Overexpression of Penicillin V Acylase from Streptomyces lavendulae and Elucidation of Its Catalytic Residues
The pva gene from Streptomyces lavendulae ATCC 13664, encoding a novel penicillin V acylase (SlPVA), has been isolated and characterized. The gene encodes an inactive precursor protein containing a secretion signal peptide that is activated by two internal autoproteolytic cleavages that release a 25...
| Autores: | , , , , , , , |
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| Tipo de recurso: | artículo |
| Fecha de publicación: | 2015 |
| País: | España |
| Institución: | Universidad Complutense de Madrid (UCM) |
| Repositorio: | Docta Complutense |
| Idioma: | inglés |
| OAI Identifier: | oai:docta.ucm.es:20.500.14352/23383 |
| Acceso en línea: | https://hdl.handle.net/20.500.14352/23383 |
| Access Level: | acceso abierto |
| Palabra clave: | 577.2 579 577.15 Microbiology Penicillin V Acylase Streptomyces lavendulae Biología Biología molecular (Biología) Microbiología (Biología) 24 Ciencias de la Vida 2415 Biología Molecular 2414 Microbiología |
| Sumario: | The pva gene from Streptomyces lavendulae ATCC 13664, encoding a novel penicillin V acylase (SlPVA), has been isolated and characterized. The gene encodes an inactive precursor protein containing a secretion signal peptide that is activated by two internal autoproteolytic cleavages that release a 25-amino-acid linker peptide and two large domains of 18.79 kDa (_-subunit) and 60.09 kDa (_-subunit). Based on sequence alignments and the three-dimensional model of SlPVA, the enzyme contains a hydrophobic pocket involved in catalytic activity, including Ser_1, His_23, Val_70, and Asn_272, which were confirmed by site-directed mutagenesis studies. The heterologous expression of pva in S. lividans led to the production of an extracellularly homogeneous heterodimeric enzyme at a 5-fold higher concentration (959 IU/liter) than in the original host and in a considerably shorter time. According to the catalytic properties of SlPVA, the enzyme must be classified as a new member of the Ntn-hydrolase superfamily, which belongs to a novel subfamily of acylases that recognize substrates with long hydrophobic acyl chains and have biotechnological applications in semisynthetic antifungal production. |
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