Study of fungal xylanases for the exploitation of lignocellulosic biomass

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Bioquímica y Biología Molecular, leída el 02-06-2017El xilano constituye la segunda mayor reserva de carbono de la biosfera, sólo precedido por la celulosa. Es un heteropolisacárido perteneciente al grupo de las hemicelulosas por lo que está presente en la mayoría de las principales fuentes de biomasa lignocelulósica. Estructuralmente, estos polímeros se caracterizan por tener una cadena principal de unidades de D-xilopiranosa unidas por enlaces β-1,4. Este esqueleto presenta frecuentes acetilaciones y está altamente ramificado, con cadenas laterales muy cortas, formadas por residuos de arabinosa o ácido glucurónico. La abundancia de cada uno de los sustituyentes depende en gran medida del tipo de biomasa vegetal. Debido a esta complejidad su hidrólisis necesita de la acción concertada de toda una batería de enzimas, de entre las cuales las endo-β-1,4-xilanasas y las β-xilosidasas desempañan un papel esencial. Las primeras hidrolizan el polisacárido atacando enlaces internos de la cadena principal, liberando como productos oligosacáridos con distinto grado de polimerización. Las β-xilosidasas son enzimas que completan la degradación, convirtiendo estos xilooligosacáridos (XOS) en xilosa. La importancia económica del aprovechamiento de este heteropolisacárido nace fundamentalmente de su gran abundancia y ha supuesto un fuerte impulso para la investigación sobre ambas enzimas xilanolíticas. La industria busca tanto la sacarificación del xilano, con vistas a la obtención de biocombustibles, como su conversión en productos de alto valor añadido. En este último campo, las endoxilanasas pueden aplicarse para la obtención de XOS, considerados actualmente prebióticos emergentes. En cuanto a las β-xilosidasas, aunque su papel más conocido es el hidrolítico, muchas presentan también la capacidad de transferir un residuo de xilosa a un compuesto aceptor, en una reacción denominada transxilosilación. De esta forma se podrían obtener glicósidos con propiedades bioactivas, abriendo un nuevo campo para la aplicación de estas enzimas...Xylan represents the second carbon reservoir in the biosphere, only preceded by cellulose. It is a heteropolysaccharide belonging to the group of hemicelluloses, therefore it is a part of most of the main sources of lignocellulosic biomass. Structurally, it is composed by a backbone of β-1,4-linked D-xylopyranosyl units, which is frequently acetylated and highly branched by short side chains of arabinose or glucuronic acid. The abundance of each of these substituents depends largely on the nature of the plant biomass. Due to its complexity, xylan hydrolysis requires the concerted action of multiple enzymes, among which two types of glycosidases, endo-β-1,4-xylanases and β-xylosidases, play the major roles. The former hydrolyze the polysaccharide by attacking internal links in the main chain, releasing oligosaccharides with different polymerization degrees. β-xylosidases end the process by converting these xylooligosaccharides (XOS) into xylose. The economical relevance of exploiting this heteropolysaccharide is based on its great abundance and has driven the research on both xylanolytic enzymes. Industry is interested both in xylan saccharification for obtaining biofuels, and in its conversion into high value-added products. Attending to the latter possibility, endoxylanases can be applied for producing XOS, which are currently considered emerging prebiotics. Regarding to β-xylosidases, many of these glycosidases display the capacity of transferring a xylosyl residue to an acceptor compound, in a reaction called transxylosylation. By this way bioactive glycosides could be obtained, opening a new field for the application of these catalysts. The xylanolytic enzymes are produced in nature mainly by bacteria and fungi for degradation of plant cell wall polysaccharides. Among these organisms, filamentous fungi are the ones which have aroused the greatest interest as producers of these enzymes. The reasons are the higher levels of xylanolytic activities displayed by fungi and the frequent secretion of the desired enzymes to the extracellular medium, which facilitates both the purification and direct use of fungal crudes for several applications...Depto. de Bioquímica y Biología MolecularFac. de Ciencias QuímicasTRUEunpu

Contribución al conocimiento del género "Anthyllis" L. (Fabaceae) en la Península Ibérica "A. plumosa sp. nov"

Se realiza un estudio morfológico, palinológico y cariológico de tres especies del género Anthyllis (sect. Oreanthyllis), describiéndose una nueva especie, A. plumosa E. Domínguez procedente de las arenas dolomíticas de la Sierra de Almijara (Málaga).In this paper a morphological, palynological and caryological study of three species of Anthyllis (sect. Oreanthyllis) is included. As a consecuence a new species A. plumosa E. Domínguez from the dolomitics sands of Sierra de Almijara (Málaga) is described

Bioactive compounds from leaf vegetables as preservatives

Trends toward a healthier diet are increasing attention to clean-label products. This has led to the search for new ingredients that avoid the use of chemical additives. Food industries are responding to these demands by incorporating natural preservatives into their products, which consumers perceive as healthy. Leafy vegetables would fit this strategy since they are common components of the diet and are associated with beneficial health effects. The objective of this chapter is to offer an overview of the large number of bioactive compounds (phenolic acids, flavonoids, anthocyanins, glucosinolates, and sulfur compounds) present in these plants, which would be responsible for their activity as potential preservatives. Its incorporation into food would improve the quality and extend the shelf life by reducing oxidative processes and inhibiting or retarding the microbial growth that occurs during processing and storage without reducing the organoleptic characteristics of the product.Axencia Galega de Innovación | Ref. IN607A2019/01CYTED | Ref. 119RT0568Ministerio de Ciencia e Innovación | Ref. IJC2020-043358-

Enzymatic fine-tuning for 2-(6-hydroxynaphthyl) β-d-xylopyranoside synthesis catalyzed by the recombinant β-xylosidase BxTW1 from Talaromyces amestolkiae

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License.-- et al.[Background]: Glycosides are compounds displaying crucial biological roles and plenty of applications. Traditionally, these molecules have been chemically obtained, but its efficient production is limited by the lack of regio- and stereo-selectivity of the chemical synthesis. As an interesting alternative, glycosidases are able to catalyze the formation of glycosides in a process considered green and highly selective. In this study, we report the expression and characterization of a fungal ß-xylosidase in Pichia pastoris. The transglycosylation potential of the enzyme was evaluated and its applicability in the synthesis of a selective anti-proliferative compound demonstrated. [Results]: The ß-xylosidase BxTW1 from the ascomycete fungus Talaromyces amestolkiae was cloned and expressed in Pichia pastoris GS115. The yeast secreted 8 U/mL of ß-xylosidase that was purified by a single step of cation-exchange chromatography. rBxTW1 in its active form is an N-glycosylated dimer of about 200 kDa. The enzyme was biochemically characterized displaying a K m and k cat against p-nitrophenyl-ß-d-xylopyranoside of 0.20 mM and 69.3 s¿1 respectively, and its maximal activity was achieved at pH 3 and 60 °C. The glycan component of rBxTW1 was also analyzed in order to interpret the observed loss of stability and maximum velocity when compared with the native enzyme. A rapid screening of aglycone specificity was performed, revealing a remarkable high number of potential transxylosylation acceptors for rBxTW1. Based on this analysis, the enzyme was successfully tested in the synthesis of 2-(6-hydroxynaphthyl) ß-d-xylopyranoside, a well-known selective anti-proliferative compound, enzymatically obtained for the first time. The application of response surface methodology, following a Box-Behnken design, enhanced this production by eightfold, fitting the reaction conditions into a multiparametric model. The naphthyl derivative was purified and its identity confirmed by NMR. [Conclusions]: A ß-xylosidase from T. amestolkiae was produced in P. pastoris and purified. The final yields were much higher than those attained for the native protein, although some loss of stability and maximum velocity was observed. rBxTW1 displayed remarkable acceptor versatility in transxylosylation, catalyzing the synthesis of a selective antiproliferative compound, 2-(6-hydroxynaphthyl) ß-d-xylopyranoside. These results evidence the interest of rBxTW1 for transxylosylation of relevant products with biotechnological interest.This work was carried out with funding from projects BIO2015-68387-R, RTC-2014-1777-3 and CTQ2015-64597-C2 from MINECO and S2013/MAE2972 from Comunidad de Madrid, as well as from the Natural Sciences and Engineering Research Council of Canada. M. Nieto-Domínguez thanks the MINECO for an FPU fellowship.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro

This work was funded by The Novo Nordisk Foundation grant to the Center for Biosustainability (NNF10CC1016517). P.I.N. was funded by grants from The Novo Nordisk Foundation (NNF20CC0035580, and LiFe, NNF18OC0034818), the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No. 814418 (SinFonia) and the Danish Council for Independent Research (SWEET, DFF-Research Project 8021-00039B). T.K. and M.N.D. were funded by fellowships from the European Union's Horizon 2020 research and innovation program under a Marie Skłodowska Curie project under grant agreement No. 713683 (COFUNDfellowsDTU).The fluorinase enzyme represents the only biological mechanism capable of forming stable C–F bonds characterized in nature thus far, offering a biotechnological route to the biosynthesis of value-added organofluorines. The fluorinase is known to operate in a hexameric form, but the consequence(s) of the oligomerization status on the enzyme activity and its catalytic properties remain largely unknown. In this work, this aspect was explored by rationally engineering trimeric fluorinase variants that retained the same catalytic rate as the wild-type enzyme. These results ruled out hexamerization as a requisite for the fluorination activity. The Michaelis constant (KM) for S-adenosyl-l-methionine, one of the substrates of the fluorinase, increased by two orders of magnitude upon hexamer disruption. Such a shift in S-adenosyl-l-methionine affinity points to a long-range effect of hexamerization on substrate binding – likely decreasing substrate dissociation and release from the active site. A practical application of trimeric fluorinase is illustrated by establishing in vitro fluorometabolite synthesis in a bacterial cell-free system.Publisher PDFPeer reviewe

Biochemical characterization and cloning of A Β-xylosidase from talaromyces amestolkiae

3 p.Peer reviewe