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1.
Chemistry ; 30(40): e202401706, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-38700372

RESUMO

Unspecific Peroxygenases (UPOs) are increasingly significant enzymes for selective oxygenations as they are stable, highly active and catalyze their reactions at the expense of only hydrogen peroxide as the oxidant. Their structural similarity to chloroperoxidase (CPO) means that UPOs can also catalyze halogenation reactions based upon the generation of hypohalous acids from halide and H2O2. Here we show that the halogenation and oxygenation modes of a UPO can be stimulated at different pH values. Using simple aromatic compounds such as thymol, we show that, at a pH of 3.0 and 6.0, either brominated or oxygenated products respectively are produced. Preparative 100 mg scale transformations of substrates were performed with 60-72 % isolated yields of brominated products obtained. A one-pot bromination-oxygenation cascade reaction on 4-ethylanisole, in which the pH was adjusted from 3.0 to 6.0 at the halfway stage, yielded sequentially brominated and oxygenated products 1-(3-bromo-4-methoxyphenyl)ethyl alcohol and 3-bromo-4-methoxy acetophenone with 82 % combined conversion. These results identify UPOs as an unusual example of a biocatalyst that is tunable for entirely different chemical reactions, dependent upon the reaction conditions.

2.
J Biotechnol ; 385: 1-12, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38428504

RESUMO

Emerging consumer demand for safer, more sustainable flavors and fragrances has created new challenges for the industry. Enzymatic syntheses represent a promising green production route, but the broad application requires engineering advancements for expanded diversity, improved selectivity, and enhanced stability to be cost-competitive with current methods. This review discusses recent advances and future outlooks for enzyme engineering in this field. We focus on carboxylic acid reductases (CARs) and unspecific peroxygenases (UPOs) that enable selective productions of complex flavor and fragrance molecules. Both enzyme types consist of natural variants with attractive characteristics for biocatalytic applications. Applying protein engineering methods, including rational design and directed evolution in concert with computational modeling, present excellent examples for property improvements to unleash the full potential of enzymes in the biosynthesis of value-added chemicals.


Assuntos
Odorantes , Oxirredutases , Oxirredutases/metabolismo , Oxigenases de Função Mista/metabolismo
3.
Biotechnol J ; 19(1): e2300421, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38044796

RESUMO

Unspecific peroxygenases (UPOs) are promising biocatalysts that catalyze oxyfunctionalization reactions without the need for costly cofactors. Pichia pastoris (reclassified as Komagataella phaffii) is considered an attractive host for heterologous expression of UPOs. However, integration of UPO-expression cassettes into the genome via a single cross-over yields recombinant Pichia transformants with different UPO gene copy numbers resulting in different expression levels. Selection of the most productive Pichia transformants by a commonly used screening in liquid medium in 96-well plates is laborious and lasts up to 5 days. In this work, we developed a simple two-step agar plate-based assay to screen P. pastoris transformants for UPO activity with less effort, within shorter time, and without automated screening devices. After cell growth and protein expression on agar plates supplemented with methanol and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), an additional top agar layer supplemented with ABTS and peroxide is added. UPO activity is visualized within 15 min by formation of green zones around UPO-secreting P. pastoris transformants. The assay was validated with two UPOs, AbrUPO from Aspergillus brasiliensis and evolved PaDa-I from Agrocybe aegerita. The assay results were confirmed in a quantitative 96-deep well plate screening in liquid medium.


Assuntos
Benzotiazóis , Oxigenases de Função Mista , Pichia , Saccharomycetales , Ácidos Sulfônicos , Ágar/metabolismo , Oxigenases de Função Mista/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
4.
Methods Enzymol ; 693: 267-306, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37977733

RESUMO

Yeast-based secretion systems are advantageous for engineering highly interesting enzymes that are not or barely producible in E. coli. The herein-presented production setup facilitates high-throughput screening as no cell lysis is required. All techniques are described in detail, with access to freely available online tools and all vectors have been made available on the non-profit plasmid repository AddGene. We describe the method for UPOs as a model enzyme, showcasing their secretion, detection, and evolution using S. cerevisiae. Additional material to transfer this to P. pastoris has been published by our group previously (Püllmann & Weissenborn, 2021).


Assuntos
Escherichia coli , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Oxigenases de Função Mista/genética , Plasmídeos/genética
5.
N Biotechnol ; 76: 63-71, 2023 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-37169331

RESUMO

Unspecific peroxygenase (UPO) presents a wide range of biotechnological applications. This study targets the use of by-products from bioethanol synthesis to produce UPO by Agrocybe aegerita. Solid-state and submerged fermentations (SSF and SmF) were evaluated, achieving the highest titers of UPO and laccase in SmF using vinasse as nutrients source. Optimized UPO production of 331 U/L was achieved in 50% (v:v) vinasse with an inoculum grown for 14 days. These conditions were scaled-up to a 4 L reactor, achieving a UPO activity of 265 U/L. Fungal proteome expression was analyzed before and after UPO activity appeared by shotgun mass spectrometry proteomics. Laccase, dye-decolorizing peroxidases (DyP), lectins and proteins involved in reactive oxygen species (ROS) production and control were detected (in addition to UPO). Interestingly, the metabolism of complex sugars and nitrogen sources had a different activity at the beginning and end of the submerged fermentation.


Assuntos
Agrocybe , Proteômica , Lacase
6.
Angew Chem Int Ed Engl ; 62(5): e202214759, 2023 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-36453718

RESUMO

A lyophilized preparation of an unspecific peroxygenase variant from Agrocybe aegerita (rAaeUPO-PaDa-I-H) is a highly effective catalyst for the oxygenation of a diverse range of N-heterocyclic compounds. Scalable biocatalytic oxygenations (27 preparative examples, ca. 100 mg scale) have been developed across a wide range of substrates, including alkyl pyridines, bicyclic N-heterocycles and indoles. H2 O2 is the only stoichiometric oxidant needed, without auxiliary electron transport proteins, which is key to the practicality of the method. Reaction outcomes can be altered depending on whether hydrogen peroxide was delivered by syringe pump or through in situ generation using an alcohol oxidase from Pichia pastoris (PpAOX) and methanol as a co-substrate. Good synthetic yields (up to 84 %), regioselectivity and enantioselectivity (up to 99 % ee) were observed in some cases, highlighting the promise of UPOs as practical, versatile and scalable oxygenation biocatalysts.


Assuntos
Oxigenases de Função Mista , Oxigenases de Função Mista/metabolismo , Catálise , Biocatálise
7.
Front Chem ; 10: 950149, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36046724

RESUMO

Chiral allylic oxidized products play an increasingly important role in the pharmaceutical, agrochemical, and pharmaceutical industries. Biocatalytic C-H oxyfunctionalization to synthesize allylic oxidized products has attracted great attention in recent years, with the ability to simplify synthetic approaches toward complex compounds. As a result, scientists have found some new enzymes and mutants through techniques of gene mining and enzyme-directed evolution in recent years. This review summarizes the recent developments in biocatalytic selective oxidation of olefins by different kinds of biocatalysts.

8.
Antioxidants (Basel) ; 11(5)2022 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-35624755

RESUMO

Unspecific peroxygenases (UPOs) are extracellular fungal enzymes of biotechnological interest as self-sufficient (and more stable) counterparts of cytochrome P450 monooxygenases, the latter being present in most living cells. Expression hosts and structural information are crucial for exploiting UPO diversity (over eight thousand UPO-type genes were identified in sequenced genomes) in target reactions of industrial interest. However, while many thousands of entries in the Protein Data Bank include molecular coordinates of P450 enzymes, only 19 entries correspond to UPO enzymes, and UPO structures from only two species (Agrocybe aegerita and Hypoxylon sp.) have been published to date. In the present study, two UPOs from the basidiomycete Marasmius rotula (rMroUPO) and the ascomycete Collariella virescens (rCviUPO) were crystallized after sequence optimization and Escherichia coli expression as active soluble enzymes. Crystals of rMroUPO and rCviUPO were obtained at sufficiently high resolution (1.45 and 1.95 Å, respectively) and the corresponding structures were solved by molecular replacement. The crystal structures of the two enzymes (and two mutated variants) showed dimeric proteins. Complementary biophysical and molecular biology studies unveiled the diverse structural bases of the dimeric nature of the two enzymes. Intermolecular disulfide bridge and parallel association between two α-helices, among other interactions, were identified at the dimer interfaces. Interestingly, one of the rCviUPO variants incorporated the ability to produce fatty acid diepoxides-reactive compounds with valuable cross-linking capabilities-due to removal of the enzyme C-terminal tail located near the entrance of the heme access channel. In conclusion, different dimeric arrangements could be described in (short) UPO crystal structures.

9.
Antioxidants (Basel) ; 11(5)2022 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-35624779

RESUMO

Vegetable oils are valuable renewable resources for the production of bio-based chemicals and intermediates, including reactive epoxides of industrial interest. Enzymes are an environmentally friendly alternative to chemical catalysis in oxygenation reactions, epoxidation included, with the added advantage of their potential selectivity. The unspecific peroxygenase of Collariella virescens is only available as a recombinant enzyme (rCviUPO), which is produced in Escherichia coli for protein engineering and analytical-scale optimization of plant lipid oxygenation. Engineering the active site of rCviUPO (by substituting one, two, or up to six residues of its access channel by alanines) improved the epoxidation of individual 18-C unsaturated fatty acids and hydrolyzed sunflower oil. The double mutation at the heme channel (F88A/T158A) enhanced epoxidation of polyunsaturated linoleic and α−linolenic acids, with the desired diepoxides representing > 80% of the products (after 99% substrate conversion). More interestingly, process optimization increased (by 100-fold) the hydrolyzate concentration, with up to 85% epoxidation yield, after 1 h of reaction time with the above double variant. Under these conditions, oleic acid monoepoxide and linoleic acid diepoxide are the main products from the sunflower oil hydrolyzate.

10.
Antioxidants (Basel) ; 11(1)2022 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-35052667

RESUMO

Unspecific peroxygenases (UPOs), whose sequences can be found in the genomes of thousands of filamentous fungi, many yeasts and certain fungus-like protists, are fascinating biocatalysts that transfer peroxide-borne oxygen (from H2O2 or R-OOH) with high efficiency to a wide range of organic substrates, including less or unactivated carbons and heteroatoms. A twice-proline-flanked cysteine (PCP motif) typically ligates the heme that forms the heart of the active site of UPOs and enables various types of relevant oxygenation reactions (hydroxylation, epoxidation, subsequent dealkylations, deacylation, or aromatization) together with less specific one-electron oxidations (e.g., phenoxy radical formation). In consequence, the substrate portfolio of a UPO enzyme always combines prototypical monooxygenase and peroxidase activities. Here, we briefly review nearly 20 years of peroxygenase research, considering basic mechanistic, molecular, phylogenetic, and biotechnological aspects.

11.
Front Bioeng Biotechnol ; 9: 705630, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34307325

RESUMO

In 2004, the fungal heme-thiolate enzyme subfamily of unspecific peroxygenases (UPOs) was first described in the basidiomycete Agrocybe aegerita. As UPOs naturally catalyze a broad range of oxidative transformations by using hydrogen peroxide as electron acceptor and thus possess a great application potential, they have been extensively studied in recent years. However, despite their versatility to catalyze challenging selective oxyfunctionalizations, the availability of UPOs for potential biotechnological applications is restricted. Particularly limiting are the identification of novel natural biocatalysts, their production, and the description of their properties. It is hence of great interest to further characterize the enzyme subfamily as well as to identify promising new candidates. Therefore, this review provides an overview of the state of the art in identification, expression, and screening approaches of fungal UPOs, challenges associated with current protein production and screening strategies, as well as potential solutions and opportunities.

12.
ACS Synth Biol ; 10(6): 1360-1372, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34075757

RESUMO

Fungal peroxygenases (UPOs) have emerged as oxyfunctionalization catalysts of tremendous interest in recent years. However, their widespread use in the field of biocatalysis is still hampered by their challenging heterologous production, substantially limiting the panel of accessible enzymes for investigation and enzyme engineering. Building upon previous work on UPO production in yeast, we have developed a combined promoter and signal peptide shuffling system for episomal high throughput UPO production in the industrially relevant, methylotrophic yeast Pichia pastoris. Eleven endogenous and orthologous promoters were shuffled with a diverse set of 17 signal peptides. Three previously described UPOs were selected as first test set, leading to the identification of beneficial promoter/signal peptide combinations for protein production. We applied the system then successfully to produce two novel UPOs: MfeUPO from Myceliophthora fergusii and MhiUPO from Myceliophthora hinnulea. To demonstrate the feasibility of the developed system to other enzyme classes, it was applied for the industrially relevant lipase CalB and the laccase Mrl2. In total, approximately 3200 transformants of eight diverse enzymes were screened and the best promoter/signal peptide combinations studied at various cofeeding, derepression, and induction conditions. High volumetric production titers were achieved by subsequent creation of stable integration lines and harnessing orthologous promoters from Hansenula polymorpha. In most cases promising yields were also achieved without the addition of methanol under derepressed conditions. To foster the use of the episomal high throughput promoter/signal peptide Pichia pastoris system, we made all plasmids available through Addgene.


Assuntos
Proteínas Fúngicas/biossíntese , Oxigenases de Função Mista/biossíntese , Pichia/enzimologia , Plasmídeos/genética , Regiões Promotoras Genéticas/genética , Engenharia de Proteínas/métodos , Sinais Direcionadores de Proteínas/genética , Saccharomycetales/enzimologia , Estudos de Viabilidade , Proteínas Fúngicas/genética , Ensaios de Triagem em Larga Escala/métodos , Microrganismos Geneticamente Modificados , Oxigenases de Função Mista/genética , Pichia/genética , Proteínas Recombinantes/biossíntese , Saccharomycetales/genética , Sordariales/enzimologia , Sordariales/genética
13.
Biotechnol Adv ; 51: 107703, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-33545329

RESUMO

Selective oxyfunctionalizations of aliphatic compounds are difficult chemical reactions, where enzymes can play an important role due to their stereo- and regio-selectivity and operation under mild reaction conditions. P450 monooxygenases are well-known biocatalysts that mediate oxyfunctionalization reactions in different living organisms (from bacteria to humans). Unspecific peroxygenases (UPOs), discovered in fungi, have arisen as "dream biocatalysts" of great biotechnological interest because they catalyze the oxyfunctionalization of aliphatic and aromatic compounds, avoiding the necessity of expensive cofactors and regeneration systems, and only depending on H2O2 for their catalysis. Here, we summarize recent advances in aliphatic oxyfunctionalization reactions by UPOs, as well as the molecular determinants of the enzyme structures responsible for their activities, emphasizing the differences found between well-known P450s and the novel fungal peroxygenases.


Assuntos
Peróxido de Hidrogênio , Oxigenases de Função Mista , Catálise , Fungos , Humanos
14.
J Agric Food Chem ; 68(19): 5375-5383, 2020 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-32292026

RESUMO

Apocarotenoids are among the most highly valued fragrance constituents, being also appreciated as synthetic building blocks. This work shows the ability of unspecific peroxygenases (UPOs, EC1.11.2.1) from several fungi, some of them being described recently, to catalyze the oxyfunctionalization of α- and ß-ionones and α- and ß-damascones. Enzymatic reactions yielded oxygenated products such as hydroxy, oxo, carboxy, and epoxy derivatives that are interesting compounds for the flavor and fragrance and pharmaceutical industries. Although variable regioselectivity was observed depending on the substrate and enzyme, oxygenation was preferentially produced at the allylic position in the ring, being especially evident in the reaction with α-ionone, forming 3-hydroxy-α-ionone and/or 3-oxo-α-ionone. Noteworthy were the reactions with damascones, in the course of which some UPOs oxygenated the terminal position of the side chain, forming oxygenated derivatives (i.e., the corresponding alcohol, aldehyde, and carboxylic acid) at C-10, which were predominant in the Agrocybe aegerita UPO reactions, and first reported here.


Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fungos/enzimologia , Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Norisoprenoides/metabolismo , Catálise , Fungos/química , Fungos/genética , Oxigenases de Função Mista/genética , Norisoprenoides/química , Especificidade por Substrato
15.
Appl Environ Microbiol ; 86(7)2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31980430

RESUMO

Unspecific peroxygenases (UPOs) constitute a new family of fungal heme-thiolate enzymes in which there is high biotechnological interest. Although several thousand genes encoding hypothetical UPO-type proteins have been identified in sequenced fungal genomes and other databases, only a few UPO enzymes have been experimentally characterized to date. Therefore, gene screening and heterologous expression from genetic databases are a priority in the search for ad hoc UPOs for oxyfunctionalization reactions of interest. Very recently, Escherichia coli production of a previously described basidiomycete UPO (as a soluble and active enzyme) has been reported. Here, we explored this convenient heterologous expression system to obtain the protein products from available putative UPO genes. In this way, two UPOs from the ascomycetes Collariella virescens (syn., Chaetomium virescens) and Daldinia caldariorum were successfully obtained, purified, and characterized. Comparison of their kinetic constants for oxidation of model substrates revealed 10- to 20-fold-higher catalytic efficiency of the latter enzyme in oxidizing simple aromatic compounds (such as veratryl alcohol, naphthalene, and benzyl alcohol). Homology molecular models of these enzymes showed three conserved and two differing residues in the distal side of the heme (the latter representing two different positions of a phenylalanine residue). Interestingly, replacement of the C. virescens UPO Phe88 by the homologous residue in the D. caldariorum UPO resulted in an F88L variant with 5- to 21-fold-higher efficiency in oxidizing these aromatic compounds.IMPORTANCE UPOs catalyze regio- and stereoselective oxygenations of both aromatic and aliphatic compounds. Similar reactions were previously described for cytochrome P450 monooxygenases, but UPOs have the noteworthy biotechnological advantage of being stable enzymes requiring only H2O2 to be activated. Both characteristics are related to the extracellular nature of UPOs as secreted proteins. In the present study, the limited repertoire of UPO enzymes available for organic synthesis and other applications is expanded with the description of two new ascomycete UPOs obtained by Escherichia coli expression of the corresponding genes as soluble and active enzymes. Moreover, directed mutagenesis in E. coli, together with enzyme molecular modeling, provided relevant structure-function information on aromatic substrate oxidation by these two new biocatalysts.


Assuntos
Chaetomium/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Oxigenases de Função Mista/genética , Xylariales/genética , Chaetomium/metabolismo , Escherichia coli/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Microrganismos Geneticamente Modificados/genética , Microrganismos Geneticamente Modificados/metabolismo , Oxigenases de Função Mista/metabolismo , Xylariales/metabolismo
16.
Microorganisms ; 6(1)2018 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-29315223

RESUMO

Enzymatic oxidation of 5-hydroxymethylfurfural (HMF) and its oxidized derivatives was studied using three fungal enzymes: wild-type aryl alcohol oxidase (AAO) from three fungal species, wild-type peroxygenase from Agrocybe aegerita (AaeUPO), and recombinant galactose oxidase (GAO). The effect of pH on different reaction steps was evaluated and apparent kinetic data (Michaelis-Menten constants, turnover numbers, specific constants) were calculated for different enzyme-substrate ratios and enzyme combinations. Finally, the target product, 2,5-furandicarboxylic acid (FDCA), was prepared in a multi-enzyme cascade reaction combining three fungal oxidoreductases at micro-scale. Furthermore, an oxidase-like reaction is proposed for heme-containing peroxidases, such as UPO, horseradish peroxidase, or catalase, causing the conversion of 5-formyl-2-furancarboxylic acid into FDCA in the absence of exogenous hydrogen peroxide.

17.
Sci Total Environ ; 610-611: 1210-1221, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-28851142

RESUMO

Willow trees are generally valuable bioindicators. Flooding during the rainy season affects the life history of Salix subfragilis, a voluntary willow species that grows in submerged wetlands, as it causes senescence of old leaves and emergence of new shoots. In this study, we assessed the potential of S. subfragilis, a Northeast Asian willow species, as a bioindicator. We also investigated seasonal variations in the concentrations of Cd, Pb, Mn, Cu, and Zn in the leaves and stems of willows growing in submerged wetlands containing low concentrations of metals, over the growing season after flooding caused by heavy rain. A significant difference in the concentrations of metals in the sediments was reflected in the leaves for Cd and Pb and in the stems for Cd, Pb, Mn, and Zn. Higher bioconcentration factors in the samples collected in spring suggest that the best monitoring time for S. subfragilis is from April to May. Whereas the concentrations of Cd, Pb, and Mn appeared to increase, especially in the leaves, the concentrations of Cu and Zn in the leaves and stems decreased toward the end of the growing season, regardless of the emergence period. Higher concentrations of Cd, Pb, and Zn were generally noted for the stems than for the leaves whereas Mn and Cu were consistently present at higher concentrations in the leaves than in the stems. These results suggest that S. subfragilis can be a useful bioindicator for Pb, as well as for Cd and Zn. Particularly, samples of new stems collected in spring could provide a good index for environmental pollution.


Assuntos
Monitoramento Ambiental , Metais Pesados/análise , Salix/química , Poluentes Químicos da Água/análise , Áreas Alagadas , Folhas de Planta/química , Caules de Planta/química , Estações do Ano
18.
Front Microbiol ; 8: 786, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28529504

RESUMO

In January 2014, a viral hemagglutinating agent named UPO216 was isolated from fecal droppings of wild birds at the UPO wetland in South Korea during an avian influenza surveillance program. Electron microscopy identified the UPO216 virus as an avian paramyxovirus (APMV). Pathogenicity tests and molecular pathotyping revealed that the virus was avirulent in chickens. The UPO216 virus was assigned to a serological group antigenically distinct from known serotypes of APMV (-1, -2, -3, -4, -6, -7, -8, and -9) by hemagglutination inhibition test, despite showing weak cross-reactivity with APMV-1 and APMV-9. The UPO216 virus RNA genome is 15,180 nucleotides (nts) in length, encodes 3'-N-P(V/W)-M-F-HN-L-5' in that order, and shows unique genetic characteristics in terms of genomic composition and evolutionary divergence (0.43 or greater from known serotypes of APMV). Phylogenetic analysis revealed that the UPO216 occupies a branch separate from APMV-1, -9, -12, and -13. Serologic surveillance of wild birds (n = 880; 15 species, five Orders) detected UPO216-reactive antibodies in 4% (20/494) of serum samples taken from five species of wild duck belonging to the Order Anseriformes. In particular, UPO216-specific antibodies showing no cross-reaction with other serotypes of APMV were detected in four species: Eurasian teal (1/36), European wigeon (1/73), mallard (4/139), and Spot-Billed duck (1/137). These results indicate that the UPO216 virus has antigenically and genetically unique characteristics distinct from known serotypes of APMV and likely has been circulating widely in wild duck species of the Order Anseriformes. Thus, we propose the UPO216 isolate as a prototype strain of a novel APMV serotype (putative APMV-15).

19.
Vínculo (São Paulo, Online) ; 13(2): 13-23, 2016.
Artigo em Português | LILACS, Index Psicologia - Periódicos | ID: biblio-846414

RESUMO

Em um Brasil no qual a população idosa será maioria nas próximas décadas, urge a criação de medidas que atendam aos aspectos subjetivos da velhice, desatrelando-a de sentidos como decrepitude, doença e exclusão. O grupo da Oficina da Linguagem, que se reúne semanalmente na cidade de Curitiba para narrar oralmente, ler e escrever histórias de vida desponta neste cenário, como uma iniciativa de promover mudanças na relação de idosos com os seus processos de envelhecimento e com a linguagem. Este artigo objetiva analisar os efeitos que as atividades dialógicas, desenvolvidas no grupo operativo da Oficina da Linguagem, exerceram na relação de idosos com a linguagem e com a velhice. Trata-se de um estudo de caso de caráter qualitativo, que se pauta na perspectiva dialógica de Mikhail Bakhtin e nos pressupostos de grupo operativo de Pichon-Rivière. Para a coleta de dados, foi aplicada uma entrevista semiestruturada junto a treze idosos que compuseram o grupo dialógico-operativo, no ano de 2012. Tais enunciados anunciam que práticas grupais perpassadas por atividades linguístico-discursivas promovem mudanças nas relações dos idosos com a maneira que se relacionarem com a própria velhice e com o outro, por meio da linguagem. Tais atividades auferiu-lhes autoestima, segurança, lugar de autoria que os instiga a sustentarem suas vozes nas mais heterogêneas cenas sociais.


In a Brazil in which the elderly population will be majority in the next decades, it is urgent to create measures that attend to the subjective aspects of old age, disregarding it of meanings such as decrepitude, disease and exclusion. The Language Workshop group, which meets weekly in the city of Curitiba to orally narrate, read and write life stories, emerges in this scenario as an initiative to promote changes in the relationship of the elderly with their aging processes and with language. This article aims to analyze the effects that the dialogic activities, developed in the operative group of the Language Workshop, exercised in the relation of the elderly with the language and with the old age. This is a case study of a qualitative nature, based on the dialogical perspective of Mikhail Bakhtin and the assumptions of the Pichon-Rivière operative group. For the data collection, a semi-structured interview was applied to thirteen elderly people who composed the dialogical-operative group, in the year 2012. Such statements announce that group practices permeated by linguistic-discursive activities promote changes in the relationships of the elderly with the way they relate to their own old age and with or the other, through the language. Such activities have earned them self-esteem, security, a place of authorship that instigates them to support their voices in the most heterogeneous social scenes.


En un Brasil donde la población de edad avanzada es más en las próximas décadas, es urgente establecer medidas que cumplan con los aspectos subjetivos de la vejez, desatando sus sentidos como la decrepitud, la enfermedad y la exclusión. El grupo Taller de Lengua, que se reúne semanalmente en la ciudad de Curitiba para narrar oral, lectura y escritura de historias de vida que surge en este escenario, como una iniciativa para promover cambios en la relación de lactancia con sus procesos y métodos de envejecimiento idioma. Este artículo tiene como objetivo analizar los efectos que las actividades desarrolladas en el dialógicas operativo Idioma taller de grupo ejercida en relación con el lenguaje y personas de edad avanzada con la vejez. Se trata de un estudio de caso cualitativo, que es guiada en la perspectiva dialógica de Mijaíl Bajtín y el grupo operativo de supuestos Pichon-Rivière. Para recopilar los datos, entrevistas semi-estructuradas con trece años de edad que compone el grupo dialógica-operatorio se aplicó en el año 2012. Dichas declaraciones anuncian que el grupo ocupado se practica actividades lingüístico-discursivas promueven cambios en las relaciones de las personas mayores con el camino que están relacionados con la edad en sí y, o bien por medio de la palabra. Tales actividades se ganaron la autoestima, la seguridad, el lugar de autoría que instiga a apoyar sus voces en las escenas sociales más heterogéneos.


Assuntos
Humanos , Masculino , Feminino , Idoso , Envelhecimento , Comunicação , Relações Interpessoais , Grupos Etários
20.
Bioorg Med Chem ; 23(15): 4324-4332, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26142319

RESUMO

Unspecific peroxygenases (UPOs, EC 1.11.2.1) have proved to be stable oxygen-transferring biocatalysts for H2O2-dependent transformation of pharmaceuticals. We have applied UPOs in a drug development program and consider the enzymatic approach in parallel to a conventional chemical synthesis of the human metabolites of the bile acid reabsorption inhibitor SAR548304. Chemical preparation of N,N-di-desmethyl metabolite was realized by a seven-step synthesis starting from a late precursor of SAR548304 and included among others palladium catalysis and laborious chromatographic purification with an overall yield of 27%. The enzymatic approach revealed that the UPO of Marasmius rotula is particularly suitable for selective N-dealkylation of the drug and enabled us to prepare both human metabolites via one-pot conversion with an overall yield of 66% N,N-di-desmethyl metabolite and 49% of N-mono-desmethylated compound in two separated kinetic-controlled reactions.


Assuntos
Glucosamina/análogos & derivados , Compostos Heterocíclicos/química , Marasmius/enzimologia , Oxigenases de Função Mista/metabolismo , Compostos de Fenilureia/síntese química , Catálise , Glucosamina/síntese química , Glucosamina/química , Glucosamina/metabolismo , Compostos Heterocíclicos/síntese química , Humanos , Peróxido de Hidrogênio/química , Paládio/química , Compostos de Fenilureia/química , Compostos de Fenilureia/metabolismo
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