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1.
Polymers (Basel) ; 15(15)2023 Jul 26.
Article in English | MEDLINE | ID: mdl-37571063

ABSTRACT

The Y509E mutant of ß-xylosidase from Geobacillus stearothermophilus (XynB2Y509E) (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. The activity expressed after immobilization by covalent bonding was 23% higher compared to the activity expressed following entrapment immobilization, with values of 122.3 and 99.4 IU.g-1, respectively. Kinetic data revealed that catalytic turnover values were decreased as compared to a free counterpart. Both biocatalysts showed increased thermal and pH stability, along with an improved storage capacity, as they retained 88% and 40% of their activity after being stored at 4 °C for two months. Moreover, XynB2Y509E immobilized by covalent binding also exhibited outstanding reusability, retaining 92% of activity after 10 cycles of reuse. In conclusion, our results suggest that the covalent bond method appears to be the best choice for XynB2Y509E immobilization.

2.
Molecules ; 26(2)2021 Jan 16.
Article in English | MEDLINE | ID: mdl-33467076

ABSTRACT

Cross-linked enzyme aggregates (CLEAs) of the Y509E mutant of glycoside hydrolase family 52 ß-xylosidase from Geobacillus stearothermophilus with dual activity of ß-xylosidase and xylanase (XynB2Y509E) 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, 12.5 mM glutaraldehyde, 3 h of cross-linking reaction at 25 °C, and pH 8.5. Under these (most effective) conditions, XynB2Y509E-CLEAs retained 92.3% of their original ß-xylosidase activity. Biochemical characterization of both crude and immobilized enzymes demonstrated that the maximum pH and temperature after immobilization remained unchanged (pH 6.5 and 65 °C). Moreover, an improvement in pH stability and thermostability was also found after immobilization. Analysis of kinetic parameters shows that the K m value of XynB2Y509E-CLEAs obtained was slightly higher than that of free XynB2Y509E (1.2 versus 0.9 mM). Interestingly, the xylanase activity developed by the mutation was also conserved after the immobilization process.


Subject(s)
Amino Acid Substitution , Bacterial Proteins/chemistry , Cross-Linking Reagents/chemistry , Geobacillus stearothermophilus/enzymology , Glutaral/chemistry , Glycoside Hydrolases/chemistry , Protein Aggregates , Bacterial Proteins/genetics , Geobacillus stearothermophilus/genetics , Glycoside Hydrolases/genetics , Mutation, Missense
3.
Mol Biotechnol ; 57(5): 454-65, 2015 May.
Article in English | MEDLINE | ID: mdl-25875730

ABSTRACT

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 Co(2+) 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.


Subject(s)
Amino Acid Isomerases/genetics , Amino Acid Isomerases/metabolism , Geobacillus stearothermophilus/enzymology , Amino Acid Isomerases/chemistry , Amino Acid Isomerases/isolation & purification , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Catalytic Domain , Cloning, Molecular , Geobacillus stearothermophilus/genetics , Mutagenesis , Protein Denaturation , Protein Multimerization
4.
Appl Microbiol Biotechnol ; 99(1): 283-91, 2015 Jan.
Article in English | MEDLINE | ID: mdl-24993356

ABSTRACT

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.


Subject(s)
Amidohydrolases/metabolism , Amino Acids/metabolism , Enzymes, Immobilized/metabolism , Amidohydrolases/chemistry , Amino Acid Isomerases/chemistry , Enzyme Stability , Geobacillus/enzymology , Temperature
5.
Biochimie ; 99: 178-88, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24333989

ABSTRACT

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. Co(2+) 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.


Subject(s)
Amidohydrolases/chemistry , Bacillus/enzymology , Bacterial Proteins/chemistry , Allantoin/chemistry , Allosteric Regulation , Amidohydrolases/antagonists & inhibitors , Amidohydrolases/genetics , Amino Acid Substitution , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Catalytic Domain , Cobalt/chemistry , Cysteine/chemistry , Enzyme Inhibitors/chemistry , Hydantoins/chemistry , Hydrogen Peroxide/chemistry , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Manganese/chemistry , Molecular Weight , Mutagenesis, Site-Directed , Protein Binding , Stereoisomerism , Substrate Specificity , Transition Temperature , Uracil/analogs & derivatives , Uracil/chemistry
6.
J Bacteriol ; 194(21): 5759-68, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22904279

ABSTRACT

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. Structural analysis of BsLcar and several members of the peptidase M20/M25/M40 family confirmed the expected conserved residues at the active site in this family, and site-directed mutagenesis revealed their relevance to substrate binding. We also found an unexpectedly conserved arginine residue (Arg(234) in BsLcar), proven to be critical for dimerization of the enzyme. The mutation of this sole residue resulted in a total loss of activity and prevented the formation of the dimer in BsLcar. Comparative studies revealed that the dimerization domain of the peptidase M20/M25/M40 family is a "small-molecule binding domain," allowing further evolutionary considerations for this enzyme family.


Subject(s)
Amidohydrolases/chemistry , Amidohydrolases/metabolism , Geobacillus stearothermophilus/enzymology , Amidohydrolases/genetics , Amino Acid Substitution , Catalytic Domain , Conserved Sequence , Crystallography, X-Ray , Geobacillus stearothermophilus/chemistry , Geobacillus stearothermophilus/genetics , Models, Molecular , Mutagenesis, Site-Directed , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Binding , Protein Conformation , Protein Multimerization , Substrate Specificity
7.
Methods Mol Biol ; 794: 87-104, 2012.
Article in English | MEDLINE | ID: mdl-21956558

ABSTRACT

Hydantoinases/dihydropyrimidinases are important biotechnological enzymes involved in the production of α- and ß-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). Several techniques are combined to obtain this goal, from cloning to activity measurement by HPLC.


Subject(s)
Amidohydrolases/metabolism , Amino Acids/biosynthesis , Protein Engineering , Base Sequence , Chromatography, High Pressure Liquid , Cyclization , DNA Primers , Polymerase Chain Reaction
8.
Appl Environ Microbiol ; 77(16): 5761-9, 2011 Aug 15.
Article in English | MEDLINE | ID: mdl-21705545

ABSTRACT

Formamidases (EC 3.5.1.49) are poorly characterized proteins. In spite of this scarce knowledge, ammonia has been described as playing a central role in the pathogenesis of human pathogens such as Helicobacter pylori, for which formamidase has been shown to participate in the nitrogen metabolic pathway. Sequence analysis has revealed that at least two different groups of formamidases are classified as EC 3.5.1.49: on the one hand, the derivatives of the FmdA-AmdA superfamily, which are the best studied to date, and on the other hand, the derivatives of Helicobacter pylori AmiF. Here we present the cloning, purification, and characterization of a recombinant formamidase from Bacillus cereus CECT 5050T (BceAmiF), the second member of the AmiF subfamily to be characterized, showing new features of the enzyme further supporting its relationship with aliphatic amidases. We also present homology modeling-based mutational studies confirming the importance of the Glu140 and Tyr191 residues in the enzymatic activities of the AmiF family. Moreover, we can conclude that a second glutamate residue is critical in several members of the nitrilase superfamily, meaning that what has consistently been identified as a C-E-K triad is in fact a C-E-E-K tetrad.


Subject(s)
Amidohydrolases/chemistry , Aminohydrolases/metabolism , Bacillus cereus/enzymology , Bacterial Proteins/chemistry , Amidohydrolases/genetics , Bacillus cereus/genetics , Bacterial Proteins/genetics , Base Sequence , Catalysis , Chromatography, Gel , Chromatography, High Pressure Liquid , Circular Dichroism/methods , Cloning, Molecular , Enzyme Activation , Enzyme Assays , Escherichia coli/chemistry , Escherichia coli/genetics , Glutamic Acid/chemistry , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Phylogeny , Protein Structure, Secondary , Recombinant Proteins/chemistry , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity
9.
Biotechnol Prog ; 26(4): 954-9, 2010.
Article in English | MEDLINE | ID: mdl-20730754

ABSTRACT

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. BsLcar showed faster hydrolysis for N-formyl-L-amino acids than for N-carbamoyl and N-acetyl-L-derivatives, with a catalytic efficiency (k(cat)/K(m)) of 8.58 x 10(5), 1.83 x 10(4), and 1.78 x 10(3) (s(-1) M(-1)), respectively, for the three precursors of L-methionine. Optimum reaction conditions for BsLcar, using the three N-substituted-L-methionine substrates, were 65 degrees C and pH 7.5. In all three cases, the metal ions Co(2+), Mn(2+), and Ni(2+) greatly enhanced BsLcar activity, whereas metal-chelating agents inhibited it, showing that BsLcar is a metalloenzyme. The Co(2+)-dependent activity profile of the enzyme showed no detectable inhibition at high metal ion concentrations.


Subject(s)
Amidohydrolases/metabolism , Geobacillus stearothermophilus/enzymology , Amidohydrolases/genetics , Geobacillus stearothermophilus/metabolism , Hydrogen-Ion Concentration , Substrate Specificity , Temperature
10.
Chem Biodivers ; 7(6): 1531-48, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20564568

ABSTRACT

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. The immense amount of information scattered throughout the literature makes it difficult to achieve a general overview of their applications. This review summarizes the state-of-the-art on D-amino acid applications and occurrence, providing both established and neophyte researchers with a comprehensive introduction to this topic.


Subject(s)
Amino Acids/metabolism , Amino Acids/chemistry , Anti-Bacterial Agents/chemistry , Enzymes/metabolism
11.
Appl Microbiol Biotechnol ; 85(3): 441-58, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19830420

ABSTRACT

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. Carbamoylase-based kinetic resolution of amino acids has been applied for the last three decades, allowing the production of optically pure D- or L-amino acids. Furthermore, this enzyme has become crucial in the industrially used multienzymatic system known as "Hydantoinase Process," where the kinetic resolution produced by coupling an enantioselective hydantoinase and the enantiospecific carbamoylase is enhanced by the enzymatic/chemical dynamic kinetic resolution of the low-rate hydrolyzed substrate. This review outlines the properties of D- and L-carbamoylases, emphasizing their biochemical/structural characteristics and their biotechnological applications. It also pinpoints new applications for the exploitation of carbamoylases over the forthcoming years.


Subject(s)
Amidohydrolases/metabolism , Amino Acids/metabolism , Amidohydrolases/chemistry , Amino Acid Sequence , Biotechnology/methods , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Sequence Homology , Stereoisomerism , Substrate Specificity
12.
J Struct Biol ; 169(2): 200-8, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19895890

ABSTRACT

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. The c-terminal tails of each subunit extend toward another monomer in a swapping-like manner, creating a hydrogen bond network which suggests its implication in protein oligomerisation. Mutational and structural evidence suggest the involvement of a conserved tyrosine in the reaction mechanism of the enzyme. SmelDhp presents both hydantoinase and dihydropyrimidinase activities, with higher affinity for the natural six-membered ring substrates. For the five-membered ring substrates, affinity was greater for those with aliphatic and apolar groups in the 5th carbon atom, with the highest rates of hydrolysis for d-5-methyl and d-5-ethyl hydantoin (k(cat)/K(m)=2736+/-380 and 944+/-52M(-1)s(-1), respectively). The optimal conditions for the enzyme activity were found to be 60 degrees C of temperature at pH 8.0. SmelDhp retains 95% of its activity after 6-hour preincubation at 60 degrees C. This is the first dihydropyrimidinase used for the hydrolytic opening of non-natural 6-monosubstituted dihydrouracils, which may be exploited for the production of beta-amino acids.


Subject(s)
Amidohydrolases/chemistry , Models, Molecular , Protein Conformation , Sinorhizobium meliloti/enzymology , Amidohydrolases/genetics , Amino Acid Sequence , Base Sequence , Evolution, Molecular , Molecular Sequence Data , Protein Binding , Sequence Alignment , Sequence Analysis, DNA , Substrate Specificity
13.
Biopolymers ; 91(9): 757-72, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19517534

ABSTRACT

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. The protein seems to be a tetramer with a rather elongated shape, as shown by AU experiments; this is further supported by the modeled structure, which keeps intact the largest tetrameric oligomerization interfaces observed in other homooctameric members of the family, but it does not maintain the octameric oligomerization interfaces. The native functional structure is mainly formed by alpha-helix, as suggested by FTIR and CD deconvoluted spectra, with similar percentages of structure to those observed in other protomers of the enolase superfamily. At low pH, the protein populates a molten-globule-like conformation. The GdmCl denaturation occurs through a monomeric intermediate, and thermal denaturation experiments indicate a high thermostability. The presence of the cofactor Co(2+) did alter slightly the secondary structure, but it did not modify substantially the stability of the protein. Thus, GkNSAAR is one of the few members of the enolase family whose conformational propensities and stability have been extensively characterized.


Subject(s)
Amino Acids , Bacterial Proteins/chemistry , Enzyme Stability , Protein Conformation , Racemases and Epimerases/chemistry , Amino Acid Sequence , Amino Acids/chemistry , Amino Acids/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cobalt/chemistry , Hot Temperature , Hydrogen-Ion Concentration , Models, Molecular , Molecular Sequence Data , Protein Denaturation , Racemases and Epimerases/genetics , Racemases and Epimerases/metabolism , Sequence Alignment , Spectroscopy, Fourier Transform Infrared , Thermodynamics
14.
Biophys Chem ; 139(1): 42-52, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19008030

ABSTRACT

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. Then, the role of the ion in the structure of the protein, and in its stability, remains to be elucidated. In this work, the stability and the structure of SmelDhp have been studied in the absence and in the presence of zinc. In its absence, the protein acquired a tetrameric functional structure at pH approximately 6.0, which is stable up to pH approximately 9.0, as concluded from fluorescence and CD. Chemical-denaturation occurred via a monomeric intermediate with non-native structure. The addition of zinc caused: (i) an increase of the helical structure, and changes in the environment of aromatic residues; and, (ii) a higher thermal stability. However, chemical-denaturation still occurred through a monomeric intermediate. This is the first hydantoinase whose changes in the stability and in the secondary structure upon addition of zinc are described and explained, and one of the few examples where the zinc exclusively alters the secondary helical structure and the environment of some aromatic residues in the protein, leaving unchanged the quaternary structure.


Subject(s)
Amidohydrolases/chemistry , Bacterial Proteins/chemistry , Sinorhizobium meliloti/enzymology , Zinc/pharmacology , Amidohydrolases/metabolism , Animals , Bacterial Proteins/metabolism , Enzyme Stability , Hydrogen-Ion Concentration , Protein Conformation , Protein Denaturation , Protein Structure, Secondary , Temperature
15.
Appl Environ Microbiol ; 75(2): 514-20, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19011069

ABSTRACT

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. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn(2+), Ni(2+), and Co(2+). The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-alpha-, -beta-, -gamma-, and -delta-amino acids, with the greatest catalytic efficiency for N-carbamoyl-beta-alanine. Beta car(At) also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the beta-amino acids taurine and ciliatine, respectively. Beta car(At) is able to produce monosubstituted beta(2)- and beta(3)-amino acids, showing better catalytic efficiency (k(cat)/K(m)) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-beta-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make beta car(At) an outstanding candidate for application in the biotechnology industry.


Subject(s)
Agrobacterium tumefaciens/enzymology , Amidohydrolases/metabolism , Amino Acids/metabolism , Bacterial Proteins/metabolism , Agrobacterium tumefaciens/genetics , Amidohydrolases/chemistry , Amidohydrolases/genetics , Amidohydrolases/isolation & purification , Bacterial Proteins/isolation & purification , Cations, Divalent/pharmacology , Chelating Agents/pharmacology , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Dimerization , Enzyme Activators/pharmacology , Enzyme Inhibitors/pharmacology , Hydrogen-Ion Concentration , Kinetics , Metals/pharmacology , Models, Molecular , Molecular Sequence Data , Molecular Weight , Oxyquinoline/analogs & derivatives , Oxyquinoline/pharmacology , Protein Structure, Tertiary , Sequence Analysis, DNA , Substrate Specificity , Temperature , beta-Alanine/analogs & derivatives , beta-Alanine/metabolism
16.
Recent Pat Biotechnol ; 2(1): 35-46, 2008.
Article in English | MEDLINE | ID: mdl-19075851

ABSTRACT

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. The specific activity of these optically pure amino acids depends on their structure, chirality and purity. There are two main approaches to obtain optically pure amino acids, namely chemical and enzymatic synthesis. Chemical synthesis gives racemic mixtures of amino acids of low yield and is not environment friendly. One of the most widely-used enzymatic method is the "Hydantoinase Process". In this cascade of reactions, the chemically synthesized D,L-5-monosubstituted hydantoin ring is first hydrolyzed by a stereoselective hydantoinase enzyme to give the corresponding N-carbamoyl alpha-amino acid that is hydrolyzed by highly enantiospecific N-carbamoyl alpha-amino acid amidohydrolase (N-carbamoylase) to yield the free amino acid. At the same time, the remaining non-hydrolyzed 5-monosubstituted hydantoin is racemized by the hydantion racemase enzyme. This process has evolved over the years from the isolation of microorganisms with one or several of these enzymes to the construction of recombinant systems for industrial application.


Subject(s)
Amidohydrolases/chemistry , Amino Acids/chemical synthesis , Amino Acids/isolation & purification , Biotechnology/trends , Chemical Industry/trends , Hydantoins/chemistry , Patents as Topic , Isomerism
17.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 64(Pt 12): 1135-8, 2008 Dec 01.
Article in English | MEDLINE | ID: mdl-19052368

ABSTRACT

N-Carbamoyl-L-amino-acid amidohydrolases (L-N-carbamoylases; EC 3.5.1.87) hydrolyze the carbon-nitrogen bond of the ureido group in N-carbamoyl-L-alpha-amino acids. These enzymes are commonly used in the production of optically pure natural and non-natural L-amino acids using the ;hydantoinase process'. Recombinant L-N-carbamoylase from Geobacillus stearothermophilus CECT43 has been expressed, purified and crystallized by hanging-drop vapour diffusion. X-ray data were collected to a resolution of 2.75 A. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 103.2, b = 211.7, c = 43.1 A and two subunits in the asymmetric unit.


Subject(s)
Amidohydrolases/chemistry , Bacterial Proteins/chemistry , Geobacillus stearothermophilus/enzymology , Amidohydrolases/genetics , Amidohydrolases/isolation & purification , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Cloning, Molecular , Crystallization , Crystallography, X-Ray , Geobacillus stearothermophilus/metabolism , Molecular Weight , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification
18.
Biochim Biophys Acta ; 1784(12): 1924-34, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18657634

ABSTRACT

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. This report describes the first extensive conformational characterization of a family 52 beta-xylosidase. The active protein was a highly hydrated dimer, whose active site was formed by the two protomers, and it probably involved aromatic residues. At low pH, the protein was not active and it populated a monomeric molten-globule-like species, which had a conformational transition with a pK(a) of approximately 4.0. Thermal and chemical-denaturations of the native protein showed hysteresis behaviour. The protein at physiological pH was formed by alpha-helix (30%) and beta-sheet (30%), as shown by CD and FTIR. Comparison with other xylosidases of the same family indicates that the percentages of secondary structure seem to be conserved among the members of the family.


Subject(s)
Bacillaceae/enzymology , Bacterial Proteins/chemistry , Endo-1,4-beta Xylanases/chemistry , Circular Dichroism/methods , Dimerization , Hydrogen-Ion Concentration , Protein Structure, Quaternary/physiology , Protein Structure, Secondary/physiology , Protein Structure, Tertiary/physiology , Spectroscopy, Fourier Transform Infrared/methods
19.
Article in English | MEDLINE | ID: mdl-18097103

ABSTRACT

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 2.17 and 1.85 A for the free and bound enzymes, respectively. Both crystals belong to space group R3 and contain two molecules of SmeHyuA per asymmetric unit. The crystals of the free and complexed SmeHyuA have unit-cell parameters a = b = 85.43, c = 152.37 A and a = b = 85.69, c = 154.38 A, crystal volumes per protein weight (V(M)) of 1.94 and 1.98 A3 Da(-1) and solvent contents of 36.7 and 37.9%, respectively.


Subject(s)
Racemases and Epimerases/chemistry , Sinorhizobium meliloti/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Binding Sites , Indicators and Reagents , Racemases and Epimerases/genetics , Racemases and Epimerases/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification
20.
Appl Environ Microbiol ; 73(5): 1525-31, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17220246

ABSTRACT

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. tumefaciens C58. Each system was formed by using a recombinant Escherichia coli strain with one plasmid harboring three genes coexpressed with one promoter in a polycistronic structure. The D-carbamoylase gene was cloned closest to the promoter in order to obtain the highest level of synthesis of the enzyme, thus avoiding intermediate accumulation, which decreases the reaction rate. Both systems were able to produce 100% conversion and 100% optically pure D-methionine, D-leucine, D-norleucine, D-norvaline, D-aminobutyric acid, D-valine, D-phenylalanine, D-tyrosine, and D-tryptophan from the corresponding hydantoin racemic mixture. For the production of almost all D-amino acids studied in this work, system 1 hydrolyzed the 5-monosubstituted hydantoins faster than system 2.


Subject(s)
Amidohydrolases/genetics , Amino Acids/biosynthesis , Escherichia coli/enzymology , Racemases and Epimerases/genetics , Recombination, Genetic , Agrobacterium tumefaciens/enzymology , Agrobacterium tumefaciens/genetics , Amidohydrolases/chemistry , Amidohydrolases/metabolism , Amino Acids/chemistry , Biotechnology/methods , Escherichia coli/genetics , Plasmids/genetics , Racemases and Epimerases/metabolism , Stereoisomerism
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