| 1. |
- Abahazi, Emese, et al.
(författare)
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Covalently immobilized Trp60Cys mutant of omega‰-transaminase from Chromobacterium violaceum for kinetic resolution of racemic amines in batch and continuous-flow modes
- 2018
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Ingår i: Biochemical engineering journal. - : Elsevier. - 1369-703X .- 1873-295X. ; 132, s. 270-278
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Tidskriftsartikel (refereegranskat)abstract
- Covalent immobilization of an engineered omega-transaminase mutant Trp60Cys from Chromobacterium violaceum (CvTAW60C) was performed on bisepoxide-activated aminoalkyl resins. Activity of the various CvTAW60C preparations was evaluated in kinetic resolution of four racemic amines (rac-1a–d). The most active EA-G-CvTAW60C preparation (CvTAW60C attached to polymeric resin with ethylamine function activated with glycerol diglycidyl ether—EA-G) could perform the kinetic resolution of racemic 4-phenylbutan-2-amine (rac-1a) over 49% conversion up to 19 consecutive reaction cycles or in media containing up to 50% v/v DMSO as cosolvent in batch mode reactions. The immobilization process of CvTAW60C onto the EA-G resin filled in stainless steel bioreactors was also tested in flow-through mode. Kinetic resolution of three racemic amines containing aromatic moieties (rac-1a-c) was performed in continuous-flow mode resulting in easy-to-separate mixture of the corresponding ketone (2a–c) and the non-converted (R)-amine in high enantiopurity (ee(R)-1a-c ≥ 96%).
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| 2. |
- Cabotaje, Princess R., et al.
(författare)
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Probing Substrate Transport Effects on Enzymatic Hydrogen Catalysis : An Alternative Proton Transfer Pathway in Putatively Sensory [FeFe] Hydrogenase
- 2023
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Ingår i: ACS Catalysis. - : American Chemical Society (ACS). - 2155-5435. ; 13:15, s. 10435-10446
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Tidskriftsartikel (refereegranskat)abstract
- [FeFe] hydrogenases, metalloenzymes catalyzing proton/dihydrogeninterconversion, have attracted intense attention due to their remarkablecatalytic properties and (bio-)technological potential for a futurehydrogen economy. In order to unravel the factors enabling their efficientcatalysis, both their unique organometallic cofactors and proteinstructural features, i.e., "outer-coordination sphere"effects have been intensively studied. These structurally diverseenzymes are divided into distinct phylogenetic groups, denoted asGroup A-D. Prototypical Group A hydrogenases display high turnoverrates (10(4)-10(5) s(-1)).Conversely, the sole characterized Group D representative, Thermoanaerobacter mathranii HydS (TamHydS), shows relatively low catalytic activity (specific activity10(-1) & mu;mol H-2 mg(-1) min(-1)) and has been proposed to serve a H-2-sensory function. The various groups of [FeFe] hydrogenaseshare the same catalytic cofactor, the H-cluster, and the structuralfactors causing the diverging reactivities of Group A and D remainto be elucidated. In the case of the highly active Group A enzymes,a well-defined proton transfer pathway (PTP) has been identified,which shuttles H+ between the enzyme surface and the activesite. In Group D hydrogenases, this conserved pathway is absent. Here,we report on the identification of highly conserved amino acid residuesin Group D hydrogenases that constitute a possible alternative PTP.We varied two proposed key amino acid residues of this pathway (E252and E289, TamHydS numbering) via site-directed mutagenesisand analyzed the resulting variants via biochemical and spectroscopicmethods. All variants displayed significantly decreased H-2-evolution and -oxidation activities. Additionally, the variantsshowed two redox states that were not characterized previously. Thesefindings provide initial evidence that these amino acid residues arecentral to the putative PTP of Group D [FeFe] hydrogenase. Since theidentified residues are highly conserved in Group D exclusively, ourresults support the notion that the PTP is not universal for differentphylogenetic groups in [FeFe] hydrogenases.
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| 3. |
- Cassimjee, Karim Engelmark, et al.
(författare)
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Chromobacterium violaceum omega-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4 '-substituted acetophenones, and follows Swain-Lupton parameterisation
- 2012
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Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 10:28, s. 5466-5470
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Tidskriftsartikel (refereegranskat)abstract
- For biocatalytic production of pharmaceutically important chiral amines the.-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum omega-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4'-substituted acetophenones (similar to 5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.
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| 4. |
- Chen, Shan, et al.
(författare)
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Stabilization of an amine transaminase for biocatalysis
- 2016
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Ingår i: Journal of Molecular Catalysis B. - : Elsevier. - 1381-1177 .- 1873-3158. ; 124, s. 20-28
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Tidskriftsartikel (refereegranskat)abstract
- The amine transaminase from Chromobacterium violaceum (Cv-ATA) is a well-known enzyme to achievechiral amines of high enantiomeric excess in laboratory scales. However, the low operational stabilityof Cv-ATA limits the enzyme applicability on larger scales. In order to improve the operational stabilityof Cv-ATA, and thereby extending its applicability, factors (additives, co-solvents, organic solvents anddifferent temperatures) targeting enzyme stability and activity were explored in order to find out how tostore and apply the enzyme. The present investigation shows that the melting point of Cv-ATA is improvedby adding sucrose or glycerol, separately. Further, by storing the enzyme at higher concentrations and inco-solvents, such as; 50% glycerol, 20% methanol or 10% DMSO, the active dimeric structure of Cv-ATAis retained. Enzyme stored in 50% glycerol at −20◦C was e.g., still fully active after 6 months. Finally,the enzyme performance was improved 5-fold by a co-lyophilization with surfactants prior to usage inisooctane.
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| 5. |
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| 6. |
- Fasano, Andrea, et al.
(författare)
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Reversible or Irreversible Catalysis of H+/H2 Conversion by FeFe Hydrogenases
- 2021
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 143:48, s. 20320-20325
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Tidskriftsartikel (refereegranskat)abstract
- Studies of molecular catalysts traditionally aim at understanding how a certain mechanism allows the reaction to be fast. A distinct question, which has only recently received attention in the case of bidirectional molecular catalysts, is how much thermodynamic driving force is required to achieve fast catalysis in either direction of the reaction. “Reversible” catalysts are bidirectional catalysts that work either way in response to even a small departure from equilibrium and thus do not waste input free energy as heat; conversely, “irreversible” catalysts require a large driving force to proceed at an appreciable rate [Fourmond et al. Nat. Rev. Chem.2021, 5, 348–360]. Numerous mechanistic rationales for these contrasting behaviors have been proposed. To understand the determinants of catalytic (ir)reversibility, we examined the steady-state, direct electron transfer voltammetry of a particular FeFe hydrogenase, from Thermoanaerobacter mathranii, which is very unusual in that it irreversibly catalyzes H2 oxidation and production: a large overpotential is required for the reaction to proceed in either direction [Land et al. Chem. Sci.2020, 11, 12789–12801]. In contrast to previous hypotheses, we demonstrate that in this particular enzyme catalytic irreversibility can be explained without invoking slow interfacial electron transfer or variations in the mechanism: the observed kinetics is fully consistent with the same catalytic pathway being used in both directions of the reaction.
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| 7. |
- Greening, Chris, et al.
(författare)
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Minimal and hybrid hydrogenases are active from archaea
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Ingår i: Cell. - 1097-4172. ; 187
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Tidskriftsartikel (refereegranskat)abstract
- Microbial hydrogen (H 2) cycling underpins the diversity and functionality of diverse anoxic ecosystems. Among the three evolutionarily distinct hydrogenase superfamilies responsible, [FeFe] hydrogenases were thought to be restricted to bacteria and eukaryotes. Here, we show that anaerobic archaea encode diverse, active, and ancient lineages of [FeFe] hydrogenases through combining analysis of existing and new genomes with extensive biochemical experiments. [FeFe] hydrogenases are encoded by genomes of nine archaeal phyla and expressed by H 2-producing Asgard archaeon cultures. We report an ultraminimal hydrogenase in DPANN archaea that binds the catalytic H-cluster and produces H 2. Moreover, we identify and characterize remarkable hybrid complexes formed through the fusion of [FeFe] and [NiFe] hydrogenases in ten other archaeal orders. Phylogenetic analysis and structural modeling suggest a deep evolutionary history of hybrid hydrogenases. These findings reveal new metabolic adaptations of archaea, streamlined H 2 catalysts for biotechnological development, and a surprisingly intertwined evolutionary history between the two major H 2-metabolizing enzymes.
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| 8. |
- Hola, Katerina, et al.
(författare)
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Carbon Dots and [FeFe] Hydrogenase Biohybrid Assemblies for Efficient Light-Driven Hydrogen Evolution
- 2020
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Ingår i: ACS Catalysis. - : AMER CHEMICAL SOC. - 2155-5435. ; 10:17, s. 9943-9952
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Tidskriftsartikel (refereegranskat)abstract
- Artificial photosynthesis is seen as a path to convert and store solar energy into chemical energy for our society. In this work, highly fluorescent aspartic acid-based carbon dots (CDs) are synthesized and employed as a photosensitizer to drive photocatalytic hydrogen evolution with an [FeFe] hydrogenase (CrHydA1). The direct interaction in CDs from L-aspartic acid (AspCDs)/CrHydA1 self-assembly systems, which is visualized from native gel electrophoresis, has been systematically investigated to understand the electron-transfer dynamics and its impact on photocatalytic efficiency. The study discloses the significant influence of the electrostatic surrounding generated by sacrificial electron donors on the intimate interplay within the oppositely charged subunits of the biohybrid assembly as well as the overall photocatalytic performance. The system reaches an external quantum efficiency of 1.7% at 420 nm and an initial activity of 1.73 mu mol(H-2) mg(-1) (hydrogenase) min(-1) under favorable electrostatic conditions. Owing to the ability of the synthesized AspCDs to operate efficiently under visible light, in contrast to other materials that require UV illumination, the stability of the biohybrid assembly in the presence of a redox mediator extends beyond 1 week.
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| 9. |
- Kätterer, Thomas, et al.
(författare)
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Växtföljders påverkan på inlagring av organiskt kol i jordbruksmark : en systematisk översikt och samhällsekonomisk analys
- 2021
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Rapport (populärvet., debatt m.m.)abstract
- Ändrade växtföljder kan göra jordbruket mer klimatsmart Genom väl avvägda växtföljder inom jordbruket kan förlusten av organiskt kol i marken minskas. Det är bra både för klimatet och markens bördighet. Vi har undersökt vad vetenskapen säger om vilka grödor och växtföljder som minskar kolförlusterna mest. I den här rapporten redovisar vi en systematisk översikt (del 1) och en samhällsekonomisk analys (del 2). Den systematiska översikten reder ut hur olika växtföljder påverkar inlagring av kol i jordbruksmark. Syftet med en systematisk översikt är att identifiera all tillgänglig och relevant kunskap, kvalitetsgranska studierna och undersöka hur olika faktorer påverkar studieresultaten. I den här systematiska översikten har vi granskat över 7500 unika sökträffar, varav 125 vetenskapliga artiklar uppfyllde de på förhand uppställda urvalskriterierna och är inkluderade i översikten. Utifrån resultaten från den systematiska översikten har vi därefter genomfört en kostnadsnyttoanalys och beräknat den samhällsekonomiska lönsamheten av olika typer av växtföljder. Vi har också genomfört en styrmedelsanalys där vi diskuterar vad som kan vara ett lämpligt styrmedel för att få till stånd en ändring av växtföljder som leder till en ökad inlagring av kol på jordbruksmark.Del 1: Övergripande evidensgraderade slutsatser av den systematiska översiktenVi är säkra på att flerårsväxter har en positiv effekt på halten av organiskt kol i jorden inom varierade växtföljderVi är säkra på att baljväxter har en positiv effekt på halten av organiskt kol i jorden inom varierade växtföljderVi kan visa att det finns en positiv effekt på halten av organiskt kol i jorden av vissa varierade växtföljder och brukningsmetoder jämfört med en upprepad monokultur.Fler och mer detaljerade slutsatser presenteras i rapporten. De skillnader i halt av organiskt kol i jorden mellan olika typer av växtföljder vi rapporterar om här framkommer tydligast när många studier vägs samman, snarare än i enskilda studier, och det statistiska underlaget därmed blir större. I den systematiska översikten har vi även identifierat några typer av växtföljder som det kan behövas mer forskning om, samt brister i hur metoder och resultat ofta rapporteras och som författare av framtida vetenskapliga artiklar bör beakta.Del 2: Resultat av den samhällsekonomiska analysenModellberäkningar visar att det är samhällsekonomiskt lönsamt att inom jordbruket praktisera växtföljder som främjar inlagring av organiskt kol i jordbruksmark även om de på kort sikt ger en lägre avkastning för den enskilde jordbrukaren.Om styrmedel i form av ekonomiska ersättningar skulle bli aktuella för att öka intresset för växtföljder som främjar inlagring av organiskt kol i jordbruksmark kan omvänd auktionering vara ett fungerande alternativ. Införande och utformning av ett eventuellt styrmedel måste dock utredas djupare och förankras brett genom sedvanligt remissförfarande.
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| 10. |
- Land, Henrik, 1987-
(författare)
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Amine Transaminases in Biocatalytic Amine Synthesis
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The use of enzymes, nature´s own catalysts, both isolated or as whole cells to perform chemical transformations is called biocatalysis. As a complement to classical chemical catalysis, biocatalysis can be an environmentally friendly and more economical option in the production and synthesis of chemicals. Research on the application of amine transaminases in synthesis of chiral amines have exploded over the last two decades and interest from the industry is increasing. Amine transaminases are promising catalysts due to their ability to perform reductive amination of ketones with excellent enantioselectivity.For a process to be efficient, high substrate specificity of the applied enzyme is an important factor. A variant of Chromobacterium violaceum amine transaminase that was obtained through rational design has an increased specific activity toward (S)-1-phenylethylamine and a set of 4´-substituted acetophenones. This result makes this variant a promising catalyst for the asymmetric synthesis of similar amines.Amine transaminase catalyzed asymmetric synthesis of amines generally suffers from unfavorable equilibrium. Two methods that include spontaneous tautomerization and biocatalytic amidation for equilibrium displacement have therefore been developed.Efficient assays and screening methods are demanded for the discovery and development of novel amine transaminases. For this purpose, a sensitive fluorescence-based assay that holds promise as a high-throughput screening method was developed.One of the major obstacles for application of enzymes in industrial processes is the instability of the enzyme toward harsh conditions. The stability of Chromobacterium violaceum amine transaminase was investigated and improved using co-solvents and other additives. Co-lyophilization with surfactants was also applied to improve the performance of the same enzyme in organic solvents.
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