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Overexpression of Penicillin V Acylase from Streptomyces lavendulae and Elucidation of Its Catalytic Residues Torres Bacete, Jesús Hormigo, Daniel Torres Guzmán, Raquel Arroyo Sánchez, Miguel Castillón, María Pilar García, José Luis Acebal Sarabia, Carmen De La Mata Riesco, Mª Isabel 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 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. Ministerio de Ciencia e Innovación (MICINN) Ministerio de Educación y Ciencia Sección Deptal. de Bioquímica y Biología Molecular (Biológicas) Fac. de Ciencias Biológicas TRUE pub 2023-06-18T05:48:28Z 2023-06-18T05:48:28Z 2015-02 journal article https://hdl.handle.net/20.500.14352/23383 0099-2240 10.1128/AEM.02352-14 eng BIO2005-07250 BIO2008- 03928 Atribución 3.0 España https://creativecommons.org/licenses/by/3.0/es/ open access application/pdf ASM