| 1. |
- Barone, Giovanni Davide, et al.
(author)
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Hints at the Applicability of Microalgae and Cyanobacteria for the Biodegradation of Plastics
- 2020
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In: Sustainability. - BASEL, SWITZERLAND : MDPI. - 2071-1050. ; 12:24
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Research review (peer-reviewed)abstract
- Massive plastic accumulation has been taking place across diverse landscapes since the 1950s, when large-scale plastic production started. Nowadays, societies struggle with continuously increasing concerns about the subsequent pollution and environmental stresses that have accompanied this plastic revolution. Degradation of used plastics is highly time-consuming and causes volumetric aggregation, mainly due to their high strength and bulky structure. The size of these agglomerations in marine and freshwater basins increases daily. Exposure to weather conditions and environmental microflora (e.g., bacteria and microalgae) can slowly corrode the plastic structure. As has been well documented in recent years, plastic fragments are widespread in marine basins and partially in main global rivers. These are potential sources of negative effects on global food chains. Cyanobacteria (e.g., Synechocystis sp. PCC 6803, and Synechococcus elongatus PCC 7942), which are photosynthetic microorganisms and were previously identified as blue-green algae, are currently under close attention for their abilities to capture solar energy and the greenhouse gas carbon dioxide for the production of high-value products. In the last few decades, these microorganisms have been exploited for different purposes (e.g., biofuels, antioxidants, fertilizers, and 'superfood' production). Microalgae (e.g., Chlamydomonas reinhardtii, and Phaeodactylum tricornutum) are also suitable for environmental and biotechnological applications based on the exploitation of solar light. Can photosynthetic bacteria and unicellular eukaryotic algae play a role for further scientific research in the bioremediation of plastics of different sizes present in water surfaces? In recent years, several studies have been targeting the utilization of microorganisms for plastic bioremediation. Among the different phyla, the employment of wild-type or engineered cyanobacteria may represent an interesting, environmentally friendly, and sustainable option.
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| 2. |
- Biundo, Antonino, et al.
(author)
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Enzyme Modification
- 2021
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In: Biocatalysis for Practitioners. - : Wiley. ; , s. 33-62
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Book chapter (other academic/artistic)abstract
- The chapter on protein engineering has introduced and discussed an overview of the available methods for the modification of enzymes. Starting with the classical directed evolution (DE) technique, which has been applied extensively throughout several different biocatalytic processes, the reader moves toward the semi-rational, rational, and de novo design of enzymes. Meanwhile, DE is clearly the current industry-leading technology; depending on the understanding of the particular enzymatic system, and on the available structural information, other techniques such as rational design are also becoming fast and efficient solutions for the development of new catalysts. This success would not be possible without the constant improvements of the computational techniques and the newly developed modeling systems for enzyme engineering.
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| 3. |
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| 4. |
- 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
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In: Enzyme and microbial technology. - : Elsevier BV. - 0141-0229 .- 1879-0909. ; 163
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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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| 5. |
- 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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| 6. |
- 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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| 7. |
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| 8. |
- 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
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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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| 9. |
- 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
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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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| 10. |
- Jönsson, Christina, et al.
(author)
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Biocatalysis in the Recycling Landscape for Synthetic Polymers and Plastics towards Circular Textiles
- 2021
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In: ChemSusChem. - : Wiley. - 1864-5631 .- 1864-564X. ; 14:19, s. 4028-4040
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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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| 11. |
- Malmström, Eva, Professor, 1966-, et al.
(author)
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Sustainable terpene-based polymeric materials
- 2019
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In: Abstracts of Papers of the American Chemical Society. - : American Chemical Society (ACS). - 0065-7727. ; 257
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Journal article (other academic/artistic)
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| 12. |
- Saenz Mendez, Patricia, et al.
(author)
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Enzyme Modification
- 2021
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In: Biocatalysis for Practitioners.. - Weinheim : Wiley-VCH Verlagsgesellschaft. - 9783527346837 ; , s. 33-51
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Book chapter (other academic/artistic)
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| 13. |
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| 14. |
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| 15. |
- Schriever, Karen, et al.
(author)
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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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In: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 143:10, s. 3794-3807
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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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| 16. |
- Schriever, Karen, et al.
(author)
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Modulating activation entropyand enthalpy of human oxidosqualene cyclase reaction by tunnel mutagenesis
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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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| 17. |
- Stamm, Arne, et al.
(author)
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A retrobiosynthesis-based route to generate pinene-derived polyesters
- 2019
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In: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 20, s. 1664-1671
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Journal article (peer-reviewed)abstract
- Significantly increased production of biobased polymers is aprerequisite to replace petroleum-based materials towardsreaching a circular bioeconomy. However, many renewablebuilding blocks from wood and other plant material are notdirectly amenable for polymerization, due to their inert backbonesand/or lack of functional group compatibility with thedesired polymerization type. Based on a retro-biosyntheticanalysis of polyesters, a chemoenzymatic route from (@)-apinenetowards a verbanone-based lactone, which is furtherused in ring-opening polymerization, is presented. Generatedpinene-derived polyesters showed elevated degradation andglass transition temperatures, compared with poly(e-decalactone),which lacks a ring structure in its backbone. Semirationalenzyme engineering of the cyclohexanone monooxygenasefrom Acinetobacter calcoaceticus enabled the biosynthesis ofthe key lactone intermediate for the targeted polyester. As aproof of principle, one enzyme variant identified from screeningin a microtiter plate was used in biocatalytic upscaling,which afforded the bicyclic lactone in 39% conversion in shakeflask scale reactions.
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| 18. |
- Stamm, Arne, et al.
(author)
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Pinene-Based Oxidative Synthetic Toolbox for Scalable Polyester Synthesis
- 2021
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In: JACS Au. - : American Chemical Society (ACS). - 2691-3704. ; 1:11, s. 1949-1960
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Journal article (peer-reviewed)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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