Gene cloning, sequence analysis, purification, and characterization of a thermostable aminoacylase from Bacillus stearothermophilus
| Autor: | Anahit Kochikyan, Alexey Savchenko, C Legrain, P Falmagne, A. Boyen, L. Desmarez, André Piérard, Vehary Sakanyan, Igor Mett, Daniel Charlier |
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| Rok vydání: | 1993 |
| Předmět: |
DNA
Bacterial DNA Complementary Protein Conformation Molecular Sequence Data Restriction Mapping Biology Molecular cloning medicine.disease_cause Applied Microbiology and Biotechnology Amidohydrolases Substrate Specificity Geobacillus stearothermophilus chemistry.chemical_compound Carboxypeptidase-G2 Enzyme Stability medicine Aromatic amino acids Amino Acid Sequence Cloning Molecular Escherichia coli Peptide sequence Aminoacylase Base Composition Ecology Base Sequence Sequence Homology Amino Acid Genetic Complementation Test Nucleic acid sequence Temperature Molecular Weight Biochemistry chemistry Genes Bacterial Food Science Biotechnology Homotetramer Plasmids Research Article |
| Zdroj: | Europe PubMed Central |
| ISSN: | 0099-2240 |
| Popis: | A genomic DNA fragment encoding aminoacylase activity of the eubacterium Bacillus stearothermophilus was cloned into Escherichia coli. Transformants expressing aminoacylase activity were selected by their ability to complement E. coli mutants defective in acetylornithine deacetylase activity, the enzyme that converts N-acetylornithine to ornithine in the arginine biosynthetic pathway. The 2.3-kb cloned fragment has been entirely sequenced. Analysis of the sequence revealed two open reading frames, one of which encoded the aminoacylase. B. stearothermophilus aminoacylase, produced in E. coli, was purified to near homogeneity in three steps, one of which took advantage of the intrinsic thermostability of the enzyme. The enzyme exists as homotetramer of 43-kDa subunits as shown by cross-linking experiments. The deacetylating capacity of purified aminoacylase varies considerably depending on the nature of the amino acid residue in the substrate. The enzyme hydrolyzes N-acyl derivatives of aromatic amino acids most efficiently. Comparison of the predicted amino acid sequence of B. stearothermophilus aminoacylase with those of eubacterial acetylornithine deacylase, succinyldiaminopimelate desuccinylase, carboxypeptidase G2, and eukaryotic aminoacylase I suggests a common origin for these enzymes. |
| Databáze: | OpenAIRE |
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