Enzymatic recycling of plastics

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Recombinant expression and purification of the 2,5-diketocamphane 1,2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007

Three different Baeyer-Villiger monooxygenases (BVMOs) were reported to be involved in the camphor metabolism by Pseudomonas putida NCIMB 10007. During (+)-camphor degradation, 2,5-diketocamphane is formed serving as substrate for the 2,5-diketocamphane 1,2-monooxygenase. This enzyme is encoded on the CAM plasmid and depends on the cofactors FMN and NADH and hence belongs to the group of type II BVMOs. We have cloned and recombinantly expressed the oxygenating subunit of the 2,5-diketocamphane 1,2-monooxygenase (2,5-DKCMO) in E. coli followed by His-tag-based affinity purification. A range of compounds representing different BVMO substrate classes were then investigated, but only bicyclic ketones were converted by 2,5-DKCMO used as crude cell extract or after purification. Interestingly, also (-)-camphor was oxidized, but conversion was about 3-fold lower compared to (+)-camphor. Moreover, activity of purified 2,5-DKCMO was observed in the absence of an NADH-dehydrogenase subunit

Enhancement of lipase selectivity by site directed mutagenesis

Lipases belong to the α/β-hydrolase fold family and naturally catalyze the hydrolysis of fats and oils into glycerol and fatty acids. This class of enzymes displays numerous features that make them useful biocatalysts, including (i) broad substrate spectrum, (ii) excellent chemo-, regio- and stereoselectivity, (iii) high stability towards harsh reaction conditions, (iv) independence of cofactors and, furthermore, (v) a wide variety of lipases is commercially available. Due to all these advantages lipases have been widely applied in industrial processes such as dairy, baking, and detergent industry. Furthermore, they can be used for the production of trans-fatty acid free margarines and biodiesel [1-3]. However, despite their great applicability, each industrial application needs particular reaction conditions (e.g. substrate selectivity or stability towards temperature, pH and/or organic solvents) that should be borne by the biocatalyst. Therefore, protein engineering can be applied in order to obtain enzymes that meet the required parameters [4]. The present study focuses on the enhancement of lipase selectivity by protein engineering and its application for the enrichment of long chain fatty acids from natural oils, which are interesting building blocks for the chemical industry. Hence, a broad spectrum of commercial lipases was screened to identify those that already displayed the desired selectivity. Furthermore, lipases with interesting structural features were selected from literature as candidates for rational design [5, 6]. The most promising candidates were overexpressed in Pichia pastoris and Escherichia coli and subsequently purified to test their hydrolytic activity towards different p-nitrophenyl fatty acid esters. The best candidate found was subjected to molecular modelling to examine the potential hotspots to perform saturation mutagenesis. Three different amino acids present in the binding pocket were identified, allowing the design and creation of three combinatorial mutant libraries. Once the libraries were transformed into E. coli, the hydrolytic activity of more than 4500 clones was screened by using the fully automatized robotic platform LARA [7]. The most selective variants were chosen and used for confirmation of their activity and selectivity towards both, different chain length p-nitrophenyl fatty acid esters and several oil fractions. Acknowledgements: The COSMOS project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 635405. [1] Bornscheuer, U. T., Eur. J. Lipid Sci. Tech., 2014, 116, 1322-1331. [2] Borrelli, G. M. et al., Int. J. Mol. Sci., 2015, 16, 20774-20840. [3] Liu, H. et al., Biotechnol. Adv., 2014, 32, 382-389. [4] Zorn, K. et al., Prog. Lipid. Res., 2016, 63, 153-164. [5] Barriuso, J. et al., Biotechnol. Adv., 2016, 34, 874-885. [6] Brundiek, H. et al., Eur J Lipid Sci Technol, 2012, 114, 1148-1153. [7] Dörr, M. et al., Biotechnol. Bioeng., 2016, 113, 1421-1432

Catalysis at the Heart of Success!

Bornscheuer, UT.; Hashmi, ASK.; García Gómez, H.; Rowan, MA. (2017). Catalysis at the Heart of Success!. ChemCatChem. 9(1):6-9. doi:10.1002/cctc.201601553S6991Bornscheuer, U. T. (2015). Biocatalysis: Successfully Crossing Boundaries. Angewandte Chemie International Edition, 55(14), 4372-4373. doi:10.1002/anie.201510042Bornscheuer, U. T. (2015). Biokatalyse: ein erfolgreicher Blick über den Tellerrand. Angewandte Chemie, 128(14), 4446-4447. doi:10.1002/ange.201510042Bornscheuer, U. T. (2009). Combined Success for Efficient Catalysis. ChemCatChem, 1(1), 5-5. doi:10.1002/cctc.200900144Weckhuysen, B. M. (2009). Crossing the Interfaces of Catalysis. ChemCatChem, 1(1), 7-7. doi:10.1002/cctc.200900146Kan, S. B. J., Lewis, R. D., Chen, K., & Arnold, F. H. (2016). Directed evolution of cytochrome c for carbon–silicon bond formation: Bringing silicon to life. Science, 354(6315), 1048-1051. doi:10.1126/science.aah621

Fatty Acids and their Derivatives as Renewable Platform Molecules for the Chemical Industry

Oils and fats of vegetable and animal origin remain an important renewable feedstock for the chemical industry. Their industrial use has increased during the last 10 years from 31 to 51 million tonnes annually. Remarkable achievements made in the field of oleochemistry in this timeframe are summarized herein, including the reduction of fatty esters to ethers, the selective oxidation and oxidative cleavage of C–C double bonds, the synthesis of alkyl-branched fatty compounds, the isomerizing hydroformylation and alkoxycarboxylation, and olefin metathesis. The use of oleochemicals for the synthesis of a great variety of polymeric materials has increased tremendously, too. In addition to lipases and phospholipases, other enzymes have found their way into biocatalytic oleochemistry. Important achievements have also generated new oil qualities in existing crop plants or by using microorganisms optimized by metabolic engineering

Re-hierarquização e Extrapolações para o Limite do Conjunto de Base Completo.

Um método sugerido previamente para calcular a energia de correlação no limite do conjunto de base completo pela redesignação dos números hierárquicos, e o uso do esquema de extrapolação unified singlet- and triplet-pair é aplicado a um conjunto de prova de 106 sistemas. A aproximação é utilizada para obter os valores extrapolados para energia de correlação, energia de atomização, anisotropia e polarizabilidade média no limite do conjunto de base completo, através de teoria de perturbação de segunda ordem de Møller-Plesset, método de coupled-cluster com excitações simples e duplas e coupled-cluster com excitações simples e duplas com correções triplas perturbativas. Uma boa concordância com as melhores estimativas disponíveis é obtida, mesmo quando o par de números hierárquicos (d, t) é usado para realizar a extrapolação. Com isso, é concebível justificar que não há razão física forte para excluir as energias dulpa-zeta em extrapolações, especialmente se a base é calibrada para obedecer ao modelo teórico. Além disso, um esquema simples de extrapolação unificado de um parâmetro é sugerido para extrapolar a energia de correlação de valência para o conjunto de base completo em espécies formadas por átomos de H até Ne. A performance do novo modelo é avaliada para a energia de correlação com um conjunto de de dados de 106 sistemas e, para polarizabilidade média, em um conjunto de 8 moléculas. Para as energias de correlação, os resultados são excelentes, na maioria das vezes melhores do que quando extrapolado com os mais populares protocolos de dois parâmetros disponíveis na literatura. Para a polarizabilidade, os resultados mostram uma melhora em relação aos valores ab initio, e uma boa concordância com os dados experimentais

Synthesis of Modified Poly(vinyl Alcohol)s and Their Degradation Using an Enzymatic Cascade

Poly(vinyl alcohol) (PVA) is a water-soluble synthetic vinyl polymer with remarkable physical properties including thermostability and viscosity. Its biodegradability, however, is low even though a large amount of PVA is released into the environment. Established physical-chemical degradation methods for PVA have several disadvantages such as high price, low efficiency, and secondary pollution. Biodegradation of PVA by microorganisms is slow and frequently involves pyrroloquinoline quinone (PQQ)-dependent enzymes, making it expensive due to the costly cofactor and hence unattractive for industrial applications. In this study, we present a modified PVA film with improved properties as well as a PQQ-independent novel enzymatic cascade for the degradation of modified and unmodified PVA. The cascade consists of four steps catalyzed by three enzymes with in situ cofactor recycling technology making this cascade suitable for industrial applications

Enzymatic degradation of polyethylene terephthalate nanoplastics analyzed in real time by isothermal titration calorimetry

Plastics are globally used for a variety of benefits. As a consequence of poor recycling or reuse, improperly disposed plastic waste accumulates in terrestrial and aquatic ecosystems to a considerable extent. Large plastic waste items become fragmented to small particles through mechanical and (photo)chemical processes. Particles with sizes ranging from millimeter (microplastics, <5 mm) to nanometer (nanoplastics, NP, <100 nm) are apparently persistent and have adverse effects on ecosystems and human health. Current research therefore focuses on whether and to what extent microorganisms or enzymes can degrade these NP. In this study, we addressed the question of what information isothermal titration calorimetry, which tracks the heat of reaction of the chain scission of a polyester, can provide about the kinetics and completeness of the degradation process. The majority of the heat represents the cleavage energy of the ester bonds in polymer backbones providing real-time kinetic information. Calorimetry operates even in complex matrices. Using the example of the cutinase-catalyzed degradation of polyethylene terephthalate (PET) nanoparticles, we found that calorimetry (isothermal titration calorimetry-ITC) in combination with thermokinetic models is excellently suited for an in-depth analysis of the degradation processes of NP. For instance, we can separately quantify i) the enthalpy of surface adsorption ∆AdsH = 129 ± 2 kJ mol−1, ii) the enthalpy of the cleavage of the ester bonds ∆EBH = −58 ± 1.9 kJ mol−1 and the apparent equilibrium constant of the enzyme substrate complex K = 0.046 ± 0.015 g L−1. It could be determined that the heat production of PET NP degradation depends to 95% on the reaction heat and only to 5% on the adsorption heat. The fact that the percentage of cleaved ester bonds (η = 12.9 ± 2.4%) is quantifiable with the new method is of particular practical importance. The new method promises a quantification of enzymatic and microbial adsorption to NP and their degradation in mimicked real-world aquatic conditions

Advanced database mining integrating sequence and structure bioinformatics with microfluidics challenges enzyme engineering

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