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.
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Evaluation of substrate promiscuity of an L-carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43.
Biotechnol Prog
December 2010
Dept. de Química-Física, Bioquímica y Química Inorgánica. Edificio C.I.T.E. I., Universidad de Almería, La Cañada de San Urbano, Almería, Spain.
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.
View Article and Find Full Text PDFCrystallization and preliminary crystallographic studies of the recombinant L-N-carbamoylase from Geobacillus stearothermophilus CECT43.
Acta Crystallogr Sect F Struct Biol Cryst Commun
December 2008
Departamento Química Física, Bioquímica y Química Inorgánica, Universidad de Almería, Almería, Spain.
N-Carbamoyl-L-amino-acid amidohydrolases (L-N-carbamoylases; EC 3.5.1.
View Article and Find Full Text PDFA novel hydantoinase process using recombinant Escherichia coli cells with dihydropyrimidinase and L-N-carbamoylase activities as biocatalyst for the production of L-homophenylalanine.
J Biotechnol
April 2008
Department of Biotechnology, Hungkuang University, Taichung 433, Taiwan.
A dihydropyrimidinase gene (pydB) was cloned from the moderate thermophilic Brevibacillus agri NCHU1002 and expressed in Escherichia coli. The purified dihydropyrimidinase exhibited strict d-enantioselectivity for D,L-p-hydroxyphenylhydantoin and D,L-5-[2-(methylthio)ethyl]hydantoin, and non-enantiospecificity for D,L-homophenylalanylhydantoin (D,L-HPAH). The hydrolytic activity of PydB was enhanced notably by Mn2+, with a maximal activity at 60 degrees C and pH 8.
View Article and Find Full Text PDFMolecular cloning and biochemical characterization of L-N-carbamoylase from Sinorhizobium meliloti CECT4114.
J Mol Microbiol Biotechnol
January 2006
Departamento de Química-Física, Bioquímica y Química Inorgánica, I. Universidad de Almería, La Cañada de San Urbano, Almería, España.
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.
View Article and Find Full Text PDF[Cloning and expression of L-N-carbamoylase gene from Arthrobacter BT801 in Escherichia coli].
Sheng Wu Gong Cheng Xue Bao
March 2003
Beijing Institute of Biotechnology, Beijing, Chinese Academy of Sciences, Shenyang, China.
Hydantoin-utility-enzyme is widely used in enzymic production of various amino acids. One of its component, carbamoylase, is responsible for the conversion of N-carbamylamino acids to corresponding amino acids, which is crucial for the stereoselectivity and rate limiting. To improve the production of the enzyme, an L-N-carbamoylase gene from Arthrobacter BT801, a hydantoinase producting strain being able to convert 5-benzylhydantoin to phenylalanine, was cloned into E.
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