High-level expression of a novel amine-synthesizing enzyme, N-substituted formamide deformylase, in Streptomyces with a strong protein expression system
| Autor: | Yoshiteru Hashimoto, Hiroshi Fukatsu, Sachio Herai, Hiroki Higashibata, Hideaki Maseda, Michihiko Kobayashi |
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| Rok vydání: | 2004 |
| Předmět: |
chemistry.chemical_classification
Expression vector biology Base Sequence Recombinant Fusion Proteins Molecular Sequence Data Arthrobacter pascens Gene Expression Regulation Bacterial biology.organism_classification medicine.disease_cause Nitrilase Streptomyces Amidohydrolases Up-Regulation Enzyme chemistry Biochemistry Arthrobacter medicine Escherichia coli Amines Biotechnology Regulator gene |
| Zdroj: | Protein expression and purification. 40(1) |
| ISSN: | 1046-5928 |
| Popis: | N-substituted formamide deformylase (NfdA) from Arthrobacter pascens F164 is a novel deformylase involved in the metabolism of isonitriles. The enzyme catalyzes the deformylation of an N-substituted formamide, which is produced from the corresponding isonitrile, to yield the corresponding amine and formate. The nfdA gene from A. pascens F164 was cloned into different types of expression vectors for Escherichia coli and Streptomyces strains. Expression in E. coli resulted in the accumulation of an insoluble protein. However, Streptomyces strains transformed with a P(nitA)-NitR system, which we very recently developed as a regulatory gene expression system for streptomycetes, allowed the heterologous overproduction of NfdA in an active form. When Streptomyces lividans TK24 transformed with pSH19-nfdA was cultured under the optimum conditions, the NfdA activity of the cell-free extract amounted to 8.5 U/mg, which was 29-fold higher than that of A. pascens F164. The enzyme also comprised approximately 20% of the total extractable cellular protein. The recombinant enzyme was purified to homogeneity and characterized. The expression system established here will allow structural analysis and mutagenesis studies of NfdA. |
| Databáze: | OpenAIRE |
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