Research Center en Biotecnologia Agroalimentaria

Almería, Spain

Research Center en Biotecnologia Agroalimentaria

Almería, Spain
SEARCH FILTERS
Time filter
Source Type

Martinez-Gomez A.I.,University of Almeria | Martinez-Gomez A.I.,Research Center en Biotecnologia Agroalimentaria | Clemente-Jimenez J.M.,University of Almeria | Clemente-Jimenez J.M.,Research Center en Biotecnologia Agroalimentaria | And 8 more authors.
Process Biochemistry | Year: 2012

Taking advantage of the catalytic promiscuity of pyrimidine-catabolism enzymes (dihydropyrimidinase (E.C. 3.5.2.2), N-carbamoyl-β-alanine amidohydrolase (E.C. 3.5.1.6)), the production of different β-alanine derivatives starting from 5- and 6-monosubstituted dihydrouracils has been evaluated using a mimesis approach. In this work, the S-enantioselective character of dihydropyrimidinase from Sinorizhobium meliloti toward 6-monosubstituted dihydrouracil derivatives has been shown. An inverted R-/S-enantioselectivity of N-carbamoyl-β-alanine amidohydrolase from Agrobacterium tumefaciens toward two different N-carbamoyl-β-amino acids has been proved. Our results have shown for the first time that this mimetic tandem constitutes an interesting biotechnological tool for the preparation of different β-alanine derivatives in an environmentally friendly way, allowing the production of enantioenriched (R)-α-phenyl-β-alanine (e.e. > 95%) and (R)-α-methyl-β-alanine (e.e. > 90%). © 2012 Elsevier Ltd.


Soriano-Maldonado P.,University of Almeria | Soriano-Maldonado P.,Research Center en Biotecnologia Agroalimentaria | Las Heras-Vazquez F.J.,University of Almeria | Las Heras-Vazquez F.J.,Research Center en Biotecnologia Agroalimentaria | And 7 more authors.
Applied Microbiology and Biotechnology | Year: 2014

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. © 2014, Springer-Verlag Berlin Heidelberg.


Martinez-Gomez A.I.,University of Almeria | Martinez-Gomez A.I.,Research Center en Biotecnologia Agroalimentaria | Soriano-Maldonado P.,University of Almeria | Soriano-Maldonado P.,Research Center en Biotecnologia Agroalimentaria | And 14 more authors.
Biochimie | Year: 2014

Allantoinases (allantoin amidohydrolase, E.C. 3.5.2.5) catalyze the hydrolysis of the amide bond of allantoin to form allantoic acid, in those organisms where allantoin is not the final product of uric acid degradation. Despite their importance in the purine catabolic pathway, sequences of microbial allantoinases with proven activity are scarce, and only the enzyme from Escherichia coli (AllEco) has been studied in detail in the genomic era. In this work, we report the cloning, purification and characterization of the recombinant allantoinase from Bacillus licheniformis CECT 20T (AllBali). The enzyme was a homotetramer with an apparent Tm of 62 ± 1 C. Optimal parameters for the enzyme activity were pH 7.5 and 50 C, showing apparent Km and kcat values of 17.7 ± 2.7 mM and 24.4 ± 1.5 s-1, respectively. Co2+ proved to be the most effective cofactor, inverting the enantioselectivity of AllBali when compared to that previously reported for other allantoinases. The common ability of different cyclic amidohydrolases to hydrolyze distinct substrates to the natural one also proved true for AllBali. The enzyme was able to hydrolyze hydantoin, dihydrouracil and 5-ethyl-hydantoin, although at relative rates 3-4 orders of magnitude lower than with allantoin. Mutagenesis experiments suggest that S292 is likely implicated in the binding of the allantoin ring through the carbonyl group of the polypeptide main chain, which is the common mechanism observed in other members of the amidohydrolase family. In addition, our results suggest an allosteric effect of H2O2 toward allantoinase. © 2013 Elsevier Masson SAS. All rights reserved.


Soriano-Maldonado P.,University of Almeria | Soriano-Maldonado P.,Research Center en Biotecnologia Agroalimentaria | Andujar-Sanchez M.,University of Almeria | Andujar-Sanchez M.,Research Center en Biotecnologia Agroalimentaria | And 9 more authors.
Molecular Biotechnology | Year: 2015

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. © 2015, Springer Science+Business Media New York.


Gonzalez-Ramirez E.,University of Almeria | Andujar-Sanchez M.,University of Almeria | Andujar-Sanchez M.,Research Center en Biotecnologia Agroalimentaria | Ortiz-Salmeron E.,University of Almeria | And 11 more authors.
Food Biophysics | Year: 2014

Phycoerythrin is the major light-harvesting pigment-protein of the red algae Porphyridium cruentum and is widely used as fluorescent probe and analytical reagent. Additionally this protein has a potential application as natural dye in food industry. Nevertheless the knowledge of the functional properties of this alga protein is limited, hindering its application as food additive. In this article we report a biophysical characterization of B-phycoerythrin from Porphyridium cruentum (B-PE) in order to study its stability and spectral properties in a broad range of pHs. This information can help in its potential application as colorant in the food industry. Spectroscopic data obtained in this work show that B-PE has a stronger functional stability in the pH range 4.0-10.0, and Size Exclusion Chromatography suggests that the protein maintains a (αβ)6-γ oligomeric structure in that range of pHs. At pH 7.0, an apparent Tm value of 77.5 ± 0.5 °C was calculated. At this pH, the protein is highly stable with a loss of only 20 % of its spectral properties (absorbance and fluorescence) after 25 days at room temperature. These results indicate that B-PE is more stable in a broad range of pHs than other phycoerythrin proteins, which would facilitate its use in the food industry. © 2014 Springer Science+Business Media New York.


Soriano-Maldonado P.,University of Almeria | Soriano-Maldonado P.,Research Center en Biotecnologia Agroalimentaria | Rodriguez-Alonso M.J.,University of Almeria | Rodriguez-Alonso M.J.,Research Center en Biotecnologia Agroalimentaria | And 10 more authors.
Process Biochemistry | Year: 2014

A bienzymatic system comprising an N-succinylamino acid racemase from Geobacillus kaustophilus CECT4264 (GkNSAAR) and an enantiospecific l-N-carbamoylase from Geobacillus stearothermophilus CECT43 (BsLcar) has been developed. This biocatalyst has been able to produce optically pure natural and non-natural l-amino acids starting from racemic mixtures of N-acetyl-, N-formyl- and N-carbamoyl-amino acids by dynamic kinetic resolution. The fastest conversion rate was found with N-formyl-amino acids, followed by N-carbamoyl- and N-acetyl-amino acids, and GkNSAAR proved to be the limiting step of the system due to its lower specific activity. Metal ion cobalt was essential for the activity of the biocatalyst and the system was optimally active when Co 2+ was added directly to the reaction mixture. The optimum pH for the biocatalyst proved to be 8.0, for both N-formyl- and N-carbamoyl-amino acid substrates, whereas optimum temperature ranges were 45-55 °C for N-formyl-amino acids and 55-70 °C for N-carbamoyl-derivatives. The bienzymatic system was equally efficient in converting aromatic and aliphatic substrates. Total conversion was also achieved using high substrate concentrations (100 and 500 mM) with no noticeable inhibition. This "Amidohydrolase Process" enables the production of both natural and non-natural l-amino acids from a broad substrate spectrum with yields of over 95%. © 2014 Elsevier Ltd.


Dahane S.,Oran University of Science and Technology - Mohamed Boudiaf | Garcia M.D.G.,University of Almeria | Garcia M.D.G.,Research Center en Biotecnologia Agroalimentaria | Moreno A.U.,University of Almeria | And 5 more authors.
Microchimica Acta | Year: 2015

We describe a MWCNT-based method for the solid-phase extraction of eight pesticides from environmental water samples. The analytes are extracted from 100 mL samples at pH 5.0 (containing 5 mmol L−1 of KCl) by passing the solution through a column filled with 20 mg of multiwalled carbon nanotubes. Following elution, the pesticides were determined by LC and electrospray ionization hybrid quadrupole linear ion trap MS. Two selected reaction monitoring transitions were monitored per compound, the most intense one being used for quantification and the second one for confirmation. In addition, an information-dependent acquisition experiment was performed for unequivocal confirmation of positive findings. Matrix effect was not found in real waters and therefore the quantitation was carried out with calibration graphs built with solvent based standards. Except for cymoxanil, the detection and quantitation limits in surface waters are in the range from 0.3 to 9.5 ng L−1 and 1.6 to 45.2 ng L−1, respectively. Recoveries from spiked ultrapure water are ~100 %, except for the most polar pesticides methomyl and cymoxanil. The same behavior is found for real water samples (except for phosalone). The relative standard deviation is <10 % in all cases. © Springer-Verlag Wien 2014.


Dahane S.,University of Almeria | Dahane S.,Oran University of Science and Technology - Mohamed Boudiaf | Martinez Galera M.,University of Almeria | Martinez Galera M.,Research Center en Biotecnologia Agroalimentaria | And 6 more authors.
Talanta | Year: 2016

This paper reports the first application of the silica based mesoporous material MCM-41 as a sorbent in solid phase extraction, to pre-concentrate pharmaceuticals of very different polarity (atenolol, nadolol, pindolol, timolol, bisoprolol, metoprolol, betaxolol, ketoprofen, naproxen, ibuprofen, diclofenac, tolfenamic acid, flufenamic acid and meclofenamic acid) in surface waters. The analytes were extracted from 100 mL water samples at pH 2.0 (containing 10-3 mol/L of sodium chloride) by passing the solution through a cartridge filled with 100 mg of MCM-41. Following elution, the pharmaceuticals were determined by micro-liquid chromatography and triple quadrupole-mass spectrometry. Two selected reaction monitoring transitions were monitored per compound, the most intense one being used for quantification and the second one for confirmation. Matrix effect was found in real waters for most analytes and was overcome using the standard addition method, which compared favorably with the matrix matched calibration method. The detection limits in solvent (acetonitrile:water 10:90, v/v) ranged from 0.01 to 1.48 μg/L and in real water extracts from 0.10 to 3.85 μg/L (0.001-0.0385 μg/L in the water samples). The quantitation limits in solvent were in the range 0.02-4.93 μg/L, whereas in real water extracts were between 0.45 and 10.00 μg/L (0.0045 and 0.1000 μg/L in the water samples). When ultrapure water samples were spiked at two concentration levels of each pharmaceutical (0.1 and 0.2 μg/L) and quantified using solvent based calibration graphs, recoveries were near 100%. However, recoveries for most pharmaceuticals were comparable or better than de described above, when river water samples (spiked at the same concentration levels) were quantified by the standard addition method and slightly worse using the matrix matched calibration method. Five real samples (two rivers, one dam and two fountain water samples) were analyzed by the developed method, atenolol, timolol, betaxolol, nadolol and diclofenac being found in some of them, at levels higher than their quantitation limits. © 2016 Elsevier B.V. All rights reserved.


Rodriguez-Alonso M.J.,University of Almeria | Rodriguez-Alonso M.J.,Research Center en Biotecnologia Agroalimentaria | Clemente-Jimenez J.M.,University of Almeria | Clemente-Jimenez J.M.,Research Center en Biotecnologia Agroalimentaria | And 4 more authors.
Biochemical Engineering Journal | Year: 2015

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 1mM), 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. © 2015 Elsevier B.V.


Arroyo-Caro J.M.,Research Center en Biotecnologia Agroalimentaria | Chileh T.,Research Center en Biotecnologia Agroalimentaria | Alonso D.L.,Research Center en Biotecnologia Agroalimentaria | Garcia-Maroto F.,Research Center en Biotecnologia Agroalimentaria
Lipids | Year: 2013

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23) catalyzes acylation of lysophosphatidylcholine (lysoPtdCho) to produce phosphatidylcholine (PtdCho), the main phospholipid in cellular membranes. This reaction is a key component of the acyl-editing process, involving recycling of the fatty acids (FA) mainly at the sn-2 position of PtdCho. Growing evidences indicate that the LPCAT reaction controls the direct entry of newly synthesized FA into PtdCho and, at least in some plant species, it has an important impact on the synthesis and composition of triacylglycerols. Here we describe the molecular characterization of the single LPCAT gene found in the genome of Ricinus communis (RcLPCAT) that is homologous to LPCAT genes of the MBOAT family previously described in Arabidopsis and Brassica. RcLPCAT is ubiquitously expressed in all organs of the castor plant. Biochemical properties have been studied by heterologous expression of RcLPCAT in the ale1 yeast mutant, defective in lysophospholipid acyltransferase activity. RcLPCAT preferentially acylates lysoPtdCho against other lysophospholipids (lysoPL) and does not discriminates the acyl chain in the acceptor, displaying a strong activity with alkyl lysoPL. Regarding the acyl-CoA donor, RcLPCAT uses monounsaturated fatty acid thioesters, such as oleoyl-CoA (18:1-CoA), as preferred donors, while it has a low activity with saturated fatty acids and shows a poor utilization of ricinoleoyl-CoA (18:1-OH-CoA). These characteristics are discussed in terms of a possible role of RcLPCAT in regulating the entry of FA into PtdCho and the exclusion from the membranes of the hydroxylated FA. © 2013 AOCS.

Loading Research Center en Biotecnologia Agroalimentaria collaborators
Loading Research Center en Biotecnologia Agroalimentaria collaborators