness and the silver amount were also determined. The silvered spacer fabrics can be used in sealing and/or cooling water systems; therefore, the silver ion release in water was analyzed. Furthermore, textile physical tests for the measurement of breaking force and elongation were carried out. No significant change in breaking force and elongation was observed after silvering of PET spacer fabric.