Procesos biocatalíticos para la producción de carbohidratos bioactivos: fructooligosacáridos y quitooligosacáridos

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 14-07-2017Esta tesis tiene embargado el acceso al texto completo hasta el 14-01-201

Síntesis enzimática de fructooligosacáridos estimuladores de la microbiota colónica

The control of the intestinal flora is one of the targets of the functional foods and nutraceuticals. A symbiotic equilibrium can be achieved by the intake of live microorganisms (probiotics) or by the so-called prebiotics (non-digested oligosaccharides). Prebiotics are selectively fermented by the human microbiota allowing specific changes and conferring benefits upon host well-being and health. Among prebiotics fructooligosaccharides (FOS) constitute one of the most established groups. Levansucrases (EC 2.4.1.10) are a family of enzymes that catalyse the transfer of the fructosyl moiety from sucrose to different acceptors such as: (1) sucrose –yielding FOS that can be further fructosylated forming levan a polymer with food and biomedical applications -; (2) water resulting in sucrose hydrolysis. In this work a levansucrase from Zymomonas mobilis was characterized and the reaction products using sucrose as substrate were analysed by High-Performance Anion-Exchange Chromatography (HPAEC-PAD). The number of FOS synthesized by the soluble enzyme was significantly higher compared with previous reports. In order to optimize the biocatalytic process the enzyme was further immobilized by entrapment in calcium alginate gel and the resulting beads were dehydrated to obtain DALGEEs (Dry ALGinate Entrapped Enzymes). Different immobilization strategies were studied to minimize enzyme loss (lixiviation) throughout the pores. The effect of enzyme immobilization on levansucrase behaviour was also analysed.El control de la microflora intestinal es uno de los objetivos de los alimentos funcionales y nutracéuticos. El equilibrio simbiótico puede lograrse mediante la ingesta de microorganismos vivos (probióticos) o de los denominados prebióticos (oligosacáridos no digeribles). Los prebióticos son fermentados selectivamente por la microbiota generando cambios específicos en su composición que producen un beneficio en la salud del hospedador. Entre los prebióticos, los fructooligosacáridos (FOS) constituyen uno de los grupos más importantes. Las levansacarasas (EC 2.4.1.10) son una familia de enzimas que catalizan la transferencia de un grupo fructosilo desde una sacarosa a diferentes aceptores, entre ellos, otra molécula de sacarosa, dando lugar a FOS sobre los que puede transferir otro grupo fructosilo para llegar a formar levano, un polímero con aplicaciones en alimentación y biomedicina. Si el grupo fructosilo se transfiere sobre una molécula de agua, da lugar a la hidrólisis de la sacarosa.  En este trabajo se caracterizó una levansacarasa de Z. mobilis  y los productos de reacción con sacarosa como sustrato se analizaron por cromatografía de intercambio aniónico con detector amperométrico de pulsos (HPAEC-PAD). Con objeto de optimizar el proceso biocatalítico, la enzima se inmovilizó por atrapamiento en geles de alginato cálcico y las esferas resultantes se deshidrataron para formar DALGEEs (Dry ALGinate Entrapped Enzymes). Se probaron diferentes estrategias de inmovilización para minimizar la pérdida de la enzima por los poros. El efecto de la inmovilización en el comportamiento de la levansacarasa fue analizado.

Continuous Packed Bed Reactor with Immobilized β-Galactosidase for Production of Galactooligosaccharides (GOS)

The β-galactosidase from Bacillus circulans was covalently attached to aldehyde-activated (glyoxal) agarose beads and assayed for the continuous production of galactooligosaccharides (GOS) in a packed-bed reactor (PBR). The immobilization was fast (1 h) and the activity of the resulting biocatalyst was 97.4 U/g measured with o-nitrophenyl-β-d-galactopyranoside (ONPG). The biocatalyst showed excellent operational stability in 14 successive 20 min reaction cycles at 45 °C in a batch reactor. A continuous process for GOS synthesis was operated for 213 h at 0.2 mL/min and 45 °C using 100 g/L of lactose as a feed solution. The efficiency of the PBR slightly decreased with time; however, the maximum GOS concentration (24.2 g/L) was obtained after 48 h of operation, which corresponded to 48.6% lactose conversion and thus to maximum transgalactosylation activity. HPAEC-PAD analysis showed that the two major GOS were the trisaccharide Gal-β(1→4)-Gal-β(1→4)-Glc and the tetrasaccharide Gal-β(1→4)-Gal-β(1→4)-Gal-β(1→4)-Glc. The PBR was also assessed in the production of GOS from milk as a feed solution. The stability of the bioreactor was satisfactory during the first 8 h of operation; after that, a decrease in the flow rate was observed, probably due to partial clogging of the column. This work represents a step forward in the continuous production of GOS employing fixed-bed reactors with immobilized β-galactosidases.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (BIO2013-48779-C4-1-R and BIO2016-76601-C3-1-R). We thank the support of COST-Action CM1303 on Systems Biocatalysis. P.S.-M. thanks the Spanish Ministry of Education for FPU Grant (FPU13/01185). We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Enzymatic synthesis and characterization of different families of chitooligosaccharides and their bioactive properties

Chitooligosaccharides (COS) are homo- or hetero-oligomers of D-glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc) that can be obtained by chitosan or chitin hydrolysis. Their enzymatic production is preferred over other methodologies (physical, chemical, etc.) due to the mild conditions required, the fewer amounts of waste and its efficiency to control product composition. By properly selecting the enzyme (chitinase, chitosanase or nonspecific enzymes) and the substrate properties (degree of deacetylation, molecular weight, etc.), it is possible to direct the synthesis towards any of the three COS types: fully acetylated (faCOS), partially acetylated (paCOS) and fully deacetylated (fdCOS). In this article, we review the main strategies to steer the COS production towards a specific group. The chemical characterization of COS by advanced techniques, e.g., high-performance anion-exchange chromatography with pulsed amperometric detection (HPAECPAD) and MALDI-TOF mass spectrometry, is critical for structure-function studies. The scaling of processes to synthesize specific COS mixtures is difficult due to the low solubility of chitin/chitosan, the heterogeneity of the reaction mixtures, and high amounts of salts. Enzyme immobilization can help to minimize such hurdles. The main bioactive properties of COS are herein reviewed. Finally, the anti-inflammatory activity of three COS mixtures was assayed in murine macrophages after stimulation with lipopolysaccharidesThis work was supported by grants from the EU EMFF-Blue Economy-2018 (FISH4FISH- 863697 project), the Spanish Ministry of Economy and Competitiveness (Grants BIO2016-76601- C3-1,2-R), the Spanish Ministry of Science and Innovation (Grants PID2019-105838RB-C31/C32), Fundación Ramón Areces (XIX Call of Research Grants in Life and Material Sciences) and by an institutional grant from Fundación Ramón Areces to the Centro de Biología Molecula

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

Regioselective synthesis of neo-erlose by the beta-fructofuranosidase from Xanthophyllomyces dendrorhous

The beta-fructofuranosidase from the yeast Xanthophyllomyces dendrorhous (Xd-INV) catalyzes the synthesis of neo-fructooligosaccharides (neo-FOS of the 6G-series), which contain a beta(2-6) linkage between a fructose and the glucosyl moiety of sucrose. In this work we demonstrate that the enzyme is also able to fructosylate other carbohydrates that contain glucose, in particular disaccharides (maltose, isomaltulose, isomaltose, trehalose) and higher oligosaccharides (maltotriose, raffinose, maltotetraose), but not monosaccharides (glucose, fructose, galactose). With maltose as acceptor, the reaction in the presence of Xd-INV proceeded with high regioselectivity; the product was purified and chemically characterized, and turned out to be 6’-O--fructosylmaltose (neo-erlose). Using 100 g/L sucrose as fructosyl donor and 300 g/L maltose as acceptor, the maximum concentration of neo-erlose was 38.3 g/L. Thus, novel hetero-fructooligosaccharides with potential applications in the functional food and pharmaceutical industries can be obtained with Xd-INV.Projects BIO2010-20508-C04-01 and BIO2010-20508-C04-04 from Spanish Ministry of Science and Innovation supported this research. We thank Fundación Ramon Areces for an institutional grant to the Centro de Biología Molecular Severo Ochoa.Peer 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

Tocilizumab in visual involvement of giant cell arteritis: a multicenter study of 471 patients

Background: Visual involvement is the most feared complication of giant cell arteritis (GCA). Information on the efficacy of tocilizumab (TCZ) for this complication is scarce and controversial. Objective: We assessed a wide series of GCA treated with TCZ, to evaluate its role in the prevention of new visual complications and its efficacy when this manifestation was already present before the initiation of TCZ. Design: This is an observational multicenter study of patients with GCA treated with TCZ. Methods: Patients were divided into two subgroups according to the presence or absence of visual involvement before TCZ onset. Visual manifestations were classified into the following categories: transient visual loss (TVL), permanent visual loss (PVL), diplopia, and blurred vision. Results: Four hundred seventy-one GCA patients (mean age, 74 +/- 9 years) were treated with TCZ. Visual manifestations were observed in 122 cases (26%), of which 81 were present at TCZ onset: PVL (n = 60; unilateral/bilateral: 48/12), TVL (n = 17; unilateral/bilateral: 11/6), diplopia (n = 2), and blurred vision (n = 2). None of the patients without previous visual involvement or with TVL had new episodes after initiation of TCZ, while only 11 out of 60 (18%) patients with PVL experienced some improvement. The two patients with diplopia and one of the two patients with blurred vision improved. Conclusion: TCZ may have a protective effect against the development of visual complications or new episodes of TVL in GCA. However, once PVL was established, only a few patients improved

Continuous production of chitooligosaccharides by an immobilized enzyme in a dual-reactor system

[EN] A chitosanolytic activity found in a commercial α-amylase from Bacillus amylolyquefaciens (BAN) was covalently immobilized onto glyoxal agarose beads (25% recovery of activity) and assessed for the continuous production of chitooligosaccharides (COS). The immobilization did not change the reaction profile (with chitotriose and chitobiose as major products, using chitosans of different polymerization and deacetylation degrees), but significantly increased the enzyme thermostability. A two-step process was proposed, in which chitosan was first hydrolyzed in a batch reactor to a viscosity that could flow through a packed-bead reactor (PBR), thus avoiding clogging of the column. The relationship between hydrolysis degree of chitosan (1% w/v) and viscosity of the solution was assessed in a batch reactor. A 50% hydrolyzed chitosan did not cause any clogging of the PBR. Under these conditions, the productivity of the PBR at the lowest dilution rate was 37 g L h, with a conversion yield of 73%. In contrast, at the highest dilution rate, the productivity was nearly 200 g L h, but the conversion yield dropped to around 40%.This work was supported by a grant from the Spanish Ministry of Economy and Competitiveness (BIO2013-48779-C4-1-R). We thank COST-Action 310 CM1303 on Systems Biocatalysts for granting P. Santos-Moriano a Short Term Scientific Mission. P. Santos-Moriano thanks the Spanish Ministry of Education for FPU gran