96 research outputs found

    Surface structure and properties of poly-(ethylene terephthalate) hydrolyzed by alkali and cutinase

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    This study was aimed at comparatively investigating the hydrolysis of crystalline and amorphous poly-(ethylene terephthalate) films by alkali and cutinase. Changes of surface properties were investigated by FTIR spectroscopy (ATR mode). The A1341/A1410 and I1120/I1100 absorbance ratios, and the full width at half maximum of the carbonyl stretching band (FWHM1715) were used to evaluate the polymer crystallinity and its changes upon hydrolysis. The effect of different treatments on chain orientation was evaluated by calculating R ratios of appropriate bands. The spectroscopic indexes showed that both alkali and enzyme treatments induced structural and conformational rearrangements with a consequent increase in crystallinity in both amorphous and crystalline films. The crystalline PET film was modified more strongly by alkali than by cutinase, while the opposite occurred for the amorphous one. The trend of the water contact angle (WCA) clearly indicates that alkali is more effective than cutinase in enhancing hydrophilicity of PET films and that the effect is stronger on amorphous than on crystalline films. The values of WCA correlate well with the FTIR indexes calculated from the spectra of hydrolyzed crystalline PET films. The mechanism of the surface hydrolysis of PET by alkali and cutinase is discussed

    Fungal Enzymes as Catalytic Tools for Polyethylene Terephthalate (PET) Degradation.

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    The ubiquitous persistence of plastic waste in diverse forms and different environmental matrices is one of the main challenges that modern societies are facing at present. The exponential utilization and recalcitrance of synthetic plastics, including polyethylene terephthalate (PET), results in their extensive accumulation, which is a significant threat to the ecosystem. The growing amount of plastic waste ending up in landfills and oceans is alarming due to its possible adverse effects on biota. Thus, there is an urgent need to mitigate plastic waste to tackle the environmental crisis of plastic pollution. With regards to PET, there is a plethora of literature on the transportation route, ingestion, environmental fate, amount, and the adverse ecological and human health effects. Several studies have described the deployment of various microbial enzymes with much focus on bacterial-enzyme mediated removal and remediation of PET. However, there is a lack of consolidated studies on the exploitation of fungal enzymes for PET degradation. Herein, an effort has been made to cover this literature gap by spotlighting the fungi and their unique enzymes, e.g., esterases, lipases, and cutinases. These fungal enzymes have emerged as candidates for the development of biocatalytic PET degradation processes. The first half of this review is focused on fungal biocatalysts involved in the degradation of PET. The latter half explains three main aspects: (1) catalytic mechanism of PET hydrolysis in the presence of cutinases as a model fungal enzyme, (2) limitations hindering enzymatic PET biodegradation, and (3) strategies for enhancement of enzymatic PET biodegradation

    In Silico Studies on Proteins for Synthetic Biology

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    Synthetic biology develops artificial biomolecules or biological systems with novel functionalities for diverse applications in research, medicine or industry. This thesis focuses on in silico studies of three proteins that are promising candidates for enzymatic plastic waste treatment and highly sensitive biosensors, respectively. The first candidate is the enzyme Fusarium solani Cutinase (Longhi and Cambillau 1999), which is able to degrade synthetic polymers, like PET. It allows for the development of an environmental friendly and sustainable solution for plastic waste treatment on an industrial scale. As the wildype enzyme loses its activity during the process of PET degradation, a rational design approach was followed, to improve the activity of this enzyme for PET as substrate. Via MD simulations and linear response theory (LRT) (Ikeguchi et al. 2005) based on coarse-grained elastic network models, the reason for the loss of activity could be identified. Based on the knownledge gained, mutants with improved activity for PET were proposed. In the context of this study, an extension for the LRT method similar to that of a previous study (Knorr 2015) was developed. The second protein system, the hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel (Santoro and Tibbs 1999), regulates the flux of ions across biological membranes by changes in the membrane voltage and binding of the ligand cAMP. Hence, it is an ideal model for studying the interplay of different domains during the gating process. Together with plenty of other ion channels, it can also serve as building blocks for the assembly of different domains to design synthetic ion channels with novel functionalities. To understand the complex mechanism of HCN gating, the extension of the LRT method was adjusted to work for a tetramer and was used to determine the conformational changes that occur upon binding of the ligand cAMP. In this context, movements in the transmembrane domains that are involved in the gating process were discovered for the first time. They provide important information on the complex gating mechanism and enable a directed planning of further experimental and theoretical investigations. Small viral pore forming proteins also enable the flux of ions across biological membranes and therefore can be seen as viral companions of ion channels. The third protein is such a pore forming protein from HIV and simian relatives SIV, called Vpu (Cohen et al. 1988). As this small protein is less complex than ion channels but also exhibits ion channel function, it is another candidate to serve as building block for the design of artificial ion channels. To consider the Vpu protein as possible building block, the formation of an ion conducting pore has to be a reliable property. In this thesis, the evolutionary conservation of ion channel formation was proved by computing the Shannon entropy (Strait and Dewey 1996) for involved residues based on a multiple sequence alignment. Although the study could not clarify the role of the ion channel function for virus release or replication, the detected evolutionary conservation serves as proof for the functional significance. Hence, this protein reliably forms an ion conducting pore and can be further considered as possible building block for the assembly of synthetic ion channels

    Enhanced hydrolysis of polyethylene terephthalate (PET) plastics by ozone and ultrasound pretreatment

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    The rapidly accumulating post-consumer polyethylene terephthalate (PET) plastics pose a great threat to our environment as they constitute one of the most used products in our day-to-day life. As a result, degradation of PET and recycling has become the focus of considerable interest in the last decade. Hydrolysis of PET is very challenging as they are extremely resistant to both biotic and abiotic degradation. A technically and economically feasible approach to degrade PET waste from the environment is highly desirable. Physicochemical pre-treatment can play an important role in making PET more degradable by changing their surface properties. Direct recycling of segregated PET has problems of contamination of additives and components used in various PET products. PET hydrolysis however can lead to recovery of the monomers terephthalic acid (TPA) and ethylene glycol (EG) as well as the dimers bis(2-hydroxyethyl) terephthalate (BHET) and mono(2- hydroxyethyl) terephthalate (MHET) which can be reused for making new PETs. This can potentially solve the difficulties associated with PET recycling and lead to a circular economy. The present study reports the effect of ozone and ultrasound pretreatment on both enzymatic and chemical hydrolysis of PET. The results showed that combination of ozone pretreatment followed by ultra-sonication during enzymatic hydrolysis using HiC cutinase enzyme resulted in almost 9-fold increase in TPA and EG recovery compared to enzymatic hydrolysis of untreated PET. However, the long reaction time in enzymatic hydrolysis prompted us to investigate chemical hydrolysis. Although, chemical hydrolysis of pretreated PET films using methanolic sodium hydroxide as solvent resulted in 80% weight loss (at 50C, atmospheric pressure), the recovery of monomers was relatively not as efficient as enzymatic hydrolysis. Size reduction of the PET films followed by chemical hydrolysis gave the highest (90%) breakdown, but it is a very energy intensive process

    Development of Computer-aided Concepts for the Optimization of Single-Molecules and their Integration for High-Throughput Screenings

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    In the field of synthetic biology, highly interdisciplinary approaches for the design and modelling of functional molecules using computer-assisted methods have become established in recent decades. These computer-assisted methods are mainly used when experimental approaches reach their limits, as computer models are able to e.g., elucidate the temporal behaviour of nucleic acid polymers or proteins by single-molecule simulations, as well as to illustrate the functional relationship of amino acid residues or nucleotides to each other. The knowledge raised by computer modelling can be used continuously to influence the further experimental process (screening), and also shape or function (rational design) of the considered molecule. Such an optimization of the biomolecules carried out by humans is often necessary, since the observed substrates for the biocatalysts and enzymes are usually synthetic (``man-made materials'', such as PET) and the evolution had no time to provide efficient biocatalysts. With regard to the computer-aided design of single-molecules, two fundamental paradigms share the supremacy in the field of synthetic biology. On the one hand, probabilistic experimental methods (e.g., evolutionary design processes such as directed evolution) are used in combination with High-Throughput Screening (HTS), on the other hand, rational, computer-aided single-molecule design methods are applied. For both topics, computer models/concepts were developed, evaluated and published. The first contribution in this thesis describes a computer-aided design approach of the Fusarium Solanie Cutinase (FsC). The activity loss of the enzyme during a longer incubation period was investigated in detail (molecular) with PET. For this purpose, Molecular Dynamics (MD) simulations of the spatial structure of FsC and a water-soluble degradation product of the synthetic substrate PET (ethylene glycol) were computed. The existing model was extended by combining it with Reduced Models. This simulation study has identified certain areas of FsC which interact very strongly with PET (ethylene glycol) and thus have a significant influence on the flexibility and structure of the enzyme. The subsequent original publication establishes a new method for the selection of High-Throughput assays for the use in protein chemistry. The selection is made via a meta-optimization of the assays to be analyzed. For this purpose, control reactions are carried out for the respective assay. The distance of the control distributions is evaluated using classical static methods such as the Kolmogorov-Smirnov test. A performance is then assigned to each assay. The described control experiments are performed before the actual experiment (screening), and the assay with the highest performance is used for further screening. By applying this generic method, high success rates can be achieved. We were able to demonstrate this experimentally using lipases and esterases as an example. In the area of green chemistry, the above-mentioned processes can be useful for finding enzymes for the degradation of synthetic materials more quickly or modifying enzymes that occur naturally in such a way that these enzymes can efficiently convert synthetic substrates after successful optimization. For this purpose, the experimental effort (consumption of materials) is kept to a minimum during the practical implementation. Especially for large-scale screenings, a prior consideration or restriction of the possible sequence-space can contribute significantly to maximizing the success rate of screenings and minimizing the total time they require. In addition to classical methods such as MD simulations in combination with reduced models, new graph-based methods for the presentation and analysis of MD simulations have been developed. For this purpose, simulations were converted into distance-dependent dynamic graphs. Based on this reduced representation, efficient algorithms for analysis were developed and tested. In particular, network motifs were investigated to determine whether this type of semantics is more suitable for describing molecular structures and interactions within MD simulations than spatial coordinates. This concept was evaluated for various MD simulations of molecules, such as water, synthetic pores, proteins, peptides and RNA structures. It has been shown that this novel form of semantics is an excellent way to describe (bio)molecular structures and their dynamics. Furthermore, an algorithm (StreAM-Tg) has been developed for the creation of motif-based Markov models, especially for the analysis of single molecule simulations of nucleic acids. This algorithm is used for the design of RNAs. The insights obtained from the analysis with StreAM-Tg (Markov models) can provide useful design recommendations for the (re)design of functional RNA. In this context, a new method was developed to quantify the environment (i.e. water; solvent context) and its influence on biomolecules in MD simulations. For this purpose, three vertex motifs were used to describe the structure of the individual water molecules. This new method offers many advantages. With this method, the structure and dynamics of water can be accurately described. For example, we were able to reproduce the thermodynamic entropy of water in the liquid and vapor phase along the vapor-liquid equilibrium curve from the triple point to the critical point. Another major field covered in this thesis is the development of new computer-aided approaches for HTS for the design of functional RNA. For the production of functional RNA (e.g., aptamers and riboswitches), an experimental, round-based HTS (like SELEX) is typically used. By using Next Generation Sequencing (NGS) in combination with the SELEX process, this design process can be studied at the nucleotide and secondary structure levels for the first time. The special feature of small RNA molecules compared to proteins is that the secondary structure (topology), with a minimum free energy, can be determined directly from the nucleotide sequence, with a high degree of certainty. Using the combination of M. Zuker's algorithm, NGS and the SELEX method, it was possible to quantify the structural diversity of individual RNA molecules under consideration of the genetic context. This combination of methods allowed the prediction of rounds in which the first ciprofloxacin-riboswitch emerged. In this example, only a simple structural comparison was made for the quantification (Levenshtein distance) of the diversity of each round. To improve this, a new representation of the RNA structure as a directed graph was modeled, which was then compared with a probabilistic subgraph isomorphism. Finally, the NGS dataset (ciprofloxacin-riboswitch) was modeled as a dynamic graph and analyzed after the occurrence of defined seven-vertex motifs. For this purpose, motif-based semantics were integrated into HTS for RNA molecules for the first time. The identified motifs could be assigned to secondary structural elements that were identified experimentally in the ciprofloxacin aptamer R10k6. Finally, all the algorithms presented were integrated into an R library, published and made available to scientists from all over the world

    Biotechnological model for ubiquitous mixed petroleum- and bio-based plastics degradation and upcycling into bacterial nanocellulose

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    Ubiquitous post-consumer plastic waste is often physically mixed combining recalcitrant petroleum-based plastics with bioplastics, forming (petro-bio)plastic streams. Finding appropriate end-of-life (EoL) strategies for mixed (petro-bio)plastic waste is highly pertinent in achieving environmental protection, sustainability for plastic value chain industries including recyclers and government policy makers worldwide. The presence of bioplastic mixed in with polyethylene terephthalate (PET) or other petroleum-based plastic streams poses a substantial drawback to mechanical recycling and strongly impedes the development of sustainable EoL routes. Here, we present a model system for the sustainable management of mixed (petro-bio)plastic waste, demonstrating a biotechnological route through synergy-promoted enzymatic degradation of PET–representing petrochemical polyester plastic–mixed with thermoplastic starch (TPS)–as a model bioplastic. Leaf-branch compost cutinase (LCCICCG) and commercial amylase (AMY) deliver effective depolymerization of this mixed (petro-bio)plastic material, with subsequent bio-upcycling of the mixed waste stream into bacterial nanocellulose (BNC) by Komagataeibacter medellinensis. Compared to LCCICCG and AMY, the LCCICCG/AMY combined treatment synergistically produced a 2.6- and 4.4-fold increase in enzymatic decomposition at 70 °C in four days, respectively, yielding sugars and terephthalic acid (TPA) as the main depolymerization building blocks. Bio-upcycling of post-enzymatic degradation hydrolysates resulted in a high BNC yield of 3 g L−1 after 10 days. This work paves the way for sustainable management routes for challenging mixed recalcitrant plastic and bioplastic waste and prepares opportunities for its participation in the circular production of sustainable eco-polymers

    Biodegradation of synthetic and biodegradable plastics by leachate microbiomes

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    Dissertação de mestrado em BiotecnologiaNos últimos anos, várias estratégias têm sido desenvolvidas para colmatar a acumulação de plásticos no ambiente, como a descoberta de novos microrganismos e enzimas que consigam eficientemente biodegradar plásticos. Neste trabalho, as comunidades microbianas de lixiviado, em aerobiose e anaerobiose em condições termófilas, foram estudadas pela sua capacidade de biodegradar polímeros não-biodegradáveis (PE (polietileno) e PET (politereftalato de etileno)) e biodegradáveis (PCL (policaprolactona e PHB/PBAT (polihidroxibutirato/poli (butileno adipato-co-tereftalato)). Esta biodegradação também foi testada utilizando sedimento marinho como inóculo, em condições aeróbicas, metanogénicas e sulfato-redutores em temperaturas mesófilas. As experiências com lixiviado demonstraram uma biodegradação completa com PCL em pó, em condições anaeróbicas e aeróbicas (103 ± 18 % e 99 ± 6 %, respetivamente), observando-se, também, uma biodegradação completa para o PCL em filme em condições anaeróbias (100 ± 0,2%), e uma biodegradação de 28 a 100% em condições aeróbias. PHB/PBAT demonstrou uma biodegradação parcial (24 % ± 0,2 %) em anaerobiose. Contudo, não se observou uma produção de metano/consumo de oxigénio significativa para o PE e PET, resultando numa baixa biodegradação. Mesmo assim, um dos ensaios demonstrou uma biodegradação aparente de 5 ± 2%, ao fim de 180 dias. As comunidades microbianas dos ensaios com PCL demonstraram ser distintas e diversas. Coprothermobacter estava presente em grande abundância nos ensaios aeróbios e anaeróbios e poderá ter estado diretamente ligado à biodegradação de PCL. Methanothermobacter demonstrou ser o microrganismo metanogénico mais abundante (mais de 55 % abundância relativa), tendo um papel importante na conversão do PCL a metano. Nos estudos com sedimento marinho, o PCL demonstrou ser biodegradado em condições aeróbias e sulfato-redutoras, mas não em condições metanogénicas. Até ao momento, a comunidade microbiana de sedimento não demonstrou ter capacidade para biodegradar PE e PET Estes resultados demostram que lixiviado e sedimento marinho são potenciais fontes de microrganismos com a capacidade de biodegradar PCL, sendo necessário mais estudos para isolar e caracterizar estas comunidades microbianas.In the last decades, various strategies have been developed to overcome the plastic waste problem, such as using biodegradable polymers, applying treatments that facilitate plastic degradation, and discovering novel microorganisms and enzymes that are capable of biodegrading complex polymers. This work explored leachate microbial communities in aerobic and anaerobic thermophilic conditions for their ability to biodegrade non-biodegradable (PE (polyethylene) and PET (polyethylene terephthalate)) and biodegradable (PCL (polycaprolactone) and PHB/PBAT (polyhydroxy butyrate/polybutylene adipate co-terephthalate blend)) polymers. Biodegradation was also tested with microbiomes from marine sediment, under aerobic, methanogenic, and sulphate-reducing mesophilic conditions. With leachate, complete biodegradation of powder PCL was observed both under anaerobic and aerobic conditions (103 ± 18 % and 99 ± 6 %, respectively). PCL films were fully converted to methane (100 ± 0,2%) under anaerobic conditions, and biodegradation under aerobic conditions ranged from 28 to 100 %. The blend PHB/PBAT was partially biodegraded under anaerobic conditions (24 ± 0,2 %). Generally, no significant methane production or oxygen consumption were detected in the assays with PE and PET, indicating no considerable biodegradation. Nevertheless, in one assay PE was apparently converted to methane (5 ± 2 % in 180 days), but further analyses are necessary to confirm this biodegradation. PCL-degrading microbial communities developed under aerobic and anaerobic assays were diverse and distinct. Coprothermobacter sp. was very abundant in aerobic and anaerobic incubations and was potentially involved in PCL biodegradation in both conditions. Methanothermobacter sp. was the most abundant methanogen (over 55 % relative abundance), being an important player during PCL conversion to methane. PCL was also biodegraded by the marine sediment, under aerobic and sulphate-reducing conditions, but not under methanogenic conditions. Thus far, the marine sediment microbiome did not biodegrade PE and PET. These results show that leachate and marine sediment microbiomes are potentially good sources of microorganisms with the ability to biodegrade PCL, and further attempts should be made to isolate key microorganisms, obtain efficient microbial consortia, facilitate microbial access to the polymers, and stimulate the activity of plastic-degrading microorganisms
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