Annually, 400 Mt of plastics are produced of which roughly 40% is discarded within a year. Current plastic waste management approaches focus on applying physical, thermal, and chemical treatments... Show moreAnnually, 400 Mt of plastics are produced of which roughly 40% is discarded within a year. Current plastic waste management approaches focus on applying physical, thermal, and chemical treatments of plastic polymers. However, these methods have severe limitations leading to the loss of valuable materials and resources. Another major drawback is the rapid accumulation of plastics into the environment causing one of the biggest environmental threats of the twenty-first century. Therefore, to complement current plastic management approaches novel routes toward plastic degradation and upcycling need to be developed. Enzymatic degradation and conversion of plastics present a promising approach toward sustainable recycling of plastics and plastics building blocks. However, the quest for novel enzymes that efficiently operate in cost-effective, large-scale plastics degradation poses many challenges. To date, a wide range of experimental set-ups has been reported, in many cases lacking a detailed investigation of microbial species exhibiting plastics degrading properties as well as of their corresponding plastics degrading enzymes. The apparent lack of consistent approaches compromises the necessary discovery of a wide range of novel enzymes. In this review, we discuss prospects and possibilities for efficient enzymatic degradation, recycling, and upcycling of plastics, in correlation with their wide diversity and broad utilization. Current methods for the identification and optimization of plastics degrading enzymes are compared and discussed. We present a framework for a standardized workflow, allowing transparent discovery and optimization of novel enzymes for efficient and sustainable plastics degradation in the future. Show less
Hoogendoorn, S.; Mock, E.D.; Strijland, A.; Donker-Koopman, W.E.; Elst, H. van den; Berg, R.J.B.H.N. van den; ... ; Overkleeft, H.S. 2015
Receptors tyrosine kinases or RTKs are cell surface receptors that regulate numerous cellular processes, but also have a critical role in the development and progression of many types of cancer.... Show moreReceptors tyrosine kinases or RTKs are cell surface receptors that regulate numerous cellular processes, but also have a critical role in the development and progression of many types of cancer. The overexpression of EphA4, a member of the RTK family, has been observed in a variety of malignant carcinomas. The aim of the research project associated with this thesis was to develop high affinity inhibitors of the tyrosine kinase EphA4. Ligand discovery was based on two complementary approaches, a computational screen and an NMR based screen using Target Immobilized NMR Screening (TINS). In addition, orthogonal biophysical methods including Surface Plasmon Resonance (SPR) and protein observed NMR were employed to analyse fragment binding. The crystal structure of the EphA4 kinase domain was solved and the structure of the kinase domain in complex with dasatinib, a well-known kinase inhibitor, was also elucidated. The in silico approach discovered a potent inhibitor of EphA4 for which the binding mode was elucidated via X-ray crystallography. Moreover, the TINS approach identified two compounds that may constitute starting points for the generation of more potent EphA4 inhibitors. Show less
Membrane proteins are an interesting class due to the variety of cellular functions and their importance as pharmaceutical targets, but they pose significant challenges for fragment-based drug... Show moreMembrane proteins are an interesting class due to the variety of cellular functions and their importance as pharmaceutical targets, but they pose significant challenges for fragment-based drug discovery approaches. Here we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. Using the recently developed Target Immobilized NMR Screening (TINS) approach, we screened 1,200 fragments for binding to the enzyme Disulphide bond forming protein B. Biochemical and biophysical validation of the 8 most potent hits revealed an IC50 range of 7 to 200 uM, which could be categorized as cofactor binding inhibitors or mixed model inhibitors of both cofactor and substrate protein interaction. Our results establish the utility of fragment-based methods in the development of inhibitors of membrane proteins, making a wide variety3of important membrane bound pharmaceutical targets amenable to such an approach. We first tested the immobilization procedure on G protein coupled receptors and ion channels. Furthermore, we used Nanodiscs, an alternative solubilization strategy, to solubilize teh protein without detergents. This resulted in less broad NMR signals, less non-specific binding issues, and identification of the binders from the original screen, proving that the nanodisc solubilization technique is compatible with TINS. Show less
This thesis describes the tropic acid enzymes which in 4 steps catalyze the metabolic pathway of the alkaloid atropine. The enzymes have been discovered in a Pseudomonas bacterium from soil... Show moreThis thesis describes the tropic acid enzymes which in 4 steps catalyze the metabolic pathway of the alkaloid atropine. The enzymes have been discovered in a Pseudomonas bacterium from soil underneath the Atropa belladonna plant, the natural producer of atropine. These 4 enzymes are highly specific and show peculiar kinetics explained by the very asymmetrical equilibrium for step 2 and a keto-enol tautomerism for the product of step 2, the 2-phenylmalonic semialdehyde. The last part concerns the regulation of enzyme synthesis by product induction and its major advantage for cell economy. This thesis was written in the Dutch Language in 1969 and at that time deposited in the Repositorium of the Library of the Technical University, Delft, The Netherlands htpps://repository.tudelft.nl/view/tno; # 268336.Only in 2019 the thesis was translated by the author in English as precise copy nearly page by page of the 1969 version (no update). The English translation is only meant to disseminate the scientific data, described in the Dutch version. It does not intend to add any new data. In case of differences in the interpretation of the intellectual property only the Dutch version counts. Show less
