Torres Bacete, JesúsHormigo Cisneros, DanielTorres Guzmán, RaquelArroyo, MiguelCastillón, María PilarGarcía, José LuisAcebal Sarabia, CarmenMata Riesco, Isabel de la2015-10-212015-10-212015Torres-Bacete, J., Hormigo, D., Torres-Gúzman, R., Arroyo, M., Castillón, M. P., García, J. L., ... & de la Mata, I. (2015). Overexpression of Penicillin V Acylase from Streptomyces lavendulae and Elucidation of Its Catalytic Residues. Applied and environmental microbiology, 81(4), 1225-1233.0099224010985336http://hdl.handle.net/11268/4436The 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 (α-subunit) and 60.09 kDa (β-subunit). Based on sequence alignments and the three-dimensional model of SlPVA, the enzyme contains a hydrophobic pocket involved in catalytic activity, including Serβ1, Hisβ23, Valβ70, and Asnβ272, 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.engjournal article10.1128/AEM.02352-14open accessBiotecnologíaEnzimasBiotecnologíaEnzima