Exploring the Kinetics and Thermodynamics of a Novel Histidine Ammonia-Lyase from .

Histidine ammonia-lyase (HAL) plays a pivotal role in the non-oxidative deamination of L-histidine to produce -urocanic, a crucial process in amino acid metabolism. This study examines the cloning, purification, and biochemical characterization of a novel HAL from (HAL) and eight active site mutants to assess their effects on substrate binding, catalysis, thermostability, and secondary structure. The HAL enzyme was successfully overexpressed and purified to homogeneity.

Efficiency Assessment between Entrapment and Covalent Bond Immobilization of Mutant β-Xylosidase onto Chitosan Support.

The Y509E mutant of β-xylosidase from (XynB2) (which also bears xylanase activity) has been immobilized in chitosan spheres through either entrapment or covalent bond formation methods. The maximum immobilization yield by entrapment was achieved by chitosan beads developed using a 2% chitosan solution after 1 h of maturation time in CFG buffer with ethanol. On the other hand, the highest value in covalent bond immobilization was observed when employing chitosan beads that were prepared from a 2% chitosan solution after 4 h of activation in 1% glutaraldehyde solution at pH 8.

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

Cross-linked enzyme aggregates (CLEAs) of the Y509E mutant of glycoside hydrolase family 52 β-xylosidase from with dual activity of β-xylosidase and xylanase (XynB2) 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.

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).

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.

Biochemical and mutational studies of allantoinase from Bacillus licheniformis CECT 20T.

Allantoinases (allantoin amidohydrolase, E.C. 3.

Mutational and structural analysis of L-N-carbamoylase reveals new insights into a peptidase M20/M25/M40 family member.

N-Carbamoyl-L-amino acid amidohydrolases (L-carbamoylases) are important industrial enzymes used in kinetic resolution of racemic mixtures of N-carbamoyl-amino acids due to their strict enantiospecificity. In this work, we report the first L-carbamoylase structure belonging to Geobacillus stearothermophilus CECT43 (BsLcar), at a resolution of 2.7 Å.

Engineering cyclic amidases for non-natural amino acid synthesis.

Hydantoinases/dihydropyrimidinases are important biotechnological enzymes involved in the production of α- and β-amino acids. Their isolation from new sources with different substrate specificities, improved activity, enantioselectivity, or higher stability continues to be of great industrial interest. Here, we provide a detailed description of how to produce high quantities of the recombinant hydantoinase/dihydropyrimidinase enzyme from Sinorhizobium meliloti CECT4114 (SmeDhp).

Biochemical and mutational studies of the Bacillus cereus CECT 5050T formamidase support the existence of a C-E-E-K tetrad in several members of the nitrilase superfamily.

Formamidases (EC 3.5.1.

N-Carbamoyl-β-alanine amidohydrolase from Agrobacterium tumefaciens C58: a promiscuous enzyme for the production of amino acids.

The availability of enzymes with a high promiscuity/specificity relationship permits the hydrolysis of several substrates with a view to obtaining a certain product or using one enzyme for several productive lines. N-Carbamoyl-β-alanine amidohydrolase from Agrobacterium tumefaciens (Atβcar) has shown high versatility to hydrolyze different N-carbamoyl-, N-acetyl- and N-formyl-amino acids to produce different α, β, γ and δ amino acids. We have calculated the promiscuity index for the enzyme, obtaining a value of 0.

Evaluation of substrate promiscuity of an L-carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43.

N-carbamoyl-amino-acid amidohydrolase (also known as N-carbamoylase) is the stereospecific enzyme responsible for the chirality of the D- or L-amino acid obtained in the "Hydantoinase Process." This process is based on the dynamic kinetic resolution of D,L-5-monosubstituted hydantoins. In this work, we have demonstrated the capability of a recombinant L-N-carbamoylase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (BsLcar) to hydrolyze N-acetyl and N-formyl-L-amino acids as well as the known N-carbamoyl-L-amino acids, thus proving its substrate promiscuity.

Natural occurrence and industrial applications of D-amino acids: an overview.

Interest in D-amino acids has increased in recent decades with the development of new analytical methods highlighting their presence in all kingdoms of life. Their involvement in physiological functions, and the presence of metabolic routes for their synthesis and degradation have been shown. Furthermore, D-amino acids are gaining considerable importance in the pharmaceutical industry.

Structure of dihydropyrimidinase from Sinorhizobium meliloti CECT4114: new features in an amidohydrolase family member.

The recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114 (SmelDhp) has been characterised and its crystal structure elucidated at 1.85A. The global architecture of the protein is reminiscent of that of the amidohydrolase superfamily, consisting of two domains; an (alpha/beta)(8) TIM-like barrel domain, where the catalytic centre is located, and a smaller beta-sheet sandwich domain of unknown function.

Carbamoylases: characteristics and applications in biotechnological processes.

Enzymatic kinetic resolution is a widely used biotechnological tool for the production of enantiomerically pure/enriched compounds. This technique takes advantage of the enantioselectivity or enantiospecificity of an enzyme for one of the enantiomers of a racemic substrate to isolate the desired isomer. N-Carbamoyl-D- and L-amino acid amidohydrolases (D- and L-carbamoylases) are model enzymes for this procedure due to their strict enantiospecificity.

Structure and conformational stability of a tetrameric thermostable N-succinylamino acid racemase.

The N-succinylamino acid racemases (NSAAR) belong to the enolase superfamily and they are large homooctameric/hexameric species that require a divalent metal ion for activity. We describe the structure and stability of NSAAR from Geobacillus kaustophilus (GkNSAAR) in the absence and in the presence of Co(2+) by using hydrodynamic and spectroscopic techniques. The Co(2+), among other assayed divalent ions, provides the maximal enzymatic activity at physiological pH.

Optically pure alpha-amino acids production by the "Hydantoinase Process".

Optically pure D- or L-amino acids are used as intermediates in several industries. D-amino acids are involved in the synthesis of antibiotics, pesticides, sweeteners and other biologically active peptides. L-amino acids are used as feed and food additives, as intermediates for pharmaceuticals, cosmetics, pesticides and as chiral synthons in organic synthesis.

Crystallization and preliminary crystallographic studies of the recombinant L-N-carbamoylase from Geobacillus stearothermophilus CECT43.

N-Carbamoyl-L-amino-acid amidohydrolases (L-N-carbamoylases; EC 3.5.1.

Potential application of N-carbamoyl-beta-alanine amidohydrolase from Agrobacterium tumefaciens C58 for beta-amino acid production.

An N-carbamoyl-beta-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (beta car(At)) has been characterized. Beta car(At) is most active at 30 degrees C and pH 8.0 with N-carbamoyl-beta-alanine as a substrate.

Metal-triggered changes in the stability and secondary structure of a tetrameric dihydropyrimidinase: a biophysical characterization.

Dihydropyrimidinase is involved in the reductive pathway of pyrimidine degradation, catalysing the reversible hydrolysis of the cyclic amide bond (-CO-NH-) of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamoyl-beta-amino acids. This enzyme is an attractive candidate for commercial production of D-aminoacids, which are used in the production of semi-synthetic beta-lactams, antiviral agents, artificial sweeteners, peptide hormones and pesticides. We have obtained the crystal structure of the dihydropyrimidinase from Sinorhizobium meliloti (SmelDhp) in the presence of zinc ions, but we have not been able to obtain good diffracting crystals in its absence.

The family 52 beta-xylosidase from Geobacillus stearothermophilus is a dimer: structural and biophysical characterization of a glycoside hydrolase.

Xylans are the most abundant polysaccharides forming the plant cell wall hemicelluloses, and they are degraded, among other proteins, by beta-xylosidase enzymes. In this work, the structural and biophysical properties of the family 52 beta-xylosidase from Geobacillus stearothermophilus, XynB2, are described. Size exclusion chromatography, analytical centrifugation, ITC, CD, fluorescence (steady state and ANS-binding) and FTIR were used to obtain the structure, the oligomerization state and the conformational changes of XynB2, as pH, chemical denaturants or temperature were modified.

Crystallization and preliminary crystallographic studies of an active-site mutant hydantoin racemase from Sinorhizobium meliloti CECT4114.

A recombinant active-site mutant of hydantoin racemase (C76A) from Sinorhizobium meliloti CECT 4114 (SmeHyuA) has been crystallized in the presence and absence of the substrate D,L-5-isopropyl hydantoin. Crystals of the SmeHyuA mutant suitable for data collection and structure determination were grown using the counter-diffusion method. X-ray data were collected to resolutions of 2.

Recombinant polycistronic structure of hydantoinase process genes in Escherichia coli for the production of optically pure D-amino acids.

Two recombinant reaction systems for the production of optically pure D-amino acids from different D,L-5-monosubstituted hydantoins were constructed. Each system contained three enzymes, two of which were D-hydantoinase and D-carbamoylase from Agrobacterium tumefaciens BQL9. The third enzyme was hydantoin racemase 1 for the first system and hydantoin racemase 2 for the second system, both from A.

Crystallization and preliminary crystallographic studies of the recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114.

Dihydropyrimidinases are involved in the reductive pathway of pyrimidine degradation, catalysing the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamoyl beta-amino acids. This enzyme has often been referred to as hydantoinase owing to its industrial application in the production of optically pure amino acids starting from racemic mixtures of 5-monosubstituted hydantoins. Recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114 (SmelDhp) has been expressed, purified and crystallized.

Site-directed mutagenesis indicates an important role of cysteines 76 and 181 in the catalysis of hydantoin racemase from Sinorhizobium meliloti.

Hydantoin racemase enzyme plays a crucial role in the reaction cascade known as "hydantoinase process." In conjunction with a stereoselective hydantoinase and a stereospecific carbamoylase, it allows the total conversion from D,L-5-monosubstituted hydantoins, with a low rate of racemization, to optically pure D- or L-amino acids. Residues Cys76 and Cys181 belonging to hydantoin racemase from Sinorhizobium meliloti (SmeHyuA) have been proved to be involved in catalysis.

Thermodynamic and mutational studies of l-N-carbamoylase from Sinorhizobium meliloti CECT 4114 catalytic centre.

Purified site-directed mutants of Sinorhizobium meliloti CECT 4114 l-N-carbamoylase (SmLcar) in which Glu132, His230, Asn279 and Arg292 were replaced have been studied by kinetic methods and isothermal titration calorimetry (ITC). The importance of His230, Asn279 and Arg292 residues in the recognition of N-carbamoyl-l-alpha-amino acids has been proved. The role of Glu132 has been confirmed in substrate hydrolysis.