Procedimiento de fermentación dirigida secuencial, nueva cepa de levadura que interviene en el mismo, y su aplicación industrial

Número de publicación: ES2222786 A1 (01.02.2005) También publicado como: ES2222786 B1 (01.04.2006) Número de Solicitud: Consulta de Expedientes OEPM (C.E.O.) P200202743 (28.11.2002)La presente invención se refiere a una nueva cepa de Pichia fermentans, CECT 11773, y a la aplicación de un nuevo procedimiento de vinificación mediante fermentación secuencial dirigida, por el cual el mosto es sembrado en tiempos diferentes por dicha cepa y por otra del género Saccharomyces. La primera da lugar a la síntesis de una gran cantidad de sustancias aromáticas y saborizantes con baja producción de etanol, que determinarán el aroma del producto final; la segunda levadura se encarga de terminar la fermentación aumentando la cantidad de alcohol acumulado hasta un 12-13% v/v.Universidad de Almerí

Sistema enzimático y procedimiento para la preparación de d-aminoacidos o derivados de los mismos

Número de publicación: ES2241394 A1 (16.10.2005) También publicado como: ES2241394 B1 (16.12.2006) Número de Solicitud: Consulta de Expedientes OEPM (C.E.O.) P200202208 (30.09.2002)La presente invención se refiere a un procedimiento para la preparación de D-aminoácidos o derivados de D-aminoácidos, a partir de mezclas racémicas de D,L-hidantoinas caracterizado por estar constituido por las enzimas hidantoín racemasa, D-hidantoinasa y D-carbomilasa; y a un sistema enzimático de utilidad en dicho procedimiento que cataliza la conversión estereoselectiva de D-5-hidantoina hasta D-aminoácido y la racemización entre los enantiómeros de la misma.Universidad de Almerí

l-Amino Acid Production by a Immobilized Double-Racemase Hydantoinase Process: Improvement and Comparison with a Free Protein System

Protein immobilization is proving to be an environmentally friendly strategy for manufacturing biochemicals at high yields and low production costs. This work describes the optimization of the so-called “double-racemase hydantoinase process,” a system of four enzymes used to produce optically pure l-amino acids from a racemic mixture of hydantoins. The four proteins were immobilized separately, and, based on their specific activity, the optimal whole relation was determined. The first enzyme, d,l-hydantoinase, preferably hydrolyzes d-hydantoins from d,l-hydantoins to N-carbamoyl-d-amino acids. The remaining l-hydantoins are racemized by the second enzyme, hydantoin racemase, and continue supplying substrate d-hydantoins to the first enzyme. N-carbamoyl-d-amino acid is racemized in turn to N-carbamoyl-l-amino acid by the third enzyme, carbamoyl racemase. Finally, the N-carbamoyl-l-amino acid is transformed to l-amino acid by the fourth enzyme, l-carbamoylase. Therefore, the product of one enzyme is the substrate of another. Perfect coordination of the four activities is necessary to avoid the accumulation of reaction intermediates and to achieve an adequate rate for commercial purposes. The system has shown a broad pH optimum of 7–9, with a maximum activity at 8 and an optimal temperature of 60 °C. Comparison of the immobilized system with the free protein system showed that the reaction velocity increased for the production of norvaline, norleucine, ABA, and homophenylalanine, while it decreased for l-valine and remained unchanged for l-methionine

Enzymatic dynamic kinetic resolution of racemic N-formyl- and N-carbamoyl-amino acids using immobilized L-N-carbamoylase and N-succinyl-amino acid racemase.

Taking advantage of the catalytic promiscuity of L-carbamoylase from Geobacillus stearothermophilus CECT43 (BsLcar) and N-succinyl-amino acid racemase from Geobacillus kaustophilus CECT4264 (GkNSAAR), we have evaluated the production of different optically pure L-α-amino acids starting from different racemic N-formyl- and N-carbamoyl-amino acids using a dynamic kinetic resolution approach. The enzymes were immobilized on two different solid supports, resulting in improved stability of the enzymes in terms of thermostability and storage when compared to the enzymes in solution. The bienzymatic system retained up to 80 % conversion efficiency after 20 weeks at 4 °C and up to 90 % after 1 week at 45 °C. The immobilization process also resulted in a great enhancement of the activity of BsLcar toward N-formyl-tryptophan, showing for the first time that substrate specificity of L-carbamoylases can be influenced by this approach. The system was effective for the biosynthesis of natural and unnatural L-amino acids (enantiomeric excess (e.e.) >99.5 %), such as L-methionine, L-alanine, L-tryptophan, L-homophenylalanine, L-aminobutyric acid, and L-norleucine, with a higher performance toward N-formyl-α-amino acid substrates. Biocatalyst reuse was studied, and after 10 reaction cycles, over 75 % activity remained.post-print1047 K

Sistema de coexpresión enzimática para la producción de D-aminoácidos

Número de publicación: ES2322418 A1 (19.06.2009) También publicado como: ES2322418 B1 (22.03.2010) Número de Solicitud: Consulta de Expedientes OEPM (C.E.O.) P200602619 (02.10.2006)La presente invención se refiere a un vector de coexpresión para la preparación de D-aminoácidos o derivados de D-aminoácidos, a partir de la mezcla racémica de la D,L-5-hidantoína correspondiente y a un sistema enzimático que da lugar a una ruta metabólica nueva de utilidad en dicho procedimiento, que cataliza la conversión estereoselectiva de D,L-5-hidantoína hasta D-aminoácido y la racemización entre los enantiómeros de la misma.Universidad de Almerí

Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.

N-Succinyl-amino acid racemase (NSAAR), long referred to as N-acyl- or N-acetyl-amino acid racemase, is an enolase superfamily member whose biotechnological potential was discovered decades ago, due to its use in the industrial dynamic kinetic resolution methodology first known as “Acylase Process”. In previous works, an extended and enhanced substrate spectrum of the NSAAR from Geobacillus kaustophilus CECT4264 toward different N-substituted amino acids was reported. In this work, we describe the cloning, purification, and characterization of the NSAAR from Geobacillus stearothermophilus CECT49 (GstNSAAR). The enzyme has been extensively characterized, showing a higher preference toward N-formyl-amino acids than to N-acetyl-amino acids, thus confirming that the use of the former substrates is more appropriate for a biotechnological application of the enzyme. The enzyme showed an apparent thermal denaturation midpoint of 77.0 ± 0.1 °C and an apparent molecular mass of 184 ± 5 kDa, suggesting a tetrameric species. Optimal parameters for the enzyme activity were pH 8.0 and 55–65 °C, with Co2+ as the most effective cofactor. Mutagenesis and binding experiments confirmed K166, D191, E216, D241, and K265 as key residues in the activity of GstNSAAR, but not indispensable for substrate binding.pre-print784 K

Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from G. stearothermoph

Cross-linked enzyme aggregates (CLEAs) of the Y509E mutant of glycoside hydrolase family 52 β-xylosidase from Geobacillus stearothermophilus with dual activity of β-xylosidase and xylanase (XynB2Y509E) were prepared. Ammonium sulfate was used as the precipitant agent, and glutaraldehyde as cross-linking agent. The optimum conditions were found to be 90% ammonium sulfate, 12.5 mM glutaraldehyde, 3 h of cross-linking reaction at 25 °C, and pH 8.5. Under these (most effective) conditions, XynB2Y509E-CLEAs retained 92.3% of their original β-xylosidase activity. Biochemical characterization of both crude and immobilized enzymes demonstrated that the maximum pH and temperature after immobilization remained unchanged (pH 6.5 and 65 °C). Moreover, an improvement in pH stability and thermostability was also found after immobilization. Analysis of kinetic parameters shows that the Km value of XynB2Y509E-CLEAs obtained was slightly higher than that of free XynB2Y509E (1.2 versus 0.9 mM). Interestingly, the xylanase activity developed by the mutation was also conserved after the immobilization process

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe

Probing effective field theory operators in the associated production of top quarks with a Z boson in multilepton final states at root s=13 TeV

Peer reviewe

Reconstitución de la 3-hidroxi-3-metilglutaril-Coa reductasa en vesicular fosfolipídicas

Reducción altaSe ha efectuado, por vez primera, la reconstitución "in vitro" de la hmg-coa reductasa, solubilizada previamente a partir de microsomas hepáticos de pollo, en vesículas fosfolipidicas de composición controlable. El procedimiento utilizado se basa en la incubación del material solubilizado en tritón x-100 con mezclas definidas de diferentes lípidos. Se ha establecido la idoneidad del procedimiento en el estudio de las interacciones lipido proteina existentes entre la hmg-coa reductasa y su entorno membranoso. Se ha descrito, por vez primera un procedimiento practico para la extracción y determinación cuantitativa y simultanea de los lípidos y las proteínas contenidas cuantitativa y simultáneamente en la misma muestra problema. Se ha obtenido una evidencia directa del efecto inhibidor que el colesterol libre incorporado a la membrana ejerce sobre la actividad hmg-coa reductasa reconstituida, y cuya magnitud oscila entre el 35% y el 50%. El efecto observado es progresivo, aumentando con la concentración de colesterol, es específico, corelacionable fisiológicamente y dependiente de la integridad de la membrana, se trata de un efecto directo, no mediado por proteolisis de la enzima. Por vez primera, se ha establecido, de un modo directo, el potencial regulador que, en respuesta a una situación fisiológica concreta, poseen los lípidos de la membrana sobre la actividad de moléculas de reductasa heterólogas.Univ. de Granada, Departamento de Bioquímica y Biología Molecular. Leída el 15-10-8