| 1. |
- Alexakis, Alexandros Efraim, et al.
(författare)
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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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Ingår i: Green Chemistry. - : Royal Society of Chemistry (RSC). - 1463-9262 .- 1463-9270. ; 23:5, s. 2113-2122
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Tidskriftsartikel (refereegranskat)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. |
- Biundo, Antonino, et al.
(författare)
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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
-
Ingår i: Enzyme and microbial technology. - : Elsevier BV. - 0141-0229 .- 1879-0909. ; 163
-
Tidskriftsartikel (refereegranskat)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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| 3. |
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| 4. |
- Farhat, Wissam, et al.
(författare)
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Lactone monomers obtained by enzyme catalysis and their use in reversible thermoresponsive networks
- 2020
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Ingår i: Journal of Applied Polymer Science. - : John Wiley and Sons Inc.. - 0021-8995 .- 1097-4628. ; 137:18
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Tidskriftsartikel (refereegranskat)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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| 5. |
- Guo, Boyang, et al.
(författare)
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Conformational Selection in Biocatalytic Plastic Degradation by PETase
- 2022
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 12:6, s. 3397-3409
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Tidskriftsartikel (refereegranskat)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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| 6. |
- Guo, Boyang, et al.
(författare)
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Fast Depolymerization of PET Bottle Mediated by Microwave Pre-Treatment and An Engineered PETase
- 2023
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Ingår i: ChemSusChem. - : Wiley. - 1864-5631 .- 1864-564X. ; 16:18
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Tidskriftsartikel (refereegranskat)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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| 7. |
- Hendrikse, Natalie, et al.
(författare)
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Ancestral lysosomal enzymes with increased activity harbor therapeutic potential for treatment of Hunter syndrome
- 2021
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Ingår i: ISCIENCE. - : Elsevier BV. - 2589-0042. ; 24:3
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Tidskriftsartikel (refereegranskat)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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| 8. |
- Hendrikse, Natalie
(författare)
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Engineering enzymes towards biotherapeutic applications using ancestral sequence reconstruction
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 9. |
- Hendrikse, Natalie M., et al.
(författare)
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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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Ingår i: Scientific Reports. - : Nature Research. - 2045-2322. ; 10:1
-
Tidskriftsartikel (refereegranskat)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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| 10. |
- Hueting, David A., et al.
(författare)
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Design, structure and plasma binding of ancestral β-CoV scaffold antigens
- 2023
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)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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| 11. |
- Hueting, David A., 1993-
(författare)
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In silico protein design for the enhancement of protein stability and function
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 12. |
- Hueting, David A., et al.
(författare)
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Thermoadaptation in an Ancestral Diterpene Cyclase by Altered Loop Stability
- 2022
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 126:21, s. 3809-3821
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Tidskriftsartikel (refereegranskat)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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| 13. |
- Hunold, A., et al.
(författare)
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Assembly of a Rieske non-heme iron oxygenase multicomponent system from Phenylobacterium immobile E DSM 1986 enables pyrazon cis-dihydroxylation in E. coli
- 2021
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media Deutschland GmbH. - 0175-7598 .- 1432-0614. ; 105:5, s. 2003-2015
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Tidskriftsartikel (refereegranskat)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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| 14. |
- Jönsson, Christina, et al.
(författare)
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Biocatalysis in the Recycling Landscape for Synthetic Polymers and Plastics towards Circular Textiles
- 2021
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Ingår i: ChemSusChem. - : Wiley. - 1864-5631 .- 1864-564X. ; 14:19, s. 4028-4040
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Tidskriftsartikel (refereegranskat)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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| 15. |
- Lopez-Lorenzo, Ximena
(författare)
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Chemoenzymatic Synthesis and Degradation of Plastics
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)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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| 16. |
- Lopez-Lorenzo, Ximena, et al.
(författare)
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Whole-cell Mediated Carboxylation of 2-Furoic Acid Towards the Production of Renewable Platform Chemicals and Biomaterials
- 2023
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Ingår i: ChemCatChem. - : Wiley. - 1867-3880 .- 1867-3899. ; 15:6
-
Tidskriftsartikel (refereegranskat)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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| 17. |
- Nebel, Bernd A., et al.
(författare)
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A Career in Catalysis : Bernhard Hauer
- 2023
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 13:13, s. 8861-8889
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Forskningsöversikt (refereegranskat)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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| 18. |
- Schriever, Karen, et al.
(författare)
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Engineering of Ancestors as a Tool to Elucidate Structure, Mechanism, and Specificity of Extant Terpene Cyclase
- 2021
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 143:10, s. 3794-3807
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Tidskriftsartikel (refereegranskat)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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| 19. |
- Schriever, Karen
(författare)
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Sequence- and structure guided engineering of proteins and enzymes for biotechnology and health applications
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are highly diverse and sophisticated biomolecules that represent a cornerstone of biological structure and function and have been exploited in man-made applications for thousands of years. Those proteins that facilitate chemical reactions at physiologically relevant time-scales are referred to as enzymes. Understanding the connections between proteins’ functions and their structures, mechanisms and evolution allows to engineer them towards desired properties for various applications. The aim of the work presented in this thesis is to assess different protein engineering approaches and workflows in the context of health and biotechnology applications. Four proteins were studied and/or engineered towards different outcomes using either sequence‑based information, structural information or a combination thereof. In paper I a sequence-based approach was applied to optimise vaccine candidates for severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2). Specifically, ancestral sequence reconstruction was used to generate highly stable and soluble antigens that could be produced in high quantities in a low-throughput and structure‑independent manner. These ancestral antigens interacted with antibodies from recovered patients and served as scaffolds to host a domain of the extant antigen to further enhance antibody engagement. Paper II and III applied enzyme engineering to terpene cyclases in a health and biocatalysis context, respectively. In paper II a structure-based approach was used to understand the fundamental principles underlying the catalytic mechanism of an enzyme in human steroid metabolism. Specifically, solvent access tunnels were identified and modified to probe the role of activation entropy in human oxidosqualene cyclase, which drastically modified the temperature dependence of catalysis. This finding may also have implications for engineering related plant enzymes for production of industrially relevant compounds in heterologous hosts. In paper III sequence- and structure based approaches were used together to engineer substrate specificity in a promiscuous bacterial terpene cyclase. Specifically, the structure of a stable reconstructed ancestor of spiroviolene synthase was determined in order to understand the molecular basis of substrate promiscuity and engineer highly selective variants that retained thermostability. The presented workflow is relevant for engineering these enzymes as biocatalysts for production of terpene-based high value compounds. In paper IV the metabolite regulation of a flux-controlling enzyme in the Calvin cycle was studied to eventually engineer it for enhanced growth of autotrophic production hosts. Specifically, interactions between a bifunctional cyanobacterial fructose‑1,6-bisphosphatase and a panel of metabolites were identified using a proteomics approach and verified by in vitro experiments. A synergistic regulation involving the enzyme’s redox state and glyceraldehyde 3‑phosphate was discovered, which has implications for integrated metabolic and enzyme engineering approaches involving this biocatalyst. In summary, the results presented herein highlight the utility of integrating several different engineering approaches for proteins used in health and biotechnology applications.
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| 20. |
- Sporre, Emil, et al.
(författare)
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Metabolite interactions in the bacterial Calvin cycle and implications for flux regulation
- 2022
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering to improve industrial phenotypes. Recently developed chemoproteomics workflows allow for genome-wide detection of metabolite-protein interactions that may regulate pathway activity. We applied limited proteolysis small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2 using the Calvin cycle. Clustering analysis of the hundreds of detected interactions showed that some metabolites interacted in a species-specific manner, such as interactions of glucose-6-phosphate in Cupriavidus necator and of glyoxylate in Synechocystis sp PCC 6803. These are interpreted in light of the different central carbon conversion pathways present. Metabolites interacting with the Calvin cycle enzymes fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) and transketolase were tested for effects on catalytic activity in vitro. The Calvin cycle intermediate glyceraldehyde-3-phosphate activated both Synechocystis and Cupriavidus F/SBPase, which suggests a feed-forward activation of the cycle in both photoautotrophs and chemolithoautotrophs. In contrast to the stimulating effect in reduced conditions, glyceraldehyde-3-phosphate inactivated the Synechocystis F/SBPase in oxidized conditions by accelerating protein aggregation. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated and may act in addition to redox regulation.
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| 21. |
- Sporre, Emil, et al.
(författare)
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Metabolite interactions in the bacterial Calvin cycle and implications for flux regulation
- 2023
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Ingår i: Communications Biology. - : Springer Nature. - 2399-3642. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Metabolite-level regulation of enzyme activity is important for microbes to cope with environmental shifts. Knowledge of such regulations can also guide strain engineering for biotechnology. Here we apply limited proteolysis-small molecule mapping (LiP-SMap) to identify and compare metabolite-protein interactions in the proteomes of two cyanobacteria and two lithoautotrophic bacteria that fix CO2 using the Calvin cycle. Clustering analysis of the hundreds of detected interactions shows that some metabolites interact in a species-specific manner. We estimate that approximately 35% of interacting metabolites affect enzyme activity in vitro, and the effect is often minor. Using LiP-SMap data as a guide, we find that the Calvin cycle intermediate glyceraldehyde-3-phosphate enhances activity of fructose-1,6/sedoheptulose-1,7-bisphosphatase (F/SBPase) from Synechocystis sp. PCC 6803 and Cupriavidus necator in reducing conditions, suggesting a convergent feed-forward activation of the cycle. In oxidizing conditions, glyceraldehyde-3-phosphate inhibits Synechocystis F/SBPase by promoting enzyme aggregation. In contrast, the glycolytic intermediate glucose-6-phosphate activates F/SBPase from Cupriavidus necator but not F/SBPase from Synechocystis. Thus, metabolite-level regulation of the Calvin cycle is more prevalent than previously appreciated.
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| 22. |
- Stamm, Arne, 1991-
(författare)
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Exploring the Use of Terpenes as Renewable Polymer Feedstock
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Rising environmental awareness and responsibility has increased the demand for novel, bio-based sustainable materials. Therefore, there is an extensive need to research and develop closed-loop materials from novel feedstocks that can be generated using benign, environmentally friendly synthetic routes. The work presented in this thesis was focused on the development of polymeric materials from biomass waste-feedstocks. Especially the use of turpentine, a classical underutilized side-stream of the forest industry, and its main component α-pinene was the key objective of this work. To showcase the inherent potential of terpenes as a source material for novel biopolymers, α-pinene was transformed into different monomers and subsequently polymerized. The synthesis and polymerization of the pinene derived sobreryl methacrylate (SobMA) was studied using different radical polymerization techniques, including free radical polymerizations and controlled procedures. Post-modifications were further demonstrated by synthesizing crosslinked thin films, utilizing the tertiary alcohol and the unsaturation present in each monomer unit. The same methacrylic monomer was further utilized for the polymerization of amphiphilic block copolymers to form cationic polymer latexes. Pinene derived polyesters were further developed via two different pathways. In the first pathway, α-pinene was oxidized into the bicyclic verbanone based lactone (VaL) and subsequently polymerized resulting in a biobased semicrystalline polyester. In the second pathway, polyesters were synthesized via polycondensation, utilizing the diol (1-(1'-hydroxyethyl)-3-(2'-hydroxyethyl)-2,2-dimethylcyclobutane (HHDC)) obtained from the oxidative cleavage of the double bond of α-pinene together with unsaturated biobased diacids. The resulting terpene based unsaturated polyester resins were afterwards crosslinked via UV-irradiation to yield polyester networks with adjustable properties. The use of enzymatic catalysis in key synthetic steps was elaborated to showcase the potential of replacing harsh chemical conditions with mild reaction conditions in aqueous environment.
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| 23. |
- Stamm, Arne, et al.
(författare)
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Pinene-Based Oxidative Synthetic Toolbox for Scalable Polyester Synthesis
- 2021
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Ingår i: JACS Au. - : American Chemical Society (ACS). - 2691-3704. ; 1:11, s. 1949-1960
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Tidskriftsartikel (refereegranskat)abstract
- Generation of renewable polymers is a long-standing goal toward reaching a more sustainable society, but building blocks in biomass can be incompatible with desired polymerization type, hampering the full implementation potential of biomaterials. Herein, we show how conceptually simple oxidative transformations can be used to unlock the inherent reactivity of terpene synthons in generating polyesters by two different mechanisms starting from the same alpha-pinene substrate. In the first pathway, alpha-pinene was oxidized into the bicyclic verbanone-based lactone and subsequently polymerized into star-shaped polymers via ring-opening polymerization, resulting in a biobased semicrystalline polyester with tunable glass transition and melting temperatures. In a second pathway, polyesters were synthesized via polycondensation, utilizing the diol 1-(1'-chydroxyethyl)-3-(2'-hydroxyethyl)-2,2-dimethylcyclobutane (HHDC) synthesized by oxidative cleavage of the double bond of alpha-pinene, together with unsaturated biobased diesters such as dimethyl maleate (DMM) and dimethyl itaconate (DMI). The resulting families of terpenebased polyesters were thereafter successfully cross-Iinked by either transetherification, utilizing the terminal hydroxyl groups of the synthesized verbanone-based materials, or by UV irradiation, utilizing the unsaturation provided by the DMM or DMI moieties within the HHDC-based copolymers. This work highlights the potential to apply an oxidative toolbox to valorize inert terpene metabolites enabling generation of biosourced polyesters and coatings thereof by complementary mechanisms.
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| 24. |
- Subramaniyan, Sathiyaraj, et al.
(författare)
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Designed for Circularity : Chemically Recyclable and Enzymatically Degradable Biorenewable Schiff Base Polyester-Imines
- 2023
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Ingår i: ACS Sustainable Chemistry and Engineering. - : American Chemical Society (ACS). - 2168-0485. ; 11:8, s. 3451-3465
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Tidskriftsartikel (refereegranskat)abstract
- Bio-based plastics potentially have several positive impacts on the environment; however, in order to make a real difference, they need to have managed and sustainable end of life. This means they should from the start be designed for chemical, mechanical, and/or organic (biological) recycling. Development of energy-efficient and selective chemical recycling processes is a necessary part in reaching truly circular plastic flows. Polyesters are generally well suited for chemical recycling due to the presence of reversible ester bonds. Utilization of dynamic covalent chemistry to include a second, even more easily reversed bond, such as Schiff base (SB, imine bond), could further facilitate chemical recycling, enabling depolymerization back to monomeric products under mild conditions. Here, we present the synthesis of three vanillin-derived SB monomers SBM1, SBM2, and SBM3 and the corresponding polymers SBP3a-b, SBP4a-b, and SBP5a-b. Three different diamines and two potentially bio-sourced diesters were utilized to yield altogether six different polyester-imines with different aliphatic/aromatic contents. All the obtained SB-based polyesters were thermally stable at ∼290-330 °C and had a high char yield during the pyrolysis, which may indicate inherent flame resistance. All the polyesters were amorphous with glass transition temperatures from 36 to 76 °C. The chemical recyclability and hydrolytic degradation of the synthesized polyesters was evaluated by using real-time 1H NMR spectroscopy. Finally, the susceptibility of the synthesized polyester-imines to enzymatic degradation by PETase was demonstrated. The experimental results were further supported by induced-fit docking experiments to theoretically evaluate the potential productive binding of the produced polyester-imines and intermediates thereof to the active site of PETase.
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| 25. |
- Syrén, Per-Olof, et al.
(författare)
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Design, structure and plasma binding of ancestral β-CoV scaffold antigens
- 2024
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The pandemic caused by Severe acute respiratory syndrome coronavirus 2 has had devastating consequences on global health and economy. Despite the success of vaccination campaigns emerging variants are of concern and novel viruses with the potential to drive future pandemics are circulating in nature. Development of vaccines can be challenging, as key viral protein antigens can be unstable or aggregate. In this study, we present the application of ancestral sequence reconstruction on coronavirus spike protein, resulting in stable and highly soluble ancestral scaffold antigens (AnSAs). The AnSAs interacted with plasma of patients recovered from COVID-19 but did not bind to the human angiotensin-converting enzyme 2 (ACE2) receptor. Cryo-EM analysis of the AnSAs yielded 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. This captured closed state is stabilised by an intricate hydrogen‑bonding network mediated by well-resolved loops, both within and across monomers, tethering the N‑terminal domain and RBD together, which determines their relative spatial orientation. Finally, we show how AnSAs are potent scaffolds by replacing the ancestral RBD with the Wuhan wild-type sequence, which restored ACE2 binding and increased the interaction with convalescent plasma. In contrast to rational antigen design depending on prior structural knowledge, our work highlights how stable and potentially interesting antigens can be generated using exclusively available sequence information.
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| 26. |
- Zokaei, Sepideh, 1991, et al.
(författare)
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Toughening of a Soft Polar Polythiophene through Copolymerization with Hard Urethane Segments
- 2021
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Ingår i: Advanced Science. - : Wiley. - 2198-3844. ; 8:2
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Tidskriftsartikel (refereegranskat)abstract
- Polar polythiophenes with oligoethylene glycol side chains are exceedingly soft materials. A low glass transition temperature and low degree of crystallinity prevents their use as a bulk material. The synthesis of a copolymer comprising 1) soft polythiophene blocks with tetraethylene glycol side chains, and 2) hard urethane segments is reported. The molecular design is contrary to that of other semiconductor-insulator copolymers, which typically combine a soft nonconjugated spacer with hard conjugated segments. Copolymerization of polar polythiophenes and urethane segments results in a ductile material that can be used as a free-standing solid. The copolymer displays a storage modulus of 25 MPa at room temperature, elongation at break of 95%, and a reduced degree of swelling due to hydrogen bonding. Both chemical doping and electrochemical oxidation reveal that the introduction of urethane segments does not unduly reduce the hole charge-carrier mobility and ability to take up charge. Further, stable operation is observed when the copolymer is used as the active layer of organic electrochemical transistors.
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