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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. 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.

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.

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.

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