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.

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.

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.

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.

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.

Enzymatic activity assay of D-hydantoinase by isothermal titration calorimetry. Determination of the thermodynamic activation parameters for the hydrolysis of several substrates.

Isothermal titration calorimetry (ITC) has been applied to the determination of the activity of D-hydantoinase (EC 3.5.2.

Binding studies of hydantoin racemase from Sinorhizobium meliloti by calorimetric and fluorescence analysis.

Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific d-carbamoylase guarantee the total conversion from d,l-5-monosubstituted hydantoins with a low velocity of racemization, to optically pure d-amino acids. Hydantoin racemase from Sinorhizobium meliloti was expressed in Escherichia coli. Calorimetric and fluorescence experiments were then carried out to obtain the thermodynamic binding parameters, deltaG, deltaH and DeltaS for the inhibitors L- and D-5-methylthioethyl-hydantoin.

Molecular cloning and biochemical characterization of L-N-carbamoylase from Sinorhizobium meliloti CECT4114.

An N-carbamoyl-L-amino acid amidohydrolase (L-N-carbamoylase) from Sinorhizobium meliloti CECT 4114 was cloned and expressed in Escherichia coli. The recombinant enzyme catalyzed the hydrolysis of N-carbamoyl alpha-amino acid to the corresponding free amino acid, and its purification has shown it to be strictly L-specific. The enzyme showed broad substrate specificity, and it is the first L-N-carbamoylase that hydrolyses N-carbamoyl-L-tryptophan as well as N-carbamoyl L-amino acids with aliphatic substituents.

Crystallographic and thermodynamic analysis of the binding of S-octylglutathione to the Tyr 7 to Phe mutant of glutathione S-transferase from Schistosoma japonicum.

Glutathione S-transferases are a family of multifunctional enzymes involved in the metabolism of drugs and xenobiotics. Two tyrosine residues, Tyr 7 and Tyr 111, in the active site of the enzyme play an important role in the binding and catalysis of substrate ligands. The crystal structures of Schistosoma japonicum glutathione S-transferase tyrosine 7 to phenylalanine mutant [SjGST(Y7F)] in complex with the substrate glutathione (GSH) and the competitive inhibitor S-octylglutathione (S-octyl-GSH) have been obtained.

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 27000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of approximately 100000 Da, suggesting that the native enzyme is a tetramer.

A monomer form of the glutathione S-transferase Y7F mutant from Schistosoma japonicum at acidic pH.

Dissociation and unfolding of homodimeric glutathione S-transferase Y7F mutant from Schistosoma japonicum (SjGST-Y7F) were investigated at equilibrium using urea as denaturant. The conserved residue Tyr7 plays a central role in the catalytic mechanism and the mutation Tyr-Phe yields an inactive enzyme that is able to bind the substrate GSH with a higher binding constant than the wild type enzyme. Mutant SjGST-Y7F is a dimer at pH 6 or higher and a stable monomer at pH 5 that binds GSH (K value of 1.