1,720,968 research outputs found
New microbial hydroxysteroid dehydrogenases and their synthetic application for the selective modification of bile acids.
Get PDFHydroxysteroid dehydrogenases (HSDHs), mainly obtained from bacterial sources, have been widely employed for the regio- and stereoselective oxidoreduction of the hydroxyl-keto groups of steroids, bile acids and their derivatives. In particular they might be suitable biocatalysts for the industrial synthesis of bile acids derivatives of pharmacological interest such as ursodeoxycholic acid (UDCA; 3α,7β-dihydroxy-5β-cholan-24-oic acid).
Aim of this work was i) to investigate a new enzymatic process for the synthesis of UDCA; ii) to develop new cofactor regeneration systems to be coupled to HSDHs-catalyzed reactions; iii) to clone and overexpress in E. coli new HSDHs suitable for the modification of bile acids up to an industrial level.
Specifically, the preparative-scale HSDHs-catalyzed one-pot enzymatic synthesis of 12-ketoursodeoxycholic acid (3,7-dihydroxy-12-oxo-5-cholanoic acid), a key intermediate for the synthesis of ursodeoxycholic acid, from cholic acid has been investigated. This goal has been achieved by alternating oxidative and reductive steps in a one-pot system and employing HSDHs with different cofactor specificity. To provide the necessary driving force to opposite reactions (i.e., oxidation and reduction) acting concurrently on different sites of the same substrate molecule, suitable cofactor regeneration systems were coupled to these reactions. However, due to a limited cofactor specificity of some of the enzymes used, an undesired reaction equilibrium was established resulting in by-products formation. This problem was overcome by uncoupling the oxidative and reductive biocatalysts (Paper I).
Moreover, a new laccase/mediator system for NAD(P)+ cofactor regeneration in HSDHs-catalyzed oxidations has been developed. This system has been successfully applied in aqueous, buffered reactions (space-time yield of 2.35 g L-1 h-1 ) and in biphasic systems (space-time yields of up to 27.47 g L-1 h-1), demonstrating to be very efficient, to have high stability, to tolerate solvents, and to be simple to employ (Paper II).
Finally, NADPH-dependent 7α- and 7β-hydroxysteroid dehydrogenases (7α-HSDH and 7β-HSDH) from Clostridium absonum were cloned and overexpressed in recombinant form in E. coli. The enzymes were further characterized from a functional and a kinetic point of view, demonstrating that both of them, in suitable bioconversion conditions, could be promising candidates for further applications in the epimerization reaction of bile acids at the C-7 position (Paper III)
Structural and biochemical insights into 7β-hydroxysteroid dehydrogenase stereoselectivity
No full textHydroxysteroid dehydrogenases are of great interest as biocatalysts for transformations involving steroid substrates. They feature a high degree of stereo-and regio-selectivity, acting on a defined atom with a specific configuration of the steroid nucleus. The crystal structure of 7 beta-hydroxysteroid dehydrogenase from Collinsella aerofaciens reveals a loop gating active-site accessibility, the bases of the specificity for NADP(+), and the general architecture of the steroid binding site. Comparison with 7 alpha-hydroxysteroid dehydrogenase provides a rationale for the opposite stereoselectivity. The presence of a C-terminal extension reshapes the substrate site of the beta-selective enzyme, possibly leading to an inverted orientation of the bound substrat
Two‐step Biocatalytic Synthesis of (1S)‐Nor(pseudo)ephedrine Catalyzed by Immobilized Enzymes
No full textNor(pseudo)ephedrines [N(P)Es] are naturally occurring compounds showing sympathomimetic activity that have many applications especially in the chemical industry and in the pharmaceutical field. Recently a two-step biocatalytic cascade for the preparation of (1S)-N(P)E was designed, consisting of a benzoin-type condensation catalyzed by the (S)-selective acetoin:dichlorophenolindophenol oxidoreductase (Ao : DCPIP OR) followed by a transamination mediated by either a (S)- or (R)-selective amine transaminase (ATA). In this study, we successfully immobilized both Ao : DCPIP OR and two ATAs of opposite enantioselectivity and used them in the biosynthetic cascade to N(P)Es. Immobilization yield, activity recovery, and stability of the immobilized enzymes, both after use (enzyme recycling) and storage (shelf-life), were assessed. Ao : DCPIP OR immobilized on glyoxyl-agarose exhibited remarkable stability under storage conditions throughout the 30-days monitoring period. Moreover, it was successfully reused in the synthesis of (S)-phenylacetylcarbinol [(S)-PAC] yielding high conversion (>94 %) and enantiomeric excess (>99 %). The (R)-selective At-ATA from Aspergillus terreus, immobilized on Eupergit® C, demonstrated a slightly higher storage stability when compared to the (S)-selective Sbv333-ATA from Streptomyces sp. Bv333 immobilized on the same carrier. Remarkably, both enzymes were effectively reused for ten reaction cycles in the synthesis of N(P)Es, starting from the enzymatically synthesized (S)-PAC, achieving complete conversions and excellent diastereoselectivity (>99 %)
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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