Nuevos catalizadores altamente estabilizados de la enzima β-galactosidasa de Kluyveromyces lactis inmovilizada sobre soportes glioxil

La presente invención se refiere a un procedimiento de estabilización de una β-galactosidasa de Kluyveromyces lactis, preferentemente inmovilizada en un soporte, que comprende unir la enzima a un polímero activado con al menos un agente conservante de estabilización de enzimas, dicho polímero activado comprendiendo grupos aldehído y/o carboxilo, y donde la unión es de tipo covalente y/o electrostática entre al menos un grupo amino de la enzima con un grupo aldehído o carboxilo del polímero activado. Es asimismo objeto de la presente invención, el catalizador de β-galactosidasa de Kluyveromyces lactis obtenido por el procedimiento anterior, así como sus diferentes usos tales como la hidrólisis de lactosa en leche y sueros ácidos, la síntesis de galactooligosacáridos o su uso en reactores de tanque agitado, de lecho fijo o de lecho fluidizadoPeer reviewedConsejo Superior de Investigaciones CientíficasA1 Solicitud de adición a la patent

Protein hydrolysis by immobilized and stabilized trypsin

El pdf del artículo es la versión pre-print.-- et al.The preparation of novel immobilized and stabilized derivatives of trypsin is reported here. The new derivatives preserved 80% of the initial catalytic activity toward synthetic substrates [benzoyl-arginine p-nitroanilide (BAPNA)] and were 50,000-fold more thermally stable than the diluted soluble enzyme in the absence of autolysis. Trypsin was immobilized on highly activated glyoxyl-Sepharose following a two-step immobilization strategy: (a) first, a multipoint covalent immobilization at pH 8.5 that only involves low pKa amino groups (e.g., those derived from the activation of trypsin from trypsinogen) is performed and (b) next, an additional alkaline incubation at pH 10 is performed to favor an intense, additional multipoint immobilization between the high concentration of proximate aldehyde groups on the support surface and the high pKa amino groups at the enzyme surface region that participated in the first immobilization step. Interestingly, the new, highly stable trypsin derivatives were also much more active in the proteolysis of high molecular weight proteins when compared with a nonstabilized derivative prepared on CNBr-activated Sepharose. In fact, all the proteins contained a cheese whey extract had been completely proteolyzed after 6 h at pH 9 and 50°C, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under these experimental conditions, the immobilized biocatalysts preserve more than 90% of their initial activity after 20 days. Analysis of the three-dimensional (3D) structure of the best immobilized trypsin derivative showed a surface region containing two amino terminal groups and five lysine (Lys) residues that may be responsible for this novel and interesting immobilization and stabilization. Moreover, this region is relatively far from the active site of the enzyme, which could explain the good results obtained for the hydrolysis of high-molecular weight proteins. © 2011 American Institute of Chemical Engineers (AIChE).Peer Reviewe

A Novel Halophilic Lipase, LipBL, Showing High Efficiency in the Production of Eicosapentaenoic Acid (EPA)

Background: Among extremophiles, halophiles are defined as microorganisms adapted to live and thrive in diverse extreme saline environments. These extremophilic microorganisms constitute the source of a number of hydrolases with great biotechnological applications. The interest to use extremozymes from halophiles in industrial applications is their resistance to organic solvents and extreme temperatures. Marinobacter lipolyticus SM19 is a moderately halophilic bacterium, isolated previously from a saline habitat in South Spain, showing lipolytic activity. Methods and Findings: A lipolytic enzyme from the halophilic bacterium Marinobacter lipolyticus SM19 was isolated. This enzyme, designated LipBL, was expressed in Escherichia coli. LipBL is a protein of 404 amino acids with a molecular mass of 45.3 kDa and high identity to class C b-lactamases. LipBL was purified and biochemically characterized. The temperature for its maximal activity was 80uC and the pH optimum determined at 25uC was 7.0, showing optimal activity without sodium chloride, while maintaining 20% activity in a wide range of NaCl concentrations. This enzyme exhibited high activity against short-medium length acyl chain substrates, although it also hydrolyzes olive oil and fish oil. The fish oil hydrolysis using LipBL results in an enrichment of free eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), relative to its levels present in fish oil. For improving the stability and to be used in industrial processes LipBL was immobilized in different supports. The immobilized derivatives CNBr-activated Sepharose were highly selective towards the release of EPA versus DHA. The enzyme is also active towards different chiral and prochiral esters. Exposure of LipBL to buffer-solvent mixtures showed that the enzyme had remarkable activity and stability in all organic solvents tested. Conclusions: In this study we isolated, purified, biochemically characterized and immobilized a lipolytic enzyme from a halophilic bacterium M. lipolyticus, which constitutes an enzyme with excellent properties to be used in the food industry, in the enrichment in omega-3 PUFAs

Identificación y caracterización de esterasas de Lactobacillus plantarum con interés en tecnología de alimentos

Resumen del trabajo presentado a la 7ª Reunión de la Red Tematica Bal: "Participación de las Bacterias Lácticas en la Salud Humana y en la Calidad Alimentaria" celebrado en Madrid del 4 al 5 de julio de 2013.Los ésteres son compuestos aromáticos que a pesar de encontrarse en niveles traza, son muy importantes en el perfil aromático de los alimentos. Pequeñas variaciones en los niveles de estos compuestos pueden tener importantes efectos en el aroma, y por lo tanto en la calidad, de los alimentos. Los ésteres de los alimentos se originan por acción de enzimas con actividad esterasa (EC 3.1.1.x) que pueden catalizar tanto reacciones de hidrólisis como de síntesis en función de las condiciones de reacción, por lo que tienen un gran interés en biotecnología. Entre estas enzimas se pueden distinguir carboxilesterasas, arilesterasas y lipasas. Las carboxilesterasas y las arilesterasas catalizan la hidrólisis de ésteres de cadena alifática corta o media solubles en agua, mientras que las lipasas presentan actividad frente a ésteres de cadena larga e insolubles en agua. Las feruloil esterasas, son un tipo de arilesterasas capaces de hidrolizar el enlace éster entre los ácidos cinámicos y los azúcares de las paredes celulares vegetales liberando compuestos fenólicos como el ácido cafeico, p-cumárico y ferúlico, los cuales presentan numerosas aplicaciones en la industria alimentaría. Lactobacillus plantarum es la especie de bacteria láctica modelo en fermentaciones de sustratos vegetales, en donde los ésteres de compuestos fenólicos, se encuentran en altas concentraciones. En el genoma de L. plantarum aparecen anotados genes que codifican “esterasas” o “lipasas” cuya funcionalidad no se ha comprobado bioquímicamente a pesar del interés biotecnológico que presentan. Por ello el objetivo de este trabajo es la identificación y caracterización de posibles esterasas en L. plantarum WCFS1. A pesar de que se han clonado los genes que codifican 14 posibles esterasas o lipasas, sólo se han podido producir y purificar correctamente las proteínas Lp_0796, Lp_0973, Lp_1002, Lp_2923, Lp_2987, Lp_3561 y Lp_3562. Utilizando ésteres derivados de p-nitrofenilo que varían en la longitud de su cadena alifática (desde acetato de p-nitrofenilo hasta palmitato de pnitrofenilo) se ha comprobado que todas las proteínas estudiadas hidrolizan mejor los ésteres de cadena corta, aunque Lp_1002, Lp_2926 y Lp_3562 también hidrolizan eficazmente palmitato de p-nitrofenilo.La proteína Lp_2987 es la única que no presenta actividad sobre ninguno de los derivados ensayados por lo que no se puede considerar como “esterasa”. La especificidad de substrato de las esterasas también se ha evaluado mediante una colección de ésteres que permite conocer su selectividad respecto a la carga del substrato, al tamaño de la cadena o al alcohol presente. Todas las proteínas purificadas, excepto Lp_2987, presentan actividad sobre acetato de fenilo, por lo que se pueden considerar como “aril esterasas”. La proteína Lp_0973 es la única que, además de acetato de fenilo, degrada triacetina y tributirina. De las esterasas estudiadas, la proteína Lp_0796 es la más interesante puesto que es una “feruloil esterasa” que hidroliza ésteres de ácidos hidroxicinámicos, siendo la primera vez que se describe una proteína con esta actividad en L. plantarum. Con objeto de mejorar la actividad de las esterasas para su posible uso industrial se han realizado experimentos de cristalización, inmovilización y evolución dirigida de alguna de ellas. Los resultados obtenidos indican que L. plantarum es una fuente adecuada de enzimas con actividad esterasa de gran influencia en el aroma de los alimentos.Peer reviewe

Effectiveness and Safety of the Sequential Use of a Second and Third Anti-TNF Agent in Patients With Inflammatory Bowel Disease: Results From the Eneida Registry

Background: The effectiveness of the switch to another anti-tumor necrosis factor (anti-TNF) agent is not known. The aim of this study was to analyze the effectiveness and safety of treatment with a second and third anti-TNF drug after intolerance to or failure of a previous anti-TNF agent in inflammatory bowel disease (IBD) patients. Methods: We included patients diagnosed with IBD from the ENEIDA registry who received another anti-TNF after intolerance to or failure of a prior anti-TNF agent. Results: A total of 1122 patients were included. In the short term, remission was achieved in 55% of the patients with the second anti-TNF. The incidence of loss of response was 19% per patient-year with the second anti-TNF. Combination therapy (hazard ratio [HR], 2.4; 95% confidence interval [CI], 1.8-3; P < 0.0001) and ulcerative colitis vs Crohn's disease (HR, 1.6; 95% CI, 1.1-2.1; P = 0.005) were associated with a higher probability of loss of response. Fifteen percent of the patients had adverse events, and 10% had to discontinue the second anti-TNF. Of the 71 patients who received a third anti-TNF, 55% achieved remission. The incidence of loss of response was 22% per patient-year with a third anti-TNF. Adverse events occurred in 7 patients (11%), but only 1 stopped the drug. Conclusions: Approximately half of the patients who received a second anti-TNF achieved remission; nevertheless, a significant proportion of them subsequently lost response. Combination therapy and type of IBD were associated with loss of response. Remission was achieved in almost 50% of patients who received a third anti-TNF; nevertheless, a significant proportion of them subsequently lost response

The evolution of the ventilatory ratio is a prognostic factor in mechanically ventilated COVID-19 ARDS patients

Background: Mortality due to COVID-19 is high, especially in patients requiring mechanical ventilation. The purpose of the study is to investigate associations between mortality and variables measured during the first three days of mechanical ventilation in patients with COVID-19 intubated at ICU admission. Methods: Multicenter, observational, cohort study includes consecutive patients with COVID-19 admitted to 44 Spanish ICUs between February 25 and July 31, 2020, who required intubation at ICU admission and mechanical ventilation for more than three days. We collected demographic and clinical data prior to admission; information about clinical evolution at days 1 and 3 of mechanical ventilation; and outcomes. Results: Of the 2,095 patients with COVID-19 admitted to the ICU, 1,118 (53.3%) were intubated at day 1 and remained under mechanical ventilation at day three. From days 1 to 3, PaO2/FiO2 increased from 115.6 [80.0-171.2] to 180.0 [135.4-227.9] mmHg and the ventilatory ratio from 1.73 [1.33-2.25] to 1.96 [1.61-2.40]. In-hospital mortality was 38.7%. A higher increase between ICU admission and day 3 in the ventilatory ratio (OR 1.04 [CI 1.01-1.07], p = 0.030) and creatinine levels (OR 1.05 [CI 1.01-1.09], p = 0.005) and a lower increase in platelet counts (OR 0.96 [CI 0.93-1.00], p = 0.037) were independently associated with a higher risk of death. No association between mortality and the PaO2/FiO2 variation was observed (OR 0.99 [CI 0.95 to 1.02], p = 0.47). Conclusions: Higher ventilatory ratio and its increase at day 3 is associated with mortality in patients with COVID-19 receiving mechanical ventilation at ICU admission. No association was found in the PaO2/FiO2 variation

Lipasa LipBL y sus aplicaciones

La presente invención se refiere al uso de la lipasa LipBL para la hidrólisis de distintos sustratos seleccionados de entre p-nitrofenoles, tributirina, Tween 80, aceites, sustratos quirales y proquirales, aceite de pescado, azúcares paracetilados o nitrocefin y a un procedimiento enzimático de monodesprotección regioselectiva de azúcares paracetilados para la obtención de azúcares monodesacteilados.Peer reviewedUniversidad de Sevilla Pabellón de Brasil, Consejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

Synthesis of sn-2 docosahexaenoyl monoacylglycerol by mild enzymatic transesterification of docosahexaenoic acid ethyl ester and glycerol in a solvent-free system

The enzymatic transesterification of docosahexaenoic acid (DHA) ethyl ester with glycerol was performed with several lipases in a solvent-free system and it involves the initial formation of sn-2 docosahexaenyl monoacylglyceride. This DHA derivative is highly relevant for improving the bioavailability of DHA and it has received increasing interest in the field of nutrition. Three commercial lipases, from Rhizomucor miehei (RML), Alcaligenes sp (QL), and Candida antarctica-fraction B (CALB) were tested. In certain cases (CALB), using an excess of DHA ethyl ester and high temperatures the transesterification reaction continues to the formation of triacylglycerides, but in other cases, sn-2 monoacylglyceride (2-MG) is the unique synthetic product even in the presence of high concentrations of DHA ethyl ester. At low temperatures (e.g. 37°C), RML derivatives synthesize only 2-MG in 15 min. These very mild conditions are very interesting for the thermal oxidative stability of the omega-3 fatty acid and for the thermal stability of the biocatalyst. Using Normal Phase HPLC-ELSD and accurate markers, the formation of the 2-MG was confirmed.This work was sponsored by the Spanish Ministry of Science and Innovation 223 [project number AGL-2009-07526], [project number BIO2012-36861].Peer reviewe

Modulation of the regioselectivity of Thermomyces lanuginosus lipase via biocatalyst engineering for the Ethanolysis of oil in fully anhydrous medium

[Background] Enzymatic ethanolysis of oils (for example, high oleic sunflower oil containing 90% of oleic acid) may yield two different reaction products depending on the regioselectivity of the immobilized lipase biocatalyst. Some lipase biocatalysts exhibit a 1,3-regioselectivity and they produced 2 mols of fatty acid ethyl ester plus 1 mol of sn2-monoacylglycerol (2-MAG) per mol of triglyceride without the release of glycerol. Other lipase biocatalysts are completely non-regioselective releasing 3 mols of fatty acid ethyl ester and 1 mol of glycerol per mol of triglyceride. Lipase from Thermomyces lanuginosus (TLL) adsorbed on hydrophobic supports is a very interesting biocatalyst for the ethanolysis of oil. Modulation of TLL regioselectivity in anhydrous medium was intended via two strategies of TLL immobilization: a. - interfacial adsorption on different hydrophobic supports and b.- interfacial adsorption on a given hydrophobic support under different experimental conditions.[Results] Immobilization of TLL on supports containing divinylbenezene moieties yielded excellent 1,3-regioselective biocatalysts but immobilization of TLL on supports containing octadecyl groups yielded non-regioselective biocatalysts. On the other hand, TLL immobilized on Purolite C18 at pH 8.5 and 30 °C in the presence of traces of CTAB yielded a biocatalyst with a perfect 1,3-regioselectivity and a very interesting activity: 2.5 μmols of oil ethanolyzed per min per gram of immobilized derivative. This activity is 10-fold higher than the one of commercial Lipozyme TL IM. Immobilization of the same enzyme on the same support, but at pH 7.0 and 25 °C, led to a biocatalyst which can hydrolyze all ester bonds in TG backbone.[Conclusions] Activity and regioselectivity of TLL in anhydrous media can be easily modulated via Biocatalysis Engineering producing very active immobilized derivatives able to catalyze the ethanolysis of triolein. When the biocatalyst was 1,3-regioselective a 33% of 2-monoolein was obtained and it may be a very interesting surfactant. When biocatalyst catalyzed the ethanolysis of the 3 positions during the reaction process, a 99% of ethyl oleate was obtained and it may be a very interesting drug-solvent and surfactant. The absence of acyl migrations under identical reaction conditions is clearly observed and hence the different activities and regioselectivities seem to be due to the different catalytic properties of different derivatives of TLL.This work was sponsored by the Spanish Ministry of Economy, Industry and Competitiveness (projects BIO2012–36861 and CTQ2015–70348). Javier Rocha-Martin is grateful for the Juan de la Cierva fellowship (IJCI-2014-19,260) funded by the Spanish Ministry of Economy, Industry and Competitiveness. We also thank Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for granting the scholarship to Erick Abreu Silveira.Peer reviewe

Capture of enzyme aggregates by covalent immobilization on solid supports. Relevant stabilization of enzymes by aggregation

In this paper, a novel procedure for the immobilization and stabilization of enzymes is proposed: the multipoint covalent attachment of bi-molecular enzyme aggregates. This immobilization protocol allows the “capture” and fixation of the enzyme aggregate on the support surface. In addition to stabilization by multipoint attachment, enzyme aggregation promotes very interesting stabilizing effects. In the presence of low concentrations of polyethylene glycol (30 %) the dimeric amine oxidase from Pisum sativum forms soluble bi-molecular aggregates. Enzyme aggregates were analyzed by Dynamic Light Scattering and by full chemical loading of a mesoporous support (10 % agarose gels activated with glyoxyl groups). The soluble aggregate was immobilized by multipoint attachment on glyoxyl- agarose at pH 8.5 though the four amino termini of the two dimeric molecules (Lys residues are not reactive at this pH). The immobilized aggregated structure cannot undergo any movement (translational or rotational) after multipoint attachment and the aggregate is “fixed” on the support surface even after the removal of PEG. The immobilized aggregate was further incubated at pH 10 in order to allow the Lys residues to react with the glyoxyl groups on the support. Enzyme aggregation has an important effect on enzyme stabilization: the aggregated derivative was 40 fold more stable than a similar derivative of the isolated enzyme and 200 fold more than native enzymes in experiments of thermal inactivation.This work was supported by the Ramon Areces Foundation by the project in the field of Sciences of Life and Matter titled “The detection and elimination of biogenic amines in foods: the design of new biosensors and new amino oxidase catalysts”. The authors thank the Ramon Areces Foundation for the pre-doctoral grant given to Paz Garcia-Garcia.Peer reviewe