A disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal stability and activity against polyethylene terephthalate

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

Effect of Tris, MOPS, and phosphate buffers on the hydrolysis of polyethylene terephthalate films by polyester hydrolases

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

Functional characterization and structural modeling of synthetic polyester degrading hydrolases from Thermomonospora curvata

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; Discussio

Efficient extracellular recombinant production and purification of a Bacillus cyclodextrin glucanotransferase in Escherichia coli

Background: Cyclodextrin glucanotransferases (CGTases) catalyze the synthesis of cyclodextrins, cyclic oligosaccharides composed of glucose monomers that find applications in the pharmaceutical, food, and cosmetic industries. An economic application of these industrially important enzymes requires their efficient production and recovery. In this study, the effect of Sec-type signal peptides on the recombinant expression of a CGTase derived from Bacillus sp. G825-6 was investigated in Escherichia coli BL21(DE3) using a codon-adapted gene. In addition, a novel purification method for the CGTase using starch adsorption was developed. Results: Expression vectors encoding N-terminal PelB, DacD, and the native Bacillus sp. G825-6 CGTase signal peptides (SP) were constructed for the recombinant CGTase. With the DacD SP derived from E. coli, a 3.9- and 3.1-fold increase in total enzyme activity was obtained compared to using the PelB and the native CGTase SP, respectively. DacD enabled a 7.3-fold increase of activity in the extracellular fraction after induction for 24 h compared to the native CGTase SP. After induction for 48 h, 75% of the total activity was detected in the extracellular fraction. By a batch wise adsorption to starch, the extracellular produced CGTase could be purified to homogeneity with a yield of 46.5% and a specific activity of 1637 U/mg. Conclusions: The signal peptide DacD promoted the high-level heterologous extracellular expression of a recombinant CGTase from Bacillus sp. G825-6 with a pET20b(+) vector in E. coli BL21(DE3). A protocol based on starch adsorption enabled a fast and efficient purification of the recombinant enzyme

Einfluss von Fehlern auf die Qualität von Streckenbeeinflussungsanlagen

Zur Sicherung der Leistungsfähigkeit der Verkehrsinfrastruktur in Deutschland soll im Bereich der kollektiven Verkehrsbeeinflussung in Zukunft das Werkzeug des prozessorientierten Qualitätsmanagements genutzt werden. Diese Forschungsarbeit hatte die Erarbeitung eines vollständigen Prozess- und Qualitätsmodells und praxistauglicher Handlungsempfehlungen für die verschiedenen Lebenszyklusphasen von Streckenbeeinflussungsanlagen (SBA) zum Ziel. Zu diesem Zweck wurden diverse Grundlagen zur Gestaltung eines auf SBA bezogenen Qualitätsmanagementsystems herangezogen. Das erarbeitete Prozessmodell umfasst 34 bewertungsrelevante Prozesse in den Lebenszyklusphasen Planung, Bau und Betrieb, einschließlich des unterstützenden Prozesses Wartung und Instandsetzung. Zur Identifikation von Lücken in der Qualitätssicherung der Prozesse wurde eine Fehlermöglichkeits- und -einflussanalyse (FMEA) durchgeführt. Anhand von Expertenwissen wurden Fehler und Fehlerursachen analysiert und die kritischsten Qualitätsprobleme zusammengestellt. Anhand der Ergebnisse des Prozessmodells wurde zur quantitativen Beschreibung der Auswirkungen möglicher Fehler ein Qualitätsmodell entwickelt. Auf Grundlage eines Bayes'schen Netzes wurden die Auswirkungen von Fehlern auf nachfolgende Prozesse im gesamten SBA-Lebenszyklus systematisch abgebildet. Das probabilistische Qualitätsmodell kann dabei außerdem als Diagnose-Werkzeug zur Untersuchung der Fehlerursachen von beobachteten Störungen einer SBA genutzt werden. Im Rahmen der Forschungsarbeit wurden anhand der Ergebnisse des Prozessmodells praxisorientierte, ressourcenschonende Handlungsempfehlungen abgeleitet und in einem Dokument für Planer und Betreiber von Streckenbeeinflussungsanlagen zusammengestellt. Zudem wurde eine Methode zur Abschätzung des Nutzens der Maßnahmen beschrieben, um die erforderlichen Investitionen zur Qualitätssicherung zu begründen. Zukünftige Forschungsarbeiten sollten sich im Zusammenhang mit der Adaption des Qualitätsmodells auf eine konkrete SBA insbesondere mit der Optimierung des Modells in Form von Anpassungen der Modelltopologie beschäftigen. Des Weiteren sollte untersucht werden, ob die in dieser Forschungsarbeit erarbeiteten Ergebnisse auf andere Typen von Verkehrsbeeinflussungs-anlagen übertragbar sind

Traffic Loads - Updating and adapting of input data for pavement design

Die Verkehrsbelastung ist einer der maßgebenden Einflussfaktoren für Straßenkonstruktionen. In diesem Bericht werden detaillierte und aktuelle Achslastverteilungen für die Dimensionierung von Straßenoberbauten zur Verfügung gestellt. Grundlage sind Messdaten der Achslastwaagen im deutschen Autobahnnetz. Diese sind nicht flächendeckend vorhanden. Es wird eine Methode zur flächendeckenden Projektion der Achslastmessstellendaten vorgestellt, die die nahezu flächendeckend vorhandenen Daten der Dauerzählstellen nutzt. Mit dieser Methode können bei vorhandenen Dauerzählstelldaten streckenspezifische Verkehrsbelastungskollektive in Form von B-Zahl und Achslastverteilungen bei semiprobabilistischer bzw. Achslastverteilungsfunktionen für probabilistische Dimensionierungs- und Substanzbewertungsverfahren bereitgestellt werden. Des Weiteren werden repräsentative Achslastverteilungen für Autobahnen des Fern-, Misch- und Nahverkehrs sowie für Straßen des nachgeordneten Netzes vorgestellt.Traffic loads are a decisive influencing factors for road structures. This report provides detailed and current axle-load-distributions for the design of road superstructures. Basis are measurements from axle-load-scales within the German motorway network. These scales exist only at selected locations. To project the identified axle-load-distributions to the whole road net-work a method was developed, which utilizes the comprehensive data of continuous traffic-counting-stations. With this method - existing continuous counting data given - route-specific traffic-load-collectives can be estimated in the form of: - load-repetition and axle-load-distributions for semi-probabilistic or - axle-load-distribution-functions for probabilistic dimensioning and substance evaluation methods. Furthermore, representative axle-load-distributions for highways of long-distance, mixed and local traffic as well as for roads of the subordinated network are presented

A disulfide bridge in the calcium binding site of a polyester hydrolase increases its thermal stability and activity against polyethylene terephthalate

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