Examinando por Autor "Rial, Daniela V."
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Ítem Acceso Abierto Cloning and characterization of the Type I Baeyer–Villiger monooxygenase from Leptospira biflexa(Springer, 2017-04-27) Ceccoli, Romina Denis; Bianchi, Dario A.; Fink, Michael J.; Mihovilovic, Marko D.; Rial, Daniela V.Ítem Acceso Abierto Flavoprotein monooxygenases for oxidative biocatalysis: recombinant expression in microbial hosts and applications(Frontiers Media, 2014-02-06) Ceccoli, Romina Denis; Bianchi, Dario A.; Rial, Daniela V.External flavoprotein monooxygenases comprise a group of flavin-dependent oxidoreductases that catalyze the insertion of one atom of molecular oxygen into an organic substrate and the second atom is reduced to water. These enzymes are involved in a great number of metabolic pathways both in prokaryotes and eukaryotes. Flavoprotein monooxygenases have attracted the attention of researchers for several decades and the advent of recombinant DNA technology caused a great progress in the field. These enzymes are subjected to detailed biochemical and structural characterization and some of them are also regarded as appealing oxidative biocatalysts for the production of fine chemicals and valuable intermediates toward active pharmaceutical ingredients due to their high chemo-, stereo-, and regioselectivity. Here, we review the most representative reactions catalyzed both in vivo and in vitro by prototype flavoprotein monooxygenases, highlighting the strategies employed to produce them recombinantly, to enhance the yield of soluble proteins, and to improve cofactor regeneration in order to obtain versatile biocatalysts. Although we describe the most outstanding features of flavoprotein monooxygenases, we mainly focus on enzymes that were cloned, expressed and used for biocatalysis during the last years.Ítem Acceso Abierto Mutations increasing cofactor affinity, improve stability and activity of a Baeyer Villiger monooxygenase(American Chemical Society, 2022-09-13) Mansouri, Hamid R.; Gracia Carmona, Oriol; Jodlbauer, Julia; Schweiger, Lorenz; Fink, Michael J.; Breslmayr, Erik; Laurent, Christophe; Feroz, Saima; Goncalves, Leticia C. P.; Rial, Daniela V.; Mihovilovic, Marko D.; Bommarius, Andreas S.; Ludwig, Roland; Oostenbrink, Chris; Rudroff, Florian; https://orcid.org/0000-0001-6560-9106; https://orcid.org/0000-0002-9944-1396; https://orcid.org/0000-0002-9112-6981; https://orcid.org/0000-0003-4658-1675; https://orcid.org/0000-0002-5058-5874; https://orcid.org/0000-0002-4232-2556; https://orcid.org/0000-0002-6680-8200The typically low thermodynamic and kinetic stability of enzymes is a bottleneck for their application in industrial synthesis. Baeyer−Villiger monooxygenases, which oxidize ketones to lactones using aerial oxygen, among other activities, suffer particularly from these instabilities. Previous efforts in protein engineering have increased thermodynamic stability but at the price of decreased activity. Here, we solved this trade-off by introducing mutations in a cyclohexanone monooxygenase from Acinetobacter sp., guided by a combination of rational and structure-guided consensus approaches. We developed variants with improved activity (1.5- to 2.5-fold) and increased thermodynamic (+5 °C Tm) and kinetic stability (8-fold). Our analysis revealed a crucial position in the cofactor binding domain, responsible for an 11-fold increase in affinity to the flavin cofactor, and explained using MD simulations. This gain in affinity was compatible with other mutations. While our study focused on a particular model enzyme, previous studies indicate that these findings are plausibly applicable to other BVMOs, and possibly to other flavin-dependent monooxygenases. These new design principles can inform the development of industrially robust, flavin-dependent biocatalysts for various oxidations.Ítem Acceso Abierto Prospecting biotechnologically-relevant monooxygenases from cold sediment metagenomes: an In silico approach(MDPI, 2017-04-09) Musumeci, Matías A.; Lozada, Mariana; Rial, Daniela V.; Mac Cormack, Walter P.; Jansson, Janet K.; Sjöling, Sara; Carroll, JoLynn; Dionisi, Hebe