Cloning, nucleotide sequence and expression of a hydantoinase and carbamoylase gene from Arthrobacter aurescens DSM 3745 in Escherichia coli and comparison with the corresponding genes from Arthrobacter aurescens DSM 3747
| Autor: | Christoph Syldatk, Josef Altenbuchner, Anja Wiese, Burkhard Wilms, Ralf Mattes |
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| Rok vydání: | 2001 |
| Předmět: |
Monosaccharide Transport Proteins
Base pair Recombinant Fusion Proteins Chitin Molecular cloning Biology medicine.disease_cause Applied Microbiology and Biotechnology Maltose-Binding Proteins Homology (biology) Amidohydrolases Substrate Specificity Enzyme Stability Escherichia coli medicine Arthrobacter Cloning Molecular Peptide sequence Gene Escherichia coli Proteins Hydantoins Temperature Nucleic acid sequence Stereoisomerism Sequence Analysis DNA General Medicine Hydrogen-Ion Concentration Molecular biology Protein Structure Tertiary Biochemistry Genes Bacterial Periplasmic Binding Proteins Dihydropyrimidinase ATP-Binding Cassette Transporters Carrier Proteins Biotechnology |
| Zdroj: | Applied Microbiology and Biotechnology. 55:750-757 |
| ISSN: | 1432-0614 0175-7598 |
| Popis: | The genes encoding hydantoinases (hyuH1) and carbamoylases (hyuC1) from Arthrobacter aurescens DSM 3745 and Arthrobacter aurescens DSM 3747 (hyuH2, hyuC2) were cloned in Escherichia coli and the nucleotide sequences determined. The hydantoinase genes comprised 1,377 base pairs and the carbamoylase genes 1,239 base pairs each. Both hydantoinases, as well as both carbamoylases, showed a high degree of nucleotide and amino acid sequence identity (96-98%). The hyuH and hyuC genes were expressed in E. coli under the control of the rhamnose promoter and the different specific activities obtained in E. coli crude extracts were compared to those produced by the original hosts. For purification the hyuH2 gene was expressed as a maltose-binding protein (MalE) and as an intein-chitin binding domain (CBD) fusion in E. coli. The expression of malE-hyuH2 resulted in the production of more soluble and active protein. With respect to temperature stability, optimal pH and optimal temperature, substrate and stereospecificity, the purified fusion enzyme exhibited properties similar to those of the wild-type enzyme. |
| Databáze: | OpenAIRE |
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