1,720,965 research outputs found
A new thermophilic ene‐reductase from the filamentous anoxygenic phototrophic bacterium Chloroflexus aggregans
Full text linkAiming at expanding the biocatalytic toolbox of ene‐reductase enzymes, we decided to explore photosynthetic extremophile microorganisms as unique reservoir of (new) biocatalytic ac-tivities. We selected a new thermophilic ene‐reductase homologue in Chloroflexus aggregans, a pe-culiar filamentous bacterium. We report here on the functional and structural characterization of this new enzyme, which we called CaOYE. Produced in high yields in recombinant form, it proved to be a robust biocatalyst showing high thermostability, good solvent tolerance and a wide range of pH optimum. In a preliminary screening, CaOYE displayed a restricted substrate spectrum (with generally lower activities compared to other ene‐reductases); however, given the amazing metabolic ductility and versatility of Chloroflexus aggregans, further investigations could pinpoint pecu-liar chemical activities. X‐ray crystal structure has been determined, revealing conserved features of Class III (or thermophilic‐like group) of the family of Old Yellow Enzymes: in the crystal pack-ing, the enzyme was found to assemble as dimer even if it behaves as a monomer in solution. The description of CaOYE catalytic properties and crystal structure provides new details useful for en-larging knowledge, development and application of this class of enzymes
Two new ene-reductases from photosynthetic extremophiles enlarge the panel of old yellow enzymes: CtOYE and GsOYE
Full text linkLooking for new ene-reductases with uncovered features beneficial for biotechnological applications, by mining genomes of photosynthetic extremophile organisms, we identified two new Old Yellow Enzyme homologues: CtOYE, deriving from the cyanobacterium Chroococcidiopsis thermalis, and GsOYE, from the alga Galdieria sulphuraria. Both enzymes were produced and purified with very good yields and displayed catalytic activity on a broad substrate spectrum by reducing α,β-unsaturated ketones, aldehydes, maleimides and nitroalkenes with good to excellent stereoselectivity. Both enzymes prefer NADPH but demonstrate a good acceptance of NADH as cofactor. CtOYE and GsOYE represent robust biocatalysts showing high thermostability, a wide range of pH optimum and good co-solvent tolerance. High resolution X-ray crystal structures of both enzymes have been determined, revealing conserved features of the classical OYE subfamily as well as unique properties, such as a very long loop entering the active site or an additional C-terminal alpha helix in GsOYE. Not surprisingly, the active site of CtOYE and GsOYE structures revealed high affinity toward anions caught from the mother liquor and trapped in the anion hole where electron-withdrawing groups such as carbonyl group are engaged. Ligands (para-hydroxybenzaldehyde and 2-methylcyclopenten- 1-one) added on purpose to study complexes of GsOYE were detected in the enzyme catalytic cavity, stacking on top of the FMN cofactor, and support the key role of conserved residues and FMN cofactor in the catalysis
Process for the preparation of (R)-beta-angelica lactone from alpha-angelica lactone employing ene-reductases
No full textA multi-enzymatic cascade reaction for the synthesis of vidarabine 5'-monophosphate
Full text linkWe here described a three-step multi-enzymatic reaction for the one-pot synthesis of vidarabine 5'-monophosphate (araA-MP), an antiviral drug, using arabinosyluracil (araU), adenine (Ade), and adenosine triphosphate (ATP) as precursors. To this aim, three enzymes involved in the biosynthesis of nucleosides and nucleotides were used in a cascade mode after immobilization: uridine phosphorylase from Clostridium perfringens (CpUP), a purine nucleoside phosphorylase from Aeromonas hydrophila (AhPNP), and deoxyadenosine kinase from Dictyostelium discoideum (DddAK). Specifically, CpUP catalyzes the phosphorolysis of araU thus generating uracil and α-d-arabinose-1-phosphate. AhPNP catalyzes the coupling between this latter compound and Ade to form araA (vidarabine). This nucleoside becomes the substrate of DddAK, which produces the 5'-mononucleotide counterpart (araA-MP) using ATP as the phosphate donor. Reaction conditions (i.e., medium, temperature, immobilization carriers) and biocatalyst stability have been balanced to achieve the highest conversion of vidarabine 5'-monophosphate (≥95.5%). The combination of the nucleoside phosphorylases twosome with deoxyadenosine kinase in a one-pot cascade allowed (i) a complete shift in the equilibrium-controlled synthesis of the nucleoside towards the product formation; and (ii) to overcome the solubility constraints of araA in aqueous medium, thus providing a new route to the highly productive synthesis of araA-MP
Stereodivergent Biocatalytic Formal Reduction of α Angelica Lactone to (R)-and (S)-γ-Valerolactone in a One Pot Cascade
No full textThe formal asymmetric and stereodivergent enzymatic reduction of α-angelica lactone to both enantiomers of γ-valerolactone was achieved in a one-pot cascade by uniting the promiscuous stereoselective isomerization activity of Old Yellow Enzymes with the their native reductase activity. In addition to running the cascade with one enzyme for each catalytic step, a bifunctional isomerase-reductase biocatalyst was designed by fusing twoOldYellow Enzymes, thereby generating an unprecedented case of an artificial enzyme catalyzing the reduction of nonactivated C=C bonds to access (R)-valerolactone in overall 41%conversion and up to 91î. The enzyme BfOYE4 could be used as single biocatalyst for both steps and delivered (S)-valerolactone in up to 84% ee and 41%overall conversion. The reducing equivalents were provided by a nicotinamide recycling system based on formate and formate dehydrogenase, added in a second step. This enzymatic system provides an asymmetric route to valuable chiral building blocks from an abundant bio-based chemical
What’s new in flow biocatalysis? A snapshot of 2020–2022
No full textFlow biocatalysis is a key enabling technology that is increasingly being applied to a wide array of reactions with the aim of achieving process intensification, better control of biotransformations, and minimization of waste stream. In this minireview, selected applications of flow biocatalysis to the preparation of food ingredients, APIs and fat- and oil-derived commodity chemicals, covering the period 2020-2022, are described
Immobilization of γ-Glutamyl Transpeptidase from Equine Kidney for the Synthesis of Kokumi Compounds
Full text linkγ-Glutamyl transpeptidase from equine kidney (ekGGT, E.C. 2.3.2.2) is an intrinsic membrane enzyme which transfers the γ-glutamyl moiety of glutathione to amino acids and peptides, thus producing γ-glutamyl derivatives. An immobilization study of ekGGT was carried out with the aim to develop a robust biocatalyst for the synthesis of γ-glutamyl amino acids which are known as kokumi compounds. Heterofunctional octyl-glyoxyl-agarose resulted in a high immobilization yield and activity recovery (93 % and 88 %, respectively). Immobilized ekGGT retained more than 95 % activity under reaction conditions (Tris-HCl, pH 9, 0.05 M) after 6 days, whereas the residual activity after 6 reaction cycles (18 days) was 85 %. The synthesis of γ-glutamylmethionine catalyzed by octyl-glyoxyl-agarose-ekGGT afforded the product in 42 % yield (101 mg). The immobilized ekGGT was characterized by Raman spectroscopy. The immobilization protocol developed for ekGGT could be of general applicability to membrane proteins
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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