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

Biochemical characterization of a novel hydantoin racemase from Agrobacterium tumefaciens C58

A novel hydantoin racemase gene of Agrobacterium tumefaciens C58 (AthyuA2) has been cloned and expressed in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure and showed an apparent molecular mass of 27,000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of approximately 100,000 Da, suggesting that the native enzyme is a tetramer. The optimum pH and temperature for hydantoin racemase activity were 7.5 and 55 °C, respectively, with L-5-ethylhydantoin as substrate. Enzyme activity was strongly inhibited by Cu2+ and Hg2+. No effect on enzyme activity was detected with any other divalent cations, EDTA or DTT, suggesting that it is not a metalloenzyme. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings

Rational re-design of the “double-racemase hydantoinase process” for optically pure production of natural and non-natural l-amino acids

The “hydantoinase process” is a well-established method for the industrial production of optically pure d-amino acids. However, due to the strict d-enantioselectivity of most hydantoinase enzymes, the process is less efficient for l-amino acid production. We present a new chemo-enzymatic cascade reaction for natural and non-natural l-amino acid production from racemic mixtures of 5-monosubstituted hydantoins. This system comprised the following enzymes: d-hydantoinase from Agrobacterium tumefaciens BQL9, hydantoin racemase 1 from A. tumefaciens C58 and l-N-carbamoylase from Geobacillus stearothermophilus CECT43, together with N-succinyl-amino acid racemase from G. kaustophilus CECT4264. This latter presents catalytic promiscuity and racemizes N-carbamoyl-amino acids. This activity avoids the accumulation of N-carbamoyl-d-amino acid in the reaction due to the strict d-enantioselectivity of the hydantoinase. The optimum pH for the system proved to be 8.0, whereas optimum temperature range was 50–65 ◦C, with the maximum reaction rate at 60 ◦C. The metal ion cobalt was added directly to the reaction mixture (end concentration 1 mM), but in the case of d-hydantoinase, overexpression in presence of 0.5mM Co2+ was also necessary. The enzymatic cascade reaction produced different optically pure l-amino acids by dynamic kinetic resolution, achieving 100% conversion even at high substrate concentrations (100mM) with no noticeable inhibition. This total conversion demonstrates that the “double-racemase hydantoinase process” upgrades the classical “hydantoinase process” for natural and non-natural l-amino acid production

Structural Characterization of β-Xylosidase XynB2 from Geobacillus stearothermophilus CECT43: A Member of the Glycoside Hydrolase Family GH52

β-xylosidases (4-β-D-xylan xylohydrolase, E.C. 3.2.1.37) are glycoside hydrolases (GH) catalyzing the hydrolysis of (1→4)-β-D-xylans, allowing for the removal of β-D-xylose residues from its non-reducing termini. Together with other xylan-degrading enzymes, β-xylosidases are involved in the enzymatic hydrolysis of lignocellulosic biomass, making them highly valuable in the biotechnological field. Whereas different GH families are deeply characterized from a structural point of view, the GH52 family has been barely described. In this work, we report the 2.25 Å resolution structure of Geobacillus stearothermophilus CECT43 XynB2, providing the second structural characterization for this GH family. A plausible dynamic loop closing the entrance of the catalytic cleft is proposed based on the comparison of the available GH52 structures, suggesting the relevance of a dimeric structure for members of this family. The glycone specificity at the −1 site for GH52 and GH116 members is also explained by our structural studies.Spanish Ministry of Science and Innovation/FEDER funds Grant PID2020-116261GB-I00/AEI/10.13039/501100011033European Regional Development Fund Andalucía 2014–2020 Grant UAL18-CTS-B032-AOwn Research and Transfer Plan 2020 of the University of Almeria Grant PPUENTE2020/00

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

Molecular Cloning, Purification, and Biochemical Characterization of Hydantoin Racemase from the Legume Symbiont Sinorhizobium meliloti CECT 4114

Hydantoin racemase from Sinorhizobium meliloti was functionally expressed in Escherichia coli. The native form of the enzyme was a homotetramer with a molecular mass of 100 kDa. The optimum temperature and pH for the enzyme were 40°C and 8.5, respectively. The enzyme showed a slight preference for hydantoins with short rather than long aliphatic side chains or those with aromatic rings. Substrates, which showed no detectable activity toward the enzyme, were found to exhibit competitive inhibition