| 1. |
- Alexakis, Alexandros Efraim, et al.
(author)
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Modification of cellulose through physisorption of cationic bio-based nanolatexes - comparing emulsion polymerization and RAFT-mediated polymerization-induced self-assembly
- 2021
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In: Green Chemistry. - : Royal Society of Chemistry (RSC). - 1463-9262 .- 1463-9270. ; 23:5, s. 2113-2122
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Journal article (peer-reviewed)abstract
- The polymerization of a bio-based terpene-derived monomer, sobrerol methacrylate (SobMA), was evaluated in the design of polymeric nanoparticles (nanolatexes). Their synthesis was accomplished by using emulsion polymerization, either by free-radical polymerization in the presence of a cationic surfactant or a cationic macroRAFT agent by employing RAFT-mediated polymerization-induced self-assembly (PISA). By tuning the length of the hydrophobic polymer, it was possible to control the nanoparticle size between 70 and 110 nm. The average size of the latexes in both wet and dry state were investigated by microscopy imaging and dynamic light scattering (DLS). Additionally, SobMA was successfully copolymerized with butyl methacrylate (BMA) targeting soft-core nanolatexes. The comparison of the kinetic profile of the cationically stabilized nanolatexes highlighted the differences of both processes. The SobMA-based nanolatexes yielded high T-g similar to 120 degrees C, while the copolymer sample exhibited a lower T-g similar to 50 degrees C, as assessed by Differential Scanning Calorimetry (DSC). Thereafter, the nanolatexes were adsorbed onto cellulose (filter paper), where they were annealed at elevated temperatures to result in polymeric coatings. Their morphologies were analysed by Field Emission Scanning Electron Microscopy (FE-SEM) and compared to a commercial sulfate polystyrene latex (PS latex). By microscopic investigation the film formation mechanism could be unravelled. Water contact angle (CA) measurements verified the transition from a hydrophilic to a hydrophobic surface after film formation had occured. The obtained results are promising for the toolbox of bio-based building blocks, focused on sobrerol-based monomers, to be used in emulsion polymerizations either for tailored PISA-latexes or facile conventional latex formation, in order to replace methyl methacrylate or other high T-g-monomers.
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| 2. |
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| 3. |
- Biundo, Antonino, et al.
(author)
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Regio- and stereoselective biocatalytic hydration of fatty acids from waste cooking oils en route to hydroxy fatty acids and bio-based polyesters
- 2023
-
In: Enzyme and microbial technology. - : Elsevier BV. - 0141-0229 .- 1879-0909. ; 163
-
Journal article (peer-reviewed)abstract
- The development of biorefinery approaches is of great relevance for the sustainable production of valuable compounds. In accordance with circular economy principles, waste cooking oils (WCOs) are renewable resources and biorefinery feedstocks, which contribute to a reduced impact on the environment. Frequently, this waste is wrongly disposed of into municipal sewage systems, thereby creating problems for the environment and increasing treatment costs in wastewater treatment plants. In this study, regenerated WCOs, which were intended for the production of biofuels, were transformed through a chemo-enzymatic approach to produce hydroxy fatty acids, which were further used in polycondensation reaction for polyester production. Escherichia coli whole cell biocatalyst containing the recombinantly produced Elizabethkingia meningoseptica Oleate hydratase (Em_OhyA) was used for the biocatalytic hydration of crude WCOs-derived unsaturated free fatty acids for the production of hydroxy fatty acids. Further hydrogenation reaction and methylation of the crude mixture allowed the pro-duction of (R)-10-hydroxystearic acid methyl ester that was further purified with a high purity (> 90%), at gram scale. The purified (R)-10-hydroxystearic acid methyl ester was polymerized through a polycondensation reaction to produce the corresponding polyester. This work highlights the potential of waste products to obtain bio-based hydroxy fatty acids and polyesters through a biorefinery approach.
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| 4. |
- Biundo, Antonino, et al.
(author)
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Switched reaction specificity in polyesterases towards amide bond hydrolysis by enzyme engineering
- 2019
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In: RSC Advances. - : Royal Society of Chemistry. - 2046-2069. ; 9:62, s. 36217-36226
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Journal article (peer-reviewed)abstract
- The recalcitrance of plastics like nylon and other polyamides contributes to environmental problems (e.g. microplastics in oceans) and restricts possibilities for recycling. The fact that hitherto discovered amidases (EC 3.5.1. and 3.5.2.) only show no, or low, activity on polyamides currently obstructs biotechnological-assisted depolymerization of man-made materials. In this work, we capitalized on enzyme engineering to enhance the promiscuous amidase activity of polyesterases. Through enzyme design we created a reallocated water network adapted for hydrogen bond formation to synthetic amide backbones for enhanced transition state stabilization in the polyester-hydrolyzing biocatalysts Humicola insolens cutinase and Thermobifida cellulosilytica cutinase 1. This novel concept enabled increased catalytic efficiency towards amide-containing soluble substrates. The afforded enhanced hydrolysis of the amide bond-containing insoluble substrate 3PA 6,6 by designed variants was aligned with improved transition state stabilization identified by molecular dynamics (MD) simulations. Furthermore, the presence of a favorable water-molecule network that interacted with synthetic amides in the variants resulted in a reduced activity on polyethylene terephthalate (PET). Our data demonstrate the potential of using enzyme engineering to improve the amidase activity for polyesterases to act on synthetic amide-containing polymers.
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| 5. |
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| 6. |
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| 7. |
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| 8. |
- Eriksson, Adam, et al.
(author)
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Protonation-Initiated Cyclization by a ClassII Terpene Cyclase Assisted by Tunneling
- 2017
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In: ChemBioChem. - : Wiley-VCH Verlagsgesellschaft. - 1439-4227 .- 1439-7633. ; 18:23, s. 2301-2305
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Journal article (peer-reviewed)abstract
- Terpenes represent one of the most diversified classes of natural products with potent biological activities. The key to the myriad of polycyclic terpene skeletons with crucial functions in organisms from all kingdoms of life are terpene cyclase enzymes. These biocatalysts enable stereospecific cyclization of relatively simple, linear, prefolded polyisoprenes by highly complex, partially concerted, electrophilic cyclization cascades that remain incompletely understood. Herein, additional mechanistic light is shed on terpene biosynthesis by kinetic studies in mixed H2O/D2O buffers of a classII bacterial ent-copalyl diphosphate synthase. Mass spectrometry determination of the extent of deuterium incorporation in the bicyclic product, reminiscent of initial carbocation formation by protonation, resulted in a large kinetic isotope effect of up to seven. Kinetic analysis at different temperatures confirmed that the isotope effect was independent of temperature, which is consistent with hydrogen tunneling.
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| 9. |
- Fagerland, Jenny, 1985-, et al.
(author)
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Template-assisted enzymatic synthesis of oligopeptides from a polylactide chain
- 2017
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In: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 18:12, s. 4271-4280
-
Journal article (peer-reviewed)abstract
- Peptides are often attached to polymer materials, as bioactive components, for the control of interactions between the material and its surrounding proteins and cells. However, synthesizing peptides and attaching them to polymers can be challenging and laborious. Herein, we describe the grafting of oligopeptides to an aliphatic polyester, using a one-step chemo-enzymatic synthesis with papain as the biocatalySt. To enable enzyme-mediated functionalization of the polyester, ethyl hept-6-enoylalaninate (grafter) was synthesized and attached to polylactide chains using thiol-ene click reactions. The oligopeptides were grafted onto the polylactide chains using two different synthetic routes: the grafting from strategy, in which the grafter was attached to the polyester prior to oligopeptide synthesis, or the grafting to strategy, in which oligopeptides were synthesized on the grafter first, then attached to the polymer chain. The final products were analyzed and their structures were confirmed using nuclear magnetic resonance (NMR). The peptide attachment was evaluated using size exclusion chromatography (SEC), contact angle measurement and energy-dispersive X-ray spectroscopy scanning electron microscopy (EDS-SEM). Furthermore, the mechanistic aspects of the synthesis of the oligopeptides on the grafter were studied using molecular dynamics (MD) simulations. The simulation revealed that hydrogen bonding (between the P1 amide nitrogen of the grafter backbone and the carbonyl oxygen of D158 in the papain) maintain the grafter in a productive conformation to stabilize the transition state of nitrogen inversion, a key step of the biocatalytic mechanism. Apart from being biologically relevant, both experimental and computational results suggest that the designed grafter is a good template for initiating chemo-enzymatic synthesis. The results also showed that the grafting to strategy was more successful compared to the grafting from strategy. Overall, a successful synthesis of predefined peptide functionalized polylactide was prepared, where the oligopeptides were grafted in an easy, time efficient, and environmentally friendly way.
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| 10. |
- Farhat, Wissam, et al.
(author)
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Biocatalysis for terpene-based polymers
- 2019
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In: Zeitschrift für Naturforschung C - A Journal of Biosciences. - : WALTER DE GRUYTER GMBH. - 0939-5075 .- 1865-7125. ; 74:3-4, s. 90-99
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Journal article (peer-reviewed)abstract
- Accelerated generation of bio-based materials is vital to replace current synthetic polymers obtained from petroleum with more sustainable options. However, many building blocks available from renewable resources mainly contain unreactive carbon-carbon bonds, which obstructs their efficient polymerization. Herein, we highlight the potential of applying biocatalysis to afford tailored functionalization of the inert carbocyclic core of multicyclic terpenes toward advanced materials. As a showcase, we unlock the inherent monomer reactivity of norcamphor, a bicyclic ketone used as a monoterpene model system in this study, to afford polyesters with unprecedented backbones. The efficiencies of the chemical and enzymatic Baeyer-Villiger transformation in generating key lactone intermediates are compared. The concepts discussed herein are widely applicable for the valorization of terpenes and other cyclic building blocks using chemoenzymatic strategies.
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| 11. |
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| 12. |
- Farhat, Wissam, et al.
(author)
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Lactone monomers obtained by enzyme catalysis and their use in reversible thermoresponsive networks
- 2020
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In: Journal of Applied Polymer Science. - : John Wiley and Sons Inc.. - 0021-8995 .- 1097-4628. ; 137:18
-
Journal article (peer-reviewed)abstract
- Enzyme-catalyzed transformations have a great potential in both the pharmaceutical and chemical industry to achieve complex and (stereo)selective synthesis under mild reaction conditions. Still, the implementation of biocatalysis in the prerequisite upgrading of inert synthons into activated monomers for polymer applications has not yet been fully realized. In this contribution, we show that scaled-up synthesis of bicyclic norcamphor lactone using an engineered Baeyer–Villiger monooxygenase (BVMO) is feasible to reach complete conversion of the corresponding ketone in 24 h in shake-flask. The lactone monomer obtained by enzyme catalysis was copolymerized with ε-caprolactone via ring-opening polymerization to study the impact of the additional ring on material properties. Moreover, four-arm star-like, homo and block copolymers were designed from ε-caprolactone, ε-decalactone, and norcamphor lactone and characterized for their structural and thermal properties. These newly explored macromolecules were functionalized with furan rings using the enzyme Candida antarctica lipase B which allowed the formation of thermolabile networks via the pericyclic reaction with bismaleimide by means of Diels–Alder chemistry. The bonding/debonding state of these star-like based materials can be tuned by a suitable selection of thermal treatment. The temperature-dependent reversibility was assessed by thermal analysis and solubility test. Our results presented here shed light on the high potential of the use of chemoenzymatic approaches in the synthesis of new functional materials with tuned physiochemical properties.
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| 13. |
- Fink, Michael J., et al.
(author)
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Redesign of water networks for efficient biocatalysis
- 2017
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In: Current opinion in chemical biology. - : ELSEVIER SCI LTD. - 1367-5931 .- 1879-0402. ; 37, s. 107-114
-
Research review (peer-reviewed)abstract
- Herein we highlight recent findings on the importance of water networks in proteins, and their redesign and reconfiguration as a new engineering strategy to generate enzymes with modulated binding affinity and improved catalytic versatility. Traditionally, enzyme engineering and drug design have focused on tailoring direct and favorable interactions between protein surfaces and ligands/transition states to achieve stronger binding, or an accelerated manufacturing of medicines, biofuels, fine chemicals and materials. In contrast, the opportunity to relocate water molecules in solvated binding pockets by protein design to improve overall energetics remains essentially unexplored, and fundamental understanding of the elusive processes involved is poor. Rewiring water networks in protein interiors impacts binding affinity, catalysis and the thermodynamic signature of biochemical processes through dynamic mechanisms, and thus has great potential to enhance binding specificity, accelerate catalysis and provide new reaction mechanisms and chemistry, that were not yet explored in nature.
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| 14. |
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| 15. |
- Guo, Boyang, et al.
(author)
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Conformational Selection in Biocatalytic Plastic Degradation by PETase
- 2022
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In: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 12:6, s. 3397-3409
-
Journal article (peer-reviewed)abstract
- Due to the steric effects imposed by bulky polymers, the formation of catalytically competent enzyme and substrate conformations is critical in the biodegradation of plastics. In poly(ethylene terephthalate) (PET), the backbone adopts different conformations, gauche and trans, coexisting to different extents in amorphous and crystalline regions. However, which conformation is susceptible to biodegradation and the extent of enzyme and substrate conformational changes required for expedient catalysis remain poorly understood. To overcome this obstacle, we utilized molecular dynamics simulations, docking, and enzyme engineering in concert with high-resolution microscopy imaging and solid-state nuclear magnetic resonance (NMR) to demonstrate the importance of conformational selection in biocatalytic plastic hydrolysis. Our results demonstrate how single-amino acid substitutions in Ideonella sakaiensis PETase can alter its conformational landscape, significantly affecting the relative abundance of productive ground-state structures ready to bind discrete substrate conformers. We experimentally show how an enzyme binds to plastic and provide a model for key residues involved in the recognition of gauche and trans conformations supported by in silico simulations. We demonstrate how enzyme engineering can be used to create a trans-selective variant, resulting in higher activity when combined with an all-trans PET-derived oligomeric substrate, stemming from both increased accessibility and conformational preference. Our work cements the importance of matching enzyme and substrate conformations in plastic hydrolysis, and we show that also the noncanonical trans conformation in PET is conducive for degradation. Understanding the contribution of enzyme and substrate conformations to biocatalytic plastic degradation could facilitate the generation of designer enzymes with increased performance.
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| 16. |
- Guo, Boyang, et al.
(author)
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Fast Depolymerization of PET Bottle Mediated by Microwave Pre-Treatment and An Engineered PETase
- 2023
-
In: ChemSusChem. - : Wiley. - 1864-5631 .- 1864-564X. ; 16:18
-
Journal article (peer-reviewed)abstract
- Recycling plastics is the key to reaching a sustainable materials economy. Biocatalytic degradation of plastics shows great promise by allowing selective depolymerization of man-made materials into constituent building blocks under mild aqueous conditions. However, insoluble plastics have polymer chains that can reside in different conformations and show compact secondary structures that offer low accessibility for initiating the depolymerization reaction by enzymes. In this work, we overcome these shortcomings by microwave irradiation as a pre-treatment process to deliver powders of polyethylene terephthalate (PET) particles suitable for subsequent biotechnology-assisted plastic degradation by previously generated engineered enzymes. An optimized microwave step resulted in 1400 times higher integral of released terephthalic acid (TPA) from high-performance liquid chromatography (HPLC), compared to original untreated PET bottle. Biocatalytic plastic hydrolysis of substrates originating from PET bottles responded to 78 % yield conversion from 2 h microwave pretreatment and 1 h enzymatic reaction at 30 °C. The increase in activity stems from enhanced substrate accessibility from the microwave step, followed by the administration of designer enzymes capable of accommodating oligomers and shorter chains released in a productive conformation.
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| 17. |
- Gustafsson, Camilla, et al.
(author)
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MD Simulations Reveal Complex Water Paths in Squalene–Hopene Cyclase: Tunnel-Obstructing Mutations Increase the Flow of Water in the Active Site
- 2017
-
In: ACS Omega. - : American Chemical Society (ACS). - 2470-1343. ; 2:11, s. 8495-8506
-
Journal article (peer-reviewed)abstract
- Squalene–hopene cyclase catalyzes the cyclization of squalene to hopanoids. A previous study has identified a network of tunnels in the protein, where water molecules have been indicated to move. Blocking these tunnels by site-directed mutagenesis was found to change the activation entropy of the catalytic reaction from positive to negative with a concomitant lowering of the activation enthalpy. As a consequence, some variants are faster and others are slower than the wild type (wt) in vitro under optimal reaction conditions for the wt. In this study, molecular dynamics (MD) simulations have been performed for the wt and the variants to investigate how the mutations affect the protein structure and the water flow in the enzyme, hypothetically influencing the activation parameters. Interestingly, the tunnel-obstructing variants are associated with an increased flow of water in the active site, particularly close to the catalytic residue Asp376. MD simulations with the substrate present in the active site indicate that the distance for the rate-determining proton transfer between Asp376 and the substrate is longer in the tunnel-obstructing protein variants than in the wt. On the basis of the previous experimental results and the current MD results, we propose that the tunnel-obstructing variants, at least partly, could operate by a different catalytic mechanism, where the proton transfer may have contributions from a Grotthuss-like mechanism.
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| 18. |
- Hammer, Stephan C., et al.
(author)
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Squalene hopene cyclases : highly promiscuous and evolvable catalysts for stereoselective CC and CX bond formation
- 2013
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In: Current opinion in chemical biology. - : Elsevier. - 1367-5931 .- 1879-0402. ; 17:2, s. 293-300
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Journal article (peer-reviewed)abstract
- A review. We review here how the inherent promiscuous nature, as well as the evolvability of terpene cyclase enzymes enables new applications in chem. We mainly focus on squalene hopene cyclases, class II triterpene synthases that use a proton-initiated cationic polycyclization cascade to form carbopolycyclic products. We highlight recent findings to demonstrate that these enzymes are capable of activating different functionalities other than the traditional terminal isoprene CC-group as well as being compatible with a wide range of nucleophiles beyond the 'ene-functionality'. Thus, squalene hopene cyclases demonstrate a great potential to be used as a toolbox for general Bronsted acid catalysis.
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| 19. |
- Hammer, Stephan C., et al.
(author)
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Stereoselective Friedel-Crafts alkylation catalyzed by squalene hopene cyclases
- 2012
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In: Tetrahedron. - : Elsevier Ltd.. - 0040-4020 .- 1464-5416. ; 68:Copyright (C) 2013 American Chemical Society (ACS). All Rights Reserved., s. 7624-7629
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Journal article (peer-reviewed)abstract
- In org. synthesis the Friedel-Crafts alkylation is of eminent importance, as it is a key reaction in many synthetic routes. A general access to enzymic Friedel-Crafts alkylations would be very beneficial due to the high selectivity of biocatalysts. We used designed polyprenyl Ph ethers to specifically address this reaction by using squalene hopene cyclases as catalysts. Polycyclic products with arom. rings constituting important biol. active compds. were obtained. Our results demonstrate that squalene hopene cyclases can be utilized for Friedel-Crafts alkylations and reveal the potential of these enzymes for chiral Bronsted acid catalysis.
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| 20. |
- Hammer, Stephan, et al.
(author)
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Substrate Pre-Folding and Water Molecule Organization Matters for Terpene Cyclase Catalyzed Conversion of Unnatural Substrates
- 2016
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In: ChemistrySelect. - : Wiley. - 2365-6549. ; 1, s. 3589-3593
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Journal article (peer-reviewed)abstract
- Terpene cyclase enzymes have recently been challenged with terpene substrate derivatives to generate additional chemical complexity beyond to what is currently found in nature. Herein, molecular dynamics and biocatalysis are used to shed light on the flexibility and inherent limitation of a triterpene cyclase in converting unnatural substrates. Our studies suggest that populating binding modes which allows for concerted reaction pathways is a key element towards an expanded substrate scope and new chemistries displayed by terpene cyclases. Additionally, we show that the spatial organization of water, which is influenced by both the substrate architecture as well as the active site geometry, controls the product selectivity. This highlights that activity and selectivity displayed by terpene cyclases acting on unnatural substrates is particularly difficult to predict, since they depend on various parameters.
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| 21. |
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| 22. |
- Hendil-Forssell, Peter, et al.
(author)
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Exploring water as building bricks in enzyme engineering
- 2015
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In: Chemical Communications. - : Royal Society of Chemistry. - 1359-7345 .- 1364-548X. ; 51:97, s. 17221-17224
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Journal article (peer-reviewed)abstract
- A novel enzyme engineering strategy for accelerated catalysis based on redesigning a water network through protein backbone deshielding is presented. Fundamental insight into the energetic consequences associated with the design is discussed in the light of experimental results and computer simulations. Using water as biobricks provides unique opportunities when transition state stabilisation is not easily attained by traditional enzyme engineering.
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| 23. |
- Hendrikse, Natalie, et al.
(author)
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Ancestral diterpene cyclases show increased thermostability and substrate acceptance
- 2018
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In: The FEBS Journal. - : Wiley-VCH Verlagsgesellschaft. - 1742-464X .- 1742-4658. ; 285:24, s. 4660-4673
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Journal article (peer-reviewed)abstract
- Bacterial diterpene cyclases are receiving increasing attention in biocatalysis and synthetic biology for the sustainable generation of complex multicyclic building blocks. Herein, we explore the potential of ancestral sequence reconstruction (ASR) to generate remodeled cyclases with enhanced stability, activity, and promiscuity. Putative ancestors of spiroviolene synthase, a bacterial class I diterpene cyclase, display an increased yield of soluble protein of up to fourfold upon expression in the model organism Escherichia coli. Two of the resurrected enzymes, with an estimated age of approximately 1.7 million years, display an upward shift in thermostability of 7-13 degrees C. Ancestral spiroviolene synthases catalyze cyclization of the natural C-20-substrate geranylgeranyl diphosphate (GGPP) and also accept C-15 farnesyl diphosphate (FPP), which is not converted by the extant enzyme. In contrast, the consensus sequence generated from the corresponding multiple sequence alignment was found to be inactive toward both substrates. Mutation of a nonconserved position within the aspartate-rich motif of the reconstructed ancestral cyclases was associated with modest effects on activity and relative substrate specificity (i.e., k(cat)/K-M for GGPP over k(cat)/K-M for FPP). Kinetic analyses performed at different temperatures reveal a loss of substrate saturation, when going from the ancestor with highest thermostability to the modern enzyme. The kinetics data also illustrate how an increase in temperature optimum of biocatalysis is reflected in altered entropy and enthalpy of activation. Our findings further highlight the potential and limitations of applying ASR to biosynthetic machineries in secondary metabolism.
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| 24. |
- Hendrikse, Natalie, et al.
(author)
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Ancestral lysosomal enzymes with increased activity harbor therapeutic potential for treatment of Hunter syndrome
- 2021
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In: ISCIENCE. - : Elsevier BV. - 2589-0042. ; 24:3
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Journal article (peer-reviewed)abstract
- We show the successful application of ancestral sequence reconstruction to enhance the activity of iduronate-2-sulfatase (IDS), thereby increasing its therapeutic potential for the treatment of Hunter syndrome-a lysosomal storage disease caused by impaired function of IDS. Current treatment, enzyme replacement therapy with recombinant human IDS, does not alleviate all symptoms, and an unmet medical need remains. We reconstructed putative ancestral sequences of mammalian IDS and compared them with extant IDS. Some ancestral variants displayed up to 2-fold higher activity than human IDS in in vitro assays and cleared more substrate in ex vivo experiments in patient fibroblasts. This could potentially allow for lower dosage or enhanced therapeutic effect in enzyme replacement therapy, thereby improving treatment outcomes and cost efficiency, as well as reducing treatment burden. In summary, we showed that ancestral sequence reconstruction can be applied to lysosomal enzymes that function in concert with modern enzymes and receptors in cells.
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| 25. |
- Hendrikse, Natalie
(author)
-
Engineering enzymes towards biotherapeutic applications using ancestral sequence reconstruction
- 2020
-
Doctoral thesis (other academic/artistic)abstract
- Enzymes are versatile biocatalysts that fulfill essential functions in all forms of life and, therefore, play an important role in health and disease. One specific application of enzymes in life science is their use as biopharmaceuticals, which typically benefits from high catalytic activity and stability. Increased stability and activity are both desirable properties for biopharmaceuticals as they are directly related to dosage, which in turn affects administration time, cost of production and potency of a drug. The aim of the work presented in this thesis is to enhance the therapeutic potential of enzymes by means of enzyme engineering, in particular using ancestral sequence reconstruction. In Paper I, we established the utility of this method in a model system and obtained ancestral terpene cyclases with increased activity, stability and substrate scope. In Paper II, we described the successful crystallization of the most stable ancestral terpene cyclase, which allowed for rational design of substrate specificity. Finally, we applied the method to two therapeutically relevant enzyme families associated with rare metabolic disorders. We obtained ancestral phenylalanine/tyrosine ammonia-lyases with substantially enhanced thermostability and long-term stability in Paper III and ancestral iduronate-2-sulfatases with increased activity in Paper IV. In summary, the results presented herein highlight the potential of ancestral sequence reconstruction as a method to obtain stable enzyme scaffolds for further engineering and to enhance therapeutic properties of enzymes.
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| 26. |
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| 27. |
- Hendrikse, Natalie M., et al.
(author)
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Exploring the therapeutic potential of modern and ancestral phenylalanine/tyrosine ammonia-lyases as supplementary treatment of hereditary tyrosinemia
- 2020
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In: Scientific Reports. - : Nature Research. - 2045-2322. ; 10:1
-
Journal article (peer-reviewed)abstract
- Phenylalanine/tyrosine ammonia-lyases (PAL/TALs) have been approved by the FDA for treatment of phenylketonuria and may harbour potential for complementary treatment of hereditary tyrosinemia Type I. Herein, we explore ancestral sequence reconstruction as an enzyme engineering tool to enhance the therapeutic potential of PAL/TALs. We reconstructed putative ancestors from fungi and compared their catalytic activity and stability to two modern fungal PAL/TALs. Surprisingly, most putative ancestors could be expressed as functional tetramers in Escherichia coli and thus retained their ability to oligomerize. All ancestral enzymes displayed increased thermostability compared to both modern enzymes, however, the increase in thermostability was accompanied by a loss in catalytic turnover. One reconstructed ancestral enzyme in particular could be interesting for further drug development, as its ratio of specific activities is more favourable towards tyrosine and it is more thermostable than both modern enzymes. Moreover, long-term stability assessment showed that this variant retained substantially more activity after prolonged incubation at 25 °C and 37 °C, as well as an increased resistance to incubation at 60 °C. Both of these factors are indicative of an extended shelf-life of biopharmaceuticals. We believe that ancestral sequence reconstruction has potential for enhancing the properties of enzyme therapeutics, especially with respect to stability. This work further illustrates that resurrection of putative ancestral oligomeric proteins is feasible and provides insight into the extent of conservation of a functional oligomerization surface area from ancestor to modern enzyme.
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| 28. |
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| 29. |
- Hueting, David A., et al.
(author)
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Design, structure and plasma binding of ancestral β-CoV scaffold antigens
- 2023
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In: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
-
Journal article (peer-reviewed)abstract
- We report the application of ancestral sequence reconstruction on coronavirus spike protein, resulting in stable and highly soluble ancestral scaffold antigens (AnSAs). The AnSAs interact with plasma of patients recovered from COVID-19 but do not bind to the human angiotensin-converting enzyme 2 (ACE2) receptor. Cryo-EM analysis of the AnSAs yield high resolution structures (2.6–2.8 Å) indicating a closed pre-fusion conformation in which all three receptor-binding domains (RBDs) are facing downwards. The structures reveal an intricate hydrogen-bonding network mediated by well-resolved loops, both within and across monomers, tethering the N-terminal domain and RBD together. We show that AnSA-5 can induce and boost a broad-spectrum immune response against the wild-type RBD as well as circulating variants of concern in an immune organoid model derived from tonsils. Finally, we highlight how AnSAs are potent scaffolds by replacing the ancestral RBD with the wild-type sequence, which restores ACE2 binding and increases the interaction with convalescent plasma.
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| 30. |
- Hueting, David A., 1993-
(author)
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In silico protein design for the enhancement of protein stability and function
- 2023
-
Doctoral thesis (other academic/artistic)abstract
- Enzymes are natures catalysts that increase the rate of a chemical reaction. The increased rate of a reaction is required to be able to sustain life. Despite the huge impact of enzymes, they are not perfect catalysts. Enzyme and protein engineering is the discipline in which proteins are characterized and engineered to have improved inherent properties. Interesting properties of an enzyme to improve include stability and activity. The aim of this work is to understand how proteins and enzymes function and use a variety of different protein engineering techniques to enhance the properties of different proteins. In this work proteins and enzymes are engineered to increase our knowledge of the target proteins for downstream biomedical applications. A mix between rational and semi-rational engineering is applied in this work. In paper I and paper II, the method used is ancestral sequence reconstruction. A method that utilizes the evolutionary relationship between homologous sequences. In paper I the method was applied to a terpene cyclase, which cyclizes a precursor terpene into potential interesting drug leads. The result was a hyperstable enzyme variant. In paper II the technique was applied to the SARS-CoV-2 Spike protein. The protein is responsible for the virus SARS-CoV-2 to enter human cells. The work yielded a stable spike protein that readily expresses and can be utilized as a vaccine lead. In paper III, the aim was to understand human oxidosqualene cyclase (hOSC). A terpene cyclase essential in cholesterol synthesis. The enzyme hOSC was rationally engineered to change the driving force of the reaction. Through targeted mutations the reaction changed from entropy driven to enthalpy driven. Finally, in paper IV, a rationally engineered PETase, which is capable of degrading PET polymers into monomers, was proven to be active in human serum and verifies the proof-of-concept of degrading plastic in human blood. To summarize, the results in this thesis show the applicability of different enzyme engineering techniques to stabilize or change the function of proteins and the potential of engineered proteins in medical applications.
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| 31. |
- Hueting, David A., et al.
(author)
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Thermoadaptation in an Ancestral Diterpene Cyclase by Altered Loop Stability
- 2022
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In: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 126:21, s. 3809-3821
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Journal article (peer-reviewed)abstract
- Thermostability is the key to maintain the structural integrity and catalytic activity of enzymes in industrial biotechnological processes, such as terpene cyclase-mediated generation of medicines, chiral synthons, and fine chemicals. However, affording a large increase in the thermostability of enzymes through site directed protein engineering techniques can constitute a challenge. In this paper, we used ancestral sequence reconstruction to create a hyperstable variant of the ent-copalyl diphosphate synthase PtmT2, a terpene cyclase involved in the assembly of antibiotics. Molecular dynamics simulations on the its timescale were performed to shed light on possible molecular mechanisms contributing to activity at an elevated temperature and the large 40 degrees C increase in melting temperature observed for an ancestral variant of PtmT2. In silico analysis revealed key differences in the flexibility of a loop capping the active site, between extant and ancestral proteins. For the modern enzyme, the loop collapses into the active site at elevated temperatures, thus preventing biocatalysis, whereas the loop remains in a productive conformation both at ambient and high temperatures in the ancestral variant. Restoring a Pro loop residue introduced in the ancestral variant to the corresponding Gly observed in the extant protein led to reduced catalytic activity at high temperatures, with only moderate effects on the melting temperature, supporting the importance of the flexibility of the capping loop in thermoadaptation. Conversely, the inverse Gly to Pro loop mutation in the modern enzyme resulted in a 3-fold increase in the catalytic rate. Despite an overall decrease in maximal activity of ancestor compared to wild type, its increased thermostability provides a robust backbone amenable for further enzyme engineering. Our work cements the importance of loops in enzyme catalysis and provides a molecular mechanism contributing to thermoadaptation in an ancestral enzyme.
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| 32. |
- Hunold, A., et al.
(author)
-
Assembly of a Rieske non-heme iron oxygenase multicomponent system from Phenylobacterium immobile E DSM 1986 enables pyrazon cis-dihydroxylation in E. coli
- 2021
-
In: Applied Microbiology and Biotechnology. - : Springer Science and Business Media Deutschland GmbH. - 0175-7598 .- 1432-0614. ; 105:5, s. 2003-2015
-
Journal article (peer-reviewed)abstract
- Abstract: Phenylobacterium immobile strain E is a soil bacterium with a striking metabolism relying on xenobiotics, such as the herbicide pyrazon, as sole carbon source instead of more bioavailable molecules. Pyrazon is a heterocyclic aromatic compound of environmental concern and its biodegradation pathway has only been reported in P. immobile. The multicomponent pyrazon oxygenase (PPO), a Rieske non-heme iron oxygenase, incorporates molecular oxygen at the 2,3 position of the pyrazon phenyl moiety as first step of degradation, generating a cis-dihydrodiendiol. The aim of this work was to identify the genes encoding for each one of the PPO components and enable their functional assembly in Escherichia coli. P. immobile strain E genome sequencing revealed genes encoding for RO components, such as ferredoxin-, reductase-, α- and β-subunits of an oxygenase. Though, P. immobile E displays three prominent differences with respect to the ROs currently characterized: (1) an operon-like organization for PPO is absent, (2) all the elements are randomly scattered in its DNA, (3) not only one, but 19 different α-subunits are encoded in its genome. Herein, we report the identification of the PPO components involved in pyrazon cis-dihydroxylation in P. immobile, its appropriate assembly, and its functional reconstitution in E. coli. Our results contributes with the essential missing pieces to complete the overall elucidation of the PPO from P. immobile. Key points: • Phenylobacterium immobile E DSM 1986 harbors the only described pyrazon oxygenase (PPO). • We elucidated the genes encoding for all PPO components. • Heterologous expression of PPO enabled pyrazon dihydroxylation in E. coli JW5510.
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| 33. |
- Jönsson, Christina, et al.
(author)
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Biocatalysis in the Recycling Landscape for Synthetic Polymers and Plastics towards Circular Textiles
- 2021
-
In: ChemSusChem. - : Wiley. - 1864-5631 .- 1864-564X. ; 14:19, s. 4028-4040
-
Journal article (peer-reviewed)abstract
- Although recovery of fibers from used textiles with retained material quality is desired, separation of individual components from polymer blends used in today's complex textile materials is currently not available at viable scale. Biotechnology could provide a solution to this pressing problem by enabling selective depolymerization of recyclable fibers of natural and synthetic origin, to isolate constituents or even recover monomers. We compiled experimental data for biocatalytic polymer degradation with a focus on synthetic polymers with hydrolysable links and calculated conversion rates to explore this path The analysis emphasizes that we urgently need major research efforts: beyond cellulose-based fibers, biotechnological-assisted depolymerization of plastics so far only works for polyethylene terephthalate, with degradation of a few other relevant synthetic polymer chains being reported. In contrast, by analyzing market data and emerging trends for synthetic fibers in the textile industry, in combination with numbers from used garment collection and sorting plants, it was shown that the use of difficult-to-recycle blended materials is rapidly growing. If the lack of recycling technology and production trend for fiber blends remains, a volume of more than 3400 Mt of waste will have been accumulated by 2030. This work highlights the urgent need to transform the textile industry from a biocatalytic perspective.
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| 34. |
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| 35. |
- Kürten, Charlotte, et al.
(author)
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Mechanism-Guided Discovery of an Esterase Scaffold with Promiscuous Amidase Activity
- 2016
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In: Catalysts. - : MDPI AG. - 2073-4344. ; 6:6
-
Journal article (peer-reviewed)abstract
- The discovery and generation of biocatalysts with extended catalytic versatilities are of immense relevance in both chemistry and biotechnology. An enhanced atomistic understanding of enzyme promiscuity, a mechanism through which living systems acquire novel catalytic functions and specificities by evolution, would thus be of central interest. Using esterase-catalyzed amide bond hydrolysis as a model system, we pursued a simplistic in silico discovery program aiming for the identification of enzymes with an internal backbone hydrogen bond acceptor that could act as a reaction specificity shifter in hydrolytic enzymes. Focusing on stabilization of the rate limiting transition state of nitrogen inversion, our mechanism-guided approach predicted that the acyl hydrolase patatin of the alpha/beta phospholipase fold would display reaction promiscuity. Experimental analysis confirmed previously unknown high amidase over esterase activity displayed by the first described esterase machinery with a protein backbone hydrogen bond acceptor to the reacting NH-group of amides. The present work highlights the importance of a fundamental understanding of enzymatic reactions and its potential for predicting enzyme scaffolds displaying alternative chemistries amenable to further evolution by enzyme engineering.
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| 36. |
- Kürten, Charlotte, 1989-
(author)
-
On Catalytic Mechanisms for Rational Enzyme Design Strategies
- 2018
-
Doctoral thesis (other academic/artistic)abstract
- Enzymes enable life by promoting chemical reactions that govern the metabolism of all living organisms. As green catalysts, they have been extensively used in industry. However, to reach their full potential, engineering is often required, which can benefit from a detailed understanding of the underlying reaction mechanism.In Paper I, we screened for an esterase with promiscuous amidase activity capitalizing on a key hydrogen bond acceptor that is able to stabilize the rate limiting nitrogen inversion. In silicoanalyses revealed the esterase patatin as promising target that indeed catalyzed amide hydrolysis when tested in vitro. While key transition state stabilizers for amide hydrolysis are known, we were interested in increasing our fundamental understanding of terpene cyclase catalysis (Paper II-V). In Paper II, kinetic studies in D2O-enriched buffers using a soluble diterpene cyclase suggested that hydrogen tunneling is part of the rate-limiting protonation step. In Paper III, we performed intense computational analyses on a bacterial triterpene cyclase to show the influence of water flow on catalysis. Water movement in the active site and in specific water channels, influencing transition state formation, was detected using streamline analysis. In Paper IV and V, we focused on the human membrane-bound triterpene cyclase oxidosqualene cyclase. We first established a bacterial expression and purification protocol in Paper IV, before performing detailed in vitroand in silicoanalyses in Paper V. Our analyses showed an entropy-driven reaction mechanism and the existence of a tunnel network in the structure of the human enzyme. The influence of water network rearrangements on the thermodynamics of the transition state formation were confirmed. Introducing mutations in the tunnel lining residues severely affected the temperature dependence of the reaction by changing the water flow and network rearrangements in the tunnels and concomitant the active site.
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| 37. |
- Kürten, Charlotte, et al.
(author)
-
Overexpression of functional human oxidosqualene cyclase in Escherichia coli
- 2015
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In: Protein Expression and Purification. - : Elsevier. - 1046-5928 .- 1096-0279. ; 115, s. 46-53
-
Journal article (peer-reviewed)abstract
- The generation of multicyclic scaffolds from linear oxidosqualene by enzymatic polycyclization catalysis constitutes a cornerstone in biology for the generation of bioactive compounds. Human oxidosqualene cyclase (hOSC) is a membrane-bound triterpene cyclase that catalyzes the formation of the tetracyclic steroidal backbone, a key step in cholesterol biosynthesis. Protein expression of hOSC and other eukaryotic oxidosqualene cyclases has traditionally been performed in yeast and insect cells, which has resulted in protein yields of 2.7 mg protein/g cells (hOSC in Pichia pastoris) after 48 h of expression. Herein we present, to the best of our knowledge, the first functional expression of hOSC in the model organism Escherichia coli. Using a codon-optimized gene and a membrane extraction procedure for which detergent is immediately added after cell lysis, a protein yield of 2.9 mg/g bacterial cells was achieved after four hours of expression. It is envisaged that the isolation of high amounts of active eukaryotic oxidosqualene cyclase in an easy to handle bacterial system will be beneficial in pharmacological, biochemical and biotechnological applications.
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| 38. |
- Kürten, Charlotte, et al.
(author)
-
Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes
- 2016
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In: Journal of Visualized Experiments. - : Journal of Visualized Experiments. - 1940-087X. ; :107
-
Journal article (peer-reviewed)abstract
- Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.
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| 39. |
- Lopez-Lorenzo, Ximena
(author)
-
Chemoenzymatic Synthesis and Degradation of Plastics
- 2024
-
Doctoral thesis (other academic/artistic)abstract
- The development of a carbon-based bioeconomy for synthesis and degradation of polymers has gained importance over the years. Research efforts have been made to develop green routes to produce bio-based material from biomass as well as environmentally friendly ways to synthesize and degrade polymers. Enzymes are biocatalysts that are capable of performing reactions beyond their intended purpose. The work presented in this thesis focused on using biocatalysts for novel reactions to produce bio-plastics as well as degrade synthetic polymers. In Paper I, a decarboxylase was used to perform the fixation of CO2 under mild conditions to produce the platform chemical 2,5-furandicarboxylic acid (FDCA). In Paper II, a closed-loop approach for the production of bio-based polyesters and their enzymatic degradation was investigated. Moreover, the difference of catalytic activity towards different polymer conformations was noted and further investigated in Paper III. Here, the conformational landscape to match enzyme to substrate was explored. The model substrate for this project was post-consumer PET bottles since is one of the most used polymer worldwide. The substrate conformation affected the catalytic activity of the enzymes significantly hence, in Paper IV, the physical and chemical characteristics of various PET-based substrates was investigated to better understand the factors that will yield a high reaction efficiency for polymer depolymerization. Finally, the results obtained so far were used in Paper V to show that plastic degrading enzymes can be used for microplastic degradation in human blood as a proof-of-concept. In summary, the work in this thesis showed the potential of using enzymes as catalysts for the production of platform chemicals through CO2 fixation and for polymer degradation initiating an attractive path to close the loop in a bio-economy for polymer materials.
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| 40. |
- Lopez-Lorenzo, Ximena, et al.
(author)
-
Conformational Selection in Enzyme-Catalyzed Depolymerization of Bio-based Polyesters
-
Other publication (other academic/artistic)abstract
- Enzymatic degradation of synthetic polymers holds promise for advancing towards a bio-based economy. However, the bulky nature of polymers presents challenges in accessibility for biocatalysts, hindering depolymerization reactions. Beyond the impact of crystallinity, polymer chains can reside in different conformations affecting binding efficiency to the enzyme active site. We previously showed that the gauche and trans chain conformers associated with crystalline and amorphous regions of the synthetic polyester polyethylene terephthalate (PET) display different affinity to PETase, thus affecting the depolymerization rate. However, structural-function relationships for biopolymers remains poorly understood in biocatalysis. In this study, we synthetized four bio-based polyesters with different properties by co-polymerizing the same rigid bicyclic terpene-derived diol with various dimethylesters spanning from semi-aromatic to aliphatic chains of varying length, and subjected them to enzymatic degradation reactions in concert with induced-fit docking (IFD) analyses- x. Our findings demonstrate the importance of conformational selection for biopolymers in which the simple 1-dimensional geometric parameter with respect to how the polymeric chain is folded into either a straight or twisted conformation has a crucial role in biocatalytic degradation by showing different affinities to enzyme ground state conformers. This work highlights how matching substrate and enzyme conformations needs to be considered to optimize degradation efficiency of biopolymers, providing valuable insights for the development of sustainable bioprocesses in polymer degradation.
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| 41. |
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| 42. |
- Lopez-Lorenzo, Ximena, et al.
(author)
-
Whole-cell Mediated Carboxylation of 2-Furoic Acid Towards the Production of Renewable Platform Chemicals and Biomaterials
- 2023
-
In: ChemCatChem. - : Wiley. - 1867-3880 .- 1867-3899. ; 15:6
-
Journal article (peer-reviewed)abstract
- 2,5-furandicarboxylic acid (FDCA) has gained great industrial interest as a renewable alternative to terephthalic acid (TPA) in the generation of bioplastics. However, chemical production of FDCA involves harsh reaction conditions not aligned with sustainable manufacturing. Herein, we demonstrate the use of whole-cell mediated synthesis of FDCA from 2-furoic acid (FA) as substrate. Our approach moves away from the use of isolated enzymes by supplementing the UbiD−UbiX system in E. coli with the gene of P. thermopropionicum HmfF (PtHmfF) known to generate FDCA. The resulting whole-cell system allows for production of FDCA under mild conditions by carboxylation of FA. We show how the enzymatically produced FDCA can be used to generate FDCA-based biopolymers along with a terpene-based diol monomer by enzymatic polycondensation catalyzed by Candida antarctica lipase B (CALB). This work high-lights how underutilized hemicellulose-derived C5 building blocks can be converted into renewable platform chemicals and materials by a simple cell factory in a CO2 sequestration process.
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| 43. |
- Malmström, Eva, Professor, 1966-, et al.
(author)
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Sustainable terpene-based polymeric materials
- 2019
-
In: Abstracts of Papers of the American Chemical Society. - : American Chemical Society (ACS). - 0065-7727. ; 257
-
Journal article (other academic/artistic)
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| 44. |
- Marton, Z., et al.
(author)
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Mutations in the stereospecificity pocket and at the entrance of the active site of Candida antarctica lipase B enhancing enzyme enantioselectivity
- 2010
-
In: Journal of Molecular Catalysis B. - : Elsevier BV. - 1381-1177 .- 1873-3158. ; 65:1-4, s. 11-17
-
Journal article (peer-reviewed)abstract
- Two different parts of Candida antarctica lipase B (stereospecificity pocket at the bottom of the active site and hydrophobic tunnel leading to the active site) were redesigned by single- or double-point mutations, in order to better control and improve enzyme enantioselectivity toward secondary alcohols. Single-point isosteric mutations of Ser47 and Thr42 situated in the stereospecificity pocket gave rise to variants with doubled enantioselectivity toward pentan-2-ol, in solid/gas reactor. Besides, the width and shape of the hydrophobic tunnel leading to the active site was modified by producing the following single-point mutants: Ile189Ala, Leu278Val and Ala282Leu. For each of these variants a significant modification of enantioselectivity was observed compared to wild-type enzyme, indicating that discrimination of the enantiomers by the enzyme could also arise from their different accessibilities from the enzyme surface to the catalytic site. (C) 2010 Elsevier B.V. All rights reserved.
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| 45. |
- Nebel, Bernd A., et al.
(author)
-
A Career in Catalysis : Bernhard Hauer
- 2023
-
In: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 13:13, s. 8861-8889
-
Research review (peer-reviewed)abstract
- On the occasion of Professor Bernhard Hauer′s (partial) retirement, we reflect on and highlight his distinguished career in biocatalysis. Bernhard, a biologist by training, has greatly influenced biocatalysis with his vision and ideas throughout his four-decade career. The development of his career went hand in hand with the evolution of biocatalysis and the application and development of enzymes for chemical processes. In this Account, we present selected examples of his early work on the development of enzymes and their application in an industrial setting, with a focus on his specific contributions to harnessing the catalytic power of enzymes for novel reactions and the understanding and engineering of flexible loops and channels on catalysis.
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| 46. |
- Pavlidis, I. V., et al.
(author)
-
Computational Techniques for Efficient Biocatalysis
- 2018
-
In: Modern Biocatalysis. - Cambridge : Royal Society of Chemistry. ; , s. 119-152
-
Book chapter (peer-reviewed)abstract
- Addressing some of the most challenging problems that we face today, including depletion of natural resources, sustainable energy production and the generation of green polymeric materials by the biocatalytic upcycling of renewable synthons, requires an expansion of the current available biochemical reaction space. Creating biocatalysts harboring novel chemistries - whether inside or outside the cell - is dependent on the discovery of novel enzymes and metabolic pathways, together with the de novo design of enzymes and directed evolution. Herein we review the high potential of using bioinformatics and in silico computer modelling tools to guide protein engineering and to enhance our fundamental understanding of biocatalysis. Following an overview of technical considerations and the current state-of-the art in sequence- and structure-based protein engineering methodologies, we highlight recent successful examples of their implementation in biocatalysis and synthetic biology. Moreover, we discuss how selected computational tools in concert with experimental biocatalysis could decipher how the sequence, structure and dynamics of proteins dictate their function. Using the methodologies discussed in this chapter, an accelerated biocatalytic manufacturing of chemicals, pharmaceuticals, biofuels and monomeric building blocks is envisioned.
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| 47. |
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| 48. |
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| 49. |
- Schriever, Karen, et al.
(author)
-
Engineering of Ancestors as a Tool to Elucidate Structure, Mechanism, and Specificity of Extant Terpene Cyclase
- 2021
-
In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 143:10, s. 3794-3807
-
Journal article (peer-reviewed)abstract
- Structural information is crucial for understanding catalytic mechanisms and to guide enzyme engineering efforts of biocatalysts, such as terpene cyclases. However, low sequence similarity can impede homology modeling, and inherent protein instability presents challenges for structural studies. We hypothesized that X-ray crystallography of engineered thermostable ancestral enzymes can enable access to reliable homology models of extant biocatalysts. We have applied this concept in concert with molecular modeling and enzymatic assays to understand the structure activity relationship of spiroviolene synthase, a class I terpene cyclase, aiming to engineer its specificity. Engineering a surface patch in the reconstructed ancestor afforded a template structure for generation of a high-confidence homology model of the extant enzyme. On the basis of structural considerations, we designed and crystallized ancestral variants with single residue exchanges that exhibited tailored substrate specificity and preserved thermostability. We show how the two single amino acid alterations identified in the ancestral scaffold can be transferred to the extant enzyme, conferring a specificity switch that impacts the extant enzyme's specificity for formation of the diterpene spiroviolene over formation of sesquiterpenes hedycaryol and farnesol by up to 25-fold. This study emphasizes the value of ancestral sequence reconstruction combined with enzyme engineering as a versatile tool in chemical biology.
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| 50. |
- Schriever, Karen, et al.
(author)
-
Modulating activation entropyand enthalpy of human oxidosqualene cyclase reaction by tunnel mutagenesis
-
Other publication (other academic/artistic)abstract
- The formation of tetracyclic lanosterol from (S)-2,3-oxidosqualene is catalyzed by oxidosqualenecyclase (OSC). Lanosterol is of high interest due to its essential role in steroid metabolism. Therefore,understanding how the inherent high entropic cost of forming a multicyclic core from a flexible linearsubstrate is energetically driven is of high interest. Enzyme mechanisms can involve a reducedhydration state of rearranging transient charges in intermediates and transition states. Often thesereactions have an unusually low entropy barrier. We studied the activation enthalpy and entropy inrelation to solvent tunnels accessing the active site in the carbocationic polycyclization cascadecatalyzed by human OSC (hOSC). We applied Eyring transition state analysis of lanosterol formationby hOSC at different temperatures, alongside Molecular Dynamics simulations and CAVER analysis.hOSC showed a high favorable entropy of activation (+6.4 kcal mol-1 at 310 K) at ambienttemperatures. The introduction of bulky residues at the interface of several water tunnels, resulted inenzyme variants with altered thermodynamic properties. One of the variants was enthalpy-driven andshowed an inversed temperature dependence of cyclization. We further biochemically characterizeddifferent enzyme libraries, in which rational tunnel-mutations combined with mutations suggested byphylogeny-guided protein design were introduced in different combinations. This approach yieldedseveral highly active hOSC variants (5-6x increased activity at 37 °C), as well as a highly active variantat colder temperature with inversed temperature dependence. In summary, the present workhighlights the importance of activation entropy in enzymes, which is often considered negligible, aswell as the challenges associated with rational protein design aiming to modify activationthermodynamic parameters.
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