Study of Thermotoga maritima ß-galactosidase: immobilization, engineering and phylogenetic analysis

La actividad de las lactasas fue descrita por Beijerink en 1889. El origen de dicha actividad era la ß-galactosidasa de la levadura Kluyveromyces lactis capaz de hidrolizar la lactosa en sus dos componentes, galactosa y glucosa. En la actualidad las ß-galactosidasas tienen dos aplicaciones principales, por un lado se utilizan para la obtención de productos libres en lactosa y por otro lado para la síntesis de galactooligosacáridos (GOS), a algunos de los cuales se les han asociado propiedades prebióticas. Esta doble capacidad de la enzima se da gracias al mecanismo de retención que comparte con otras proteínas del clan GH-A en la clasificación del CAZy.En este trabajo hemos explorado la posibilidad del uso de una ß-galactosidasa termoresistente (TmLac) procedente de una bacteria extremófila, Thermotoga maritima. Utilizando esta enzima como herramienta de estudio se han desarrollado tres objetivos distintos: 1- La inmovilización de la enzima a soportes para facilitar su uso industrial. Para ello se han utilizado dos aproximaciones distintas. Por un lado la inmovilización covalente a esferas magnéticas activadas con residuos epoxi. Por otro, la inmovilización a celulosa y quitina basada en el etiquetado de la TmLac con dominios de unión a carbohidratos (CBMs). La TmLac se ha logrado inmovilizar mediante ambos métodos permitiendo su reciclado. 2- Ingeniería enzimática para la mejora de las propiedades transglicosilantes. Mediante el modelado estructural de la TmLac se ha estudiado qué residuos de la proteína podrían ser determinantes en el proceso de transglicosilación. Mediante mutagénesis dirigida se han hecho un total de 12 substituciones de residuos del centro catalítico. En el mejor de los casos se ha conseguido duplicar la síntesis del GOS mayoritario producido por esta enzima, la ß-3’-galactosil-lactosa. 3- Estudio filogenético de la familia GH2 del CAZy. Algunas de las ß-galactosidasas mejor descritas y más utilizadas pertenecen a esta familia, al igual que la TmLac. Estudios prospectivos han demostrado que el extremo N-terminal de estas enzimas se encuentra muy conservado, pero existe una enorme variabilidad en el extremo C-terminal. Es por ello que mediante el uso de herramientas bioinformáticas se ha realizado un estudio detallado de la arquitectura de dominios, así como un estudio filogenético que podrían ayudar en un futuro a la búsqueda de nuevas enzimas candidatas para la síntesis de GOS

Structural Dissection of the Active Site of Thermotoga maritima β-Galactosidase Identifies Key Residues for Transglycosylating Activity

Manuscript of the article published in print 13 April 2016. The Supporting Information is available free of charge on the ACS Publications website at DOI:10.1021/acs.jafc.6b00222 .Glycoside hydrolases, specifically β-galactosidases, can be used to synthesize galacto-oligosaccharides (GOS) due to the transglycosylating (secondary) activity of these enzymes. Site-directed mutagenesis of a thermoresistant β-galactosidase from Thermotoga maritima has been carried out to study the structural basis of transgalactosylation and to obtain enzymatic variants with better performance for GOS biosynthesis. Rational design of mutations was based on homologous sequence analysis and structural modeling. Analysis of mutant enzymes indicated that residue W959, or an alternative aromatic residue at this position, is critical for the synthesis of β-3′-galactosyl-lactose, the major GOS obtained with the wild-type enzyme. Mutants W959A and W959C, but not W959F, showed an 80% reduced synthesis of this GOS. Other substitutions, N574S, N574A, and F571L, increased the synthesis of β-3′-galactosyl-lactose about 40%. Double mutants F571L/N574S and F571L/N574A showed an increase of about 2-fold.This work was funded by grant BIO2013-48779-C4-3-R, from Spain's 'Secretaría de Estado de Investigación, Desarrollo e Innovación'. D T-P was supported by a FPU fellowship from 'Ministerio de Economía y Competitividad'.Peer reviewe

Fixation of bioactive compounds to the cuticle of Artemia

Artemia is extensively used in aquaculture to feed early stages of cultured marine species. A problem associated with this practice is that Artemia fails to supply some essential nutrients. As a possible solution, we have devised a procedure to make Artemia a vehicle for exogenous nutrients and other bioactive compounds. It consists of the construction of chimeric proteins composed of a chitin-binding domain, which binds to the cuticle of Artemia, and a carrier domain that conveys a functional property. As confirmatory examples, we describe the successful fixation to Artemia's metanauplii of two hybrid proteins: a β-galactosidase from the thermophilic bacterium Thermotoga maritima and the jellyfish green fluorescent protein (GFP), both linked to the CBM2 chitin-binding domain from the hyperthermophilic archaeon Pyrococcus furiosus. Positive results of experiments carried out ex vivo and in vivo show the validity of this approach. The methodology used could become a general procedure for the attachment of different kinds of bioactive compounds, such as enzymes, hormones, antibiotics, etc., to the cuticle of Artemia as well as other arthropods. Statement of relevance: Our results overcome shortcomings of Artemia as a feedstock.En prensa2,04

Structural Basis of the Inhibition of GH1 β-glucosidases by Multivalent Pyrrolidine Iminosugars

The synthesis of multivalent pyrrolidine iminosugars via CuAAC click reaction between different pyrrolidine-azide derivatives and tri- or hexavalent alkynyl scaffolds is reported. The new multimeric compounds, together with the monomeric reference, were evaluated as inhibitors against two homologous GH1 β-glucosidases (BglA and BglB from Paenibacillus polymyxa). The multivalent inhibitors containing an aromatic moiety in the linker between the pyrrolidine and the scaffold inhibited the octameric BglA (µM range) but did not show affinity against the monomeric BglB, despite the similarity between the active site of both enzymes. A modest multivalent effect (rp/n = 12) was detected for the hexavalent inhibitor 12. Structural analysis of the complexes between the monomeric and the trimeric iminosugar inhibitors (4 and 10) and BglA showed the insertion of the inhibitors at the active site of BglA, confirming a competitive mode of inhibition as indicated by enzyme kinetics. Additionally, structural comparison of the BglA/4 complex with the reported BglB/2F-glucose complex illustrates the key determinants responsible for the inhibitory effect and explains the reasons of the inhibition of BglA and the no inhibition of BglB. Potential inhibition of other β-glucosidases with therapeutic relevance is discussed under the light of these observations.Ministerio de Economía y Competitividad CTQ2016-77270-R, BIO2016-76601-C3-3-R, AGL2016-75245-RJunta de Andalucía FQM-345Fondazione CR Firenze 2016/084

Use of a Novel Extremophilic Xylanase for an Environmentally Friendly Industrial Bleaching of Kraft Pulps

Xylanases can boost pulp bleachability in Elemental Chlorine Free (ECF) processes, but their industrial implementation for producing bleached kraft pulps is not straightforward. It requires enzymes to be active and stable at the extreme conditions of alkalinity and high temperature typical of this industrial process; most commercial enzymes are unable to withstand these conditions. In this work, a novel highly thermo and alkaline-tolerant xylanase from Pseudothermotoga thermarum was overproduced in E. coli and tested as a bleaching booster of hardwood kraft pulps to save chlorine dioxide (ClO2) during ECF bleaching. The extremozyme-stage (EXZ) was carried out at 90 °C and pH 10.5 and optimised at lab scale on an industrial oxygen-delignified eucalyptus pulp, enabling us to save 15% ClO2 to reach the mill brightness, and with no detrimental effect on paper properties. Then, the EXZ-assisted bleaching sequence was validated at pilot scale under industrial conditions, achieving 25% ClO2 savings and reducing the generation of organochlorinated compounds (AOX) by 18%, while maintaining pulp quality and papermaking properties. Technology reproducibility was confirmed with another industrial kraft pulp from a mix of hardwoods. The new enzymatic technology constitutes a realistic step towards environmentally friendly production of kraft pulps through industrial integration of biotechnology.This work was supported by WoodZymes project funded by the Bio-based Industries Joint Undertaking (BBI JU) under GA 792070. The BBI JU received support from the EU’s H2020 research and innovation programme and the Bio Based Industries Consortium.Peer reviewe

Extremozymes for wood-based building blocks: from pulp mill to board and insulation products – WoodZymes project

4 páginas.- 4 referencias.- Comunicación oral presentada en el 16th European Workshop on Lignocellulosics and Pulp (EWLP) Gothenburg, Sweden, June 28 – July 1, 2022Enzymes can substitute harsh and energy-demanding chemical treatments for production of bio-based building blocks and products from wood processing. However, their properties need to be adapted to the extreme operation conditions (such as high T and pH) commonly used by these industries. Here, we summarize the main results obtained during the WoodZymes European Project (www.woodzymes.eu), which aimed to provide tailor-made extremozymes and extremozyme-based processes never assayed before in wood biorefineries. Novel extremophilic enzymes active on kraft lignin (laccases) and xylan (xylanases) were developed and produced at pilot or industrial scales. The enzymatic fractionation of kraft lignins using the METNINTM lignin refining technology, and the extremozyme-aided delignification and bleaching of kraft pulps were demonstrated at pilot scale. The resulting lignin and hemicellulose derived compounds were chemically characterized and applied as components of phenol-(lignin)-formaldehyde resins for wood panels and of polyurethane foams, or as papermaking additives. The new extremozymes were also applied to improve some of the latter applications. The techno-economic and environmental assessment of the new materials and processes, developed in WoodZymes project, showed that extremozyme-based processes led to clear benefits in energy savings during the refining of pulp or wood fibres, enabled lower addition of harsh chemicals (e.g. ClO2 during pulp bleaching), and resulted in a lower carbon footprint of the new bio-based products by substitution of fossil-derived components.WoodZymes project was funded by the Bio-based Industries Joint Undertaking (BBI JU) under GA 792070. The BBI JU received support from the EU’s H2020 research and innovation programme and the Bio Based Industries ConsortiumN

Biogenmol

Fecha de consulta: 2016-05-05En este blog iré colocando, siempre que el tiempo me lo permita, cosas interesantes acerca de la biología, y sobre todo del campo de la biología molecular, bioquímica y la genética, así como poner a vuestra disposición los trabajos que vaya realizando a lo largo de mis estudios.N

Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase

Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme's thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme's specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described.This work was funded by grant BIO2013-48779-C4-3-R from Spain's "Secretaría de Estado de Investigación, Desarrollo e Innovación". NR was supported by a Marie Curie ITN fellowship from the European Union within the Project Leangreenfood. DTP was supported by a FPU fellowship from "Ministerio de Economía y Competitividad".USD 1,495 APC fee funded by the EC FP7 Post-Grant Open Access PilotPeer reviewe

In silico screening and experimental analysis of family GH11 xylanases for applications under conditions of alkaline pH and high temperature

Background: Xylanases are one of the most extensively used enzymes for biomass digestion. However, in many instances, their use is limited by poor performance under the conditions of pH and temperature required by the industry. Therefore, the search for xylanases able to function efficiently at alkaline pH and high temperature is an important objective for different processes that use lignocellulosic substrates, such as the production of paper pulp and biofuels. Results: A comprehensive in silico analysis of family GH11 sequences from the CAZY database allowed their phylogenetic classification in a radial cladogram in which sequences of known or presumptive thermophilic and alkalophilic xylanases appeared in three clusters. Eight sequences from these clusters were selected for experimental analysis. The coding DNA was synthesized, cloned and the enzymes were produced in E. coli. Some of these showed high xylanolytic activity at pH values > 8.0 and temperature > 80 °C. The best enzymes corresponding to sequences from Dictyoglomus thermophilum (Xyn5) and Thermobifida fusca (Xyn8). The addition of a carbohydrate-binding module (CBM9) to Xyn5 increased 4 times its activity at 90 °C and pH > 9.0. The combination of Xyn5 and Xyn8 was proved to be efficient for the saccharification of alkali pretreated rice straw, yielding xylose and xylooligosaccharides. Conclusions: This study provides a fruitful approach for the selection of enzymes with suitable properties from the information contained in extensive databases. We have characterized two xylanases able to hydrolyze xylan with high efficiency at pH > 8.0 and temperature > 80 °C.This work has been carried out as part of the European Project WOODZYMES, funded by the Bio Based Industries Joint Undertaking, under the European Union’s Horizon 2020 research and innovation program (Grant Agreement H2020-BBI-JU-792070)Peer reviewe

Enzyme xylanase ayant une thermostabilité et une stabilité alcaline extrêmes

The present invention relates to the use of an isolated polypeptide characterized in that it comprises at least 70% identity with the amino acid sequence SEQ ID NO: 1 or the nucleotide encoding thereof for the hydrolysis of xylan, wherein the hydrolysis reaction occurs at a temperature of at least 65 °C and at a pH of at least 8.0. This polypeptide shows extreme thermostability and alkaline stability which allow its use in different process such as paper making, fodder production, fermenting composting, improving dough handling, extraction of coffee, extraction of plant oils, extraction of starch, agriculture silage, clarification of juices and degumming of plant fiber sources.NoConsejo Superior de Investigaciones Científicas (CSIC)A1 Solicitud de patente con informe sobre el estado de la técnic