Estudio cristalográfico de los mecanismos de especificidad y diseño molecular de glicosidasas para la producción de oligosacáridos prebióticos y derivados bioactivos

Según diversos estudios epidemiológicos, la alimentación juega un papel determinante en la salud humana. Se ha demostrado que la dieta tiene un efecto directo sobre la composición y el equilibrio de la microbiota intestinal y, ésta, sobre la salud del individuo. Por ello, se buscan nuevos alimentos “funcionales”, como los oligosacáridos prebióticos, que son capaces de estimular el crecimiento de bacterias beneficiosas. Por otra parte, la glicosilación de distintos compuestos químicos mejora sus propiedades y su biodisponibilidad. Estos nuevos compuestos bioactivos aún no están tan desarrollados como los prebióticos, pero tienen una gran proyección de futuro en la industria farmacéutica. La investigación sobre la biosíntesis de estos productos conlleva tanto el estudio de nuevos compuestos con mejores propiedades funcionales como la búsqueda de formas más eficaces de producirlos. En este aspecto, la Biotecnología Enzimática se ha impuesto frente a otras metodologías de química tradicional gracias a su excelente regio-­‐, quimio-­‐ y estéreo-­‐especificidad. Y para profundizar en esta metodología, la Cristalografía de rayos X genera la información estructural básica para el diseño racional en la ingeniería de proteínas. Por todo ello, esta tesis se ha centrado en el estudio cristalográfico de glicosil hidrolasas (GH) que son las enzimas capaces de metabolizar carbohidratos, los biocompuestos estructuralmente más diversos en la naturaleza. Estas enzimas, altamente específicas y eficientes, intervienen en multitud de procesos biológicos esenciales por lo que un conocimiento detallado de su función a nivel molecular es clave para entender dichos procesos y controlar numerosas enfermedades, además de permitir optimizar su aplicación biotecnológica. En este manuscrito, se presentan cuatro trabajos de investigación realizados con enzimas productoras de carbohidratos prebióticos del tipo FOS (fructooligosacáridos), IMOS (isomaltooligosacáridos) o β-­‐glucanos, además de explorar su capacidad para sintetizar otros compuestos bioactivos. El objetivo general de la investigación es avanzar en el esclarecimiento de los mecanismos de reconocimiento proteína-­‐carbohidrato que modulan la especificidad de glicosidasas, mediante la Cristalografía de proteínas. Además de las implicaciones fundamentales para entender los procesos en los que estas enzimas intervienen, este conocimiento estructural es la base fundamental para realizar la ingeniería molecular de enzimas. El fin último es producir oligosacáridos con propiedades prebióticas, diseñados específicamente para manipular la microbiota intestinal del individuo, lo que abre la puerta a una futura medicina preventiva personalizada..

Use of chitin and chitosan to produce new chitooligosaccharides by chitinase Chit42: enzymatic activity and structural basis of protein specificity

Background Chitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is related to their size and acetylation degree. Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-d-glucosamine (GlcNAc) units. Gene chit42 was previously characterized, and according to its sequence, the encoded protein included in the structural Glycoside Hydrolase family GH18. Results Chit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. Protein heterologously expressed showed similar biochemical properties to those expressed by the natural producer (42 kDa, optima pH 5.5–6.5 and 30–40 °C). In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Chit42 hydrolysed colloidal chitin at least 10-times more efficiently (defined by the kcat/Km ratio) than any of the assayed chitosan. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. Masses corresponding to (d-glucosamine)1–8-GlcNAc were identified from the hydrolysis of different substrates. Crystals from Chit42 were grown and the 3D structure determined at 1.8 Å resolution, showing the expected folding described for other GH18 chitinases, and a characteristic groove shaped substrate-binding site, able to accommodate at least six sugar units. Detailed structural analysis allows depicting the features of the Chit42 specificity, and explains the chemical nature of the partially acetylated molecules obtained from analysed substrates. Conclusions Chitinase Chit42 was expressed in a heterologous system to levels never before achieved. The enzyme produced small partially acetylated chitooligosaccharides, which have enormous biotechnological potential in medicine and food. Chit42 3D structure was characterized and analysed. Production and understanding of how the enzymes generating bioactive chito-oligomers work is essential for their biotechnological application, and paves the way for future work to take advantage of chitinolytic activities. Electronic supplementary material The online version of this article (10.1186/s12934-018-0895-x) contains supplementary material, which is available to authorized users.España, MINECO BIO2013‑48779‑ C4‑1/‑2/‑4, BIO2016‑76601‑ C3‑1/‑2/‑3

Molecular characterization and heterologous expression of a Xanthophyllomyces dendrorhous ¿-glucosidase with potential for prebiotics production

Abstract Basidiomycetous yeast Xanthophyllomyces dendrorhous expresses an α-glucosidase with strong transglycosylation activity producing prebiotic sugars such as panose and an unusual tetrasaccharides mixture including α–(1–6) bonds as major products, which makes it of biotechnological interest. Initial analysis pointed to a homodimeric protein of 60 kDa subunit as responsible for this activity. In this study, the gene Xd-AlphaGlu was characterized. The 4131-bp-long gene is interrupted by 13 short introns and encodes a protein of 990 amino acids (Xd-AlphaGlu). The N-terminal sequence of the previously detected 60 kDa protein resides in this larger protein at residues 583–602. Functionality of the gene was proved in Saccharomyces cerevisiae, which produced a protein of about 130 kDa containing Xd-AlphaGlu sequences. All properties of the heterologously expressed protein, including thermal and pH profiles, activity on different substrates, and ability to produce prebiotic sugars were similar to that of the α-glucosidase produced in X. dendrorhous. No activity was detected in S. cerevisiae containing exclusively the 1256-bp from gene Xd-AlphaGlu that would encode synthesis of the 60 kDa protein previously detected. Data were compatible with an active monomeric α-glucosidase of 990 amino acids and an inactive hydrolysis product of 60 kDa. Protein Xd-AlphaGlu contained most of the elements characteristic of α-glucosidases included in the glycoside hydrolases family GH31 and its structural model based on the homologous human maltase-lucoamylase was obtained. Remarkably, the Xd-AlphaGlu C-terminal domain presents an unusually long 115-residue insertion that could be involved in this enzyme’s activity against long-size substrates such as maltoheptaose and soluble starch.Spanish Ministry of Economy and Competitiveness supported this research. We thank Fundación Ramón Areces for the institutional grant to the Centro de Biología Molecular Severo OchoaPeer Reviewe

Overproduction of a Trichoderma harzianum chitinase and analysis of its biotechnological potential to produce chitooligosaccharides

Trabajo presentado en la 7ª ed. del congreso internacional "FEMS" organizado por la Sociedad Española de Microbiología y la Federación Europea de Sociedades Microbiológicas en el Centro de Convenciones Feria Valencia (Valencia, España) durante los días 9 al 13 de julio de 2017.BACKGROUNDS: Chitooligosaccharides (COS) are β-(1,4)-linked oligomers of N-acetyl-glucosamine (GlcNAc) and glucosamine (GlcN) formed by chemical or enzymatic hydrolysis of chitosan or chitin. The growing biotechnological interest of COS in fields such as food or health increases the demand of the producing enzymes as well as their characterization and functional improvement. | OBJETIVES: Express a chitinase of 42 kDa from Trichoderma harzianum in a heterologous system, obtain protein levels compatible with its crystallization for the future protein structural resolution and evaluate the ability of the recombinant protein to produce COS. | METHODS: The chitinase gene cDNA from T. harzianum was expressed in Pichia pastoris using a restriction-free cloning strategy, production of heterologous protein was analysed and escalated up to a 5 L fermenter level. Recombinant protein was purified and some crystals were obtained which allows undertake the protein structural resolution. Synthesis of oligosaccharides from different substrates were evaluated and optimized using the recombinant enzyme. HPAEC-PAD on a Dionex ICS3000 system and Mass Spectrometry were used in the reaction studies and product characterization. | CONCLUSIONS: A chitinase of 42 kDa from T. harzianum was overexpressed in P. pastoris, the recombinant protein was purified, characterized and crystallized for the protein structural resolution. Production of COS mediated by this enzyme was evaluated and some of the molecules formed were characterized.N

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3

ObjectivesCARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.MethodsIn total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC &gt; 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsIn total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).ConclusionThis study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3

Structural analysis of the reducing-end xylose-releasing exo-oligoxylanase Rex8A from Paenibacillus barcinonensis BP-23 deciphers its molecular specificity

13 pags., 6 figs., 2 tabs.Reducing-end xylose-releasing exo-oligoxylanases (Rex) are GH8 enzymes that depolymerize xylooligosaccharides complementing xylan degradation by endoxylanases in an exo manner. We have studied Paenibacillus barcinonensis Rex8A and showed the release of xylose from xylooligomers decorated with methylglucuronic acid (UXOS) or with arabinose (AXOS). This gives the enzyme a distinctive trait among known Rex, which show activity only on linear xylooligosaccharides. The structure of the enzyme has been solved by X-ray crystallography showing a (α/α) folding common to GH8 enzymes. Analysis of inactived Rex8A-E70A complexed with xylotetraose revealed the existence of at least four binding subsites in Rex8A, with the oligosaccharide occupying subsites −3 to +1. The enzyme shows an extended Leu320-His321-Pro322 loop, common to other Rex, which blocks the binding of longer substrates to positive subsites further than +1 and seems responsible for the lack or diminished activity of Rex enzymes on xylan. Mutants with smaller residues in this loop failed to increase Rex8A activity on the polymer. Analysis of the complexes with AXOS showed the accommodation of arabinose at subsite −2, which cannot be allocated at subsite −1. Arabinose substitutions at the xylose O2 or O3 are accommodated by hydrophobic interaction and seem tolerated rather than recognized by Rex8A. A strained binding of the branch is facilitated by the lack of direct polar interactions of the xylose occupying this subsite, its water-mediated links allowing some conformational flexibility of the sugar. The plasticity of Rex8A is a notable property of the enzyme for its application in xylan deconstruction and upgrading. Database: Structural data are available in PDB database under the accession numbers 6SRD (native form), 6TPP (E70A mutant in complex with EDO), 6TOW (E70A in complex with Xyl4), 6SUD (L320A mutant in complex with xylose), 6SHY (L320A/H321S double mutant in complex with EDO), 6TO0 (E70A in complex with AX3), and 6TRH (E70A in complex with AX4).This work was supported by grants from the Spanish Ministry of Economy and Competitiveness through throughgrants BIO2016-76601-C3-3-R, BIOFABCEL CTQ2017-84966-C2-2-R. We are grateful to the staff of the Syn-chrotron Radiation Sources at Alba (Barcelona, Spain)and ESRF (Grenoble, France) for providing accessand for technical assistance at BL13-XALOC and Massif beamlines, and to the ‘Xarxa de Referencia en Biotecnologia’ (XRB)

Fructosilación de polioles con una ß-fructofuranosidasa de Schwanniomyces occidentalis, una aproximación a las bases moleculares de la especificidad enzimática

Trabajo presentado en la 33ª ed. de la reunión científica regional "ZYMOMADRID" organizada por la Sra. Catedrática en Microbiología y Pasitología Dra. dña. María Molina Martín (Dpto. Microbiología II, Fac. Farmacia, UCM) en el Campus de Moncloa (UCM, Madrid, España) durante el día 18 de marzo de 2019.En la actualidad, la síntesis enzimática de productos específicos con un valor añadido está adquiriendo un mayor peso en la industria debido a su bajo coste y respeto hacia el medio ambiente. Por ello, la búsqueda de nuevas enzimas en levaduras no convencionales permite alternativas a los métodos químicos convencionales. La levadura Schwanniomyces occidentalis expresa una β-fructofuranosidasa (Ffase, EC 3.2.1.26) que, además de hidrolizar sacarosa, es capaz de transferir moléculas de fructosa al disacárido y generar pequeños fructooligosacáridos (FOS) con enlaces β-(2→6) y β-(2→1), básicamente 6-kestosa y 1-kestosa, respectivamente. La enzima puede fructosilar también distintos monosácaridos y compuestos hidroxilados, entre ellos polioles como el manitol y eritritol. La estructura 3D de la Ffase fue ya previamente caracterizada, se obtuvieron distintos complejos proteína-sustratos/productos y los determinantes estructurales implicados en la capacidad hidrolasa y transferasa de la proteína fueron también determinados. En este trabajo, se han analizado las bases moleculares de la selectividad del proceso de producción de fructosil-eritritol/manitol de Ffase. Para ello, se purificaron los correspondientes polioles fructosilados y se cristalizaron en complejo con la variante inactiva de la proteína Ffase-D50A, previamente expresada en Saccharomyces cerevisiae. El análisis cristalográfico de los complejos reveló que los residuos Q176 y Q228 de la proteína tenían un papel fundamental en el reconocimiento de manitol, y la posición N254 en el del eritritol. Se realizaron reacciones de fructosilación de polioles utilizando variantes mutantes de la Ffase en las que las posiciones referenciadas habían sido sustituidas por diferentes aminoácidos y se evaluaron los productos obtenidos utilizando técnicas cromatográficas (HPLC-ELSD). Los resultados verificaron el papel de estos residuos en el proceso de producción de polioles fructosilados, y en uno de los mutantes utilizados se logró mejorar la producción de fructosil-eritritol en un 20%.N

New insights into the molecular mechanism behind mannitol and erythritol fructosylation by β-fructofuranosidase from Schwanniomyces occidentalis

12 pags., 5 figs., 2 tabs.The β-fructofuranosidase from Schwanniomyces occidentalis (Ffase) is a useful biotechnological tool for the fructosylation of different acceptors to produce fructooligosaccharides (FOS) and fructo-conjugates. In this work, the structural determinants of Ffase involved in the transfructosylating reaction of the alditols mannitol and erythritol have been studied in detail. Complexes with fructosyl-erythritol or sucrose were analyzed by crystallography and the effect of mutational changes in positions Gln-176, Gln-228, and Asn-254 studied to explore their role in modulating this biocatalytic process. Interestingly, N254T variant enhanced the wild-type protein production of fructosyl-erythritol and FOS by ∼ 30% and 48%, respectively. Moreover, it produced neokestose, which represented ∼ 27% of total FOS, and yielded 31.8 g l blastose by using glucose as exclusive fructosyl-acceptor. Noteworthy, N254D and Q176E replacements turned the specificity of Ffase transferase activity towards the synthesis of the fructosylated polyols at the expense of FOS production, but without increasing the total reaction efficiency. The results presented here highlight the relevance of the pair Gln-228/Asn-254 for Ffase donor-sucrose binding and opens new windows of opportunity for optimizing the generation of fructosyl-derivatives by this enzyme enhancing its biotechnological applicability.This work was supported by the Spanish Ministries of Economy and Competitiveness (BIO2016- 76601-C3-1/-2/-3) and of Science and Innovation (PID2019-105838RB-C3-1/-2/-3) as well as Fundación Ramón Areces (XIX Call of Research Grants in Life and Materials Sciences). We appreciate Fundación Ramón Areces for providing an institutional grant to the Centro de Biología Molecular Severo Ochoa. D. Piedrabuena was a recipient of a doctoral fellowship from the Spanish Ministry of Education, Culture, and Sports (FPU014/01004). We thank the staf members of the Synchrotron Radiation Source at Barcelona (ALBA, Spain) for providing access and technical assistance at BL13-XALOC beamline

Structural features of Aspergillus niger β-galactosidase define its activity against glycoside linkages

β-Galactosidases are biotechnologically interesting enzymes that catalyze the hydrolysis or transgalactosylation of β-galactosides. Among them, the Aspergillus niger β-galactosidase (AnβGal) belongs to the glycoside hydrolase family 35 (GH35) and is widely used in the industry due to its high hydrolytic activity which is used to degrade lactose. We present here its three-dimensional structure in complex with different oligosaccharides, to illustrate the structural determinants of the broad specificity of the enzyme against different glycoside linkages. Remarkably, the residues Phe264, Tyr304, and Trp806 make a dynamic hydrophobic platform that accommodates the sugar at subsite +1 suggesting a main role on the recognition of structurally different substrates. Moreover, complexes with the trisaccharides show two potential subsites +2 depending on the substrate type. This feature and the peculiar shape of its wide cavity suggest that AnβGal might accommodate branched substrates from the complex net of polysaccharides composing the plant material in its natural environment. Relevant residues were selected and mutagenesis analyses were performed to evaluate their role in the catalytic performance and the hydrolase/transferase ratio of AnβGal. Thus, we generated mutants with improved transgalactosylation activity. In particular, the variant Y304F/Y355H/N357G/W806F displays a higher level of galacto-oligosaccharides production than the Aspergillus oryzae β-galactosidase, which is the preferred enzyme in the industry owing to its high transferase activity. Our results provide new knowledge on the determinants modulating specificity and the catalytic performance of fungal GH35 β-galactosidases. In turn, this fundamental background gives novel tools for the future improvement of these enzymes, which represent an interesting target for rational design. Database: Structural data are available in PDB database under the accession numbers 5IFP (native form), 5IHR (in complex with 6GalGlu), 5IFT (in complex with 3GalGlu), 5JUV (in complex with 6GalGal), 5MGC (in complex with 4GalLac), and 5MGD (in complex with 6GalLac).Peer Reviewe