bThe 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. P enicillin acylase (PA; penicillin amidohydrolase; EC 3.5.1.11) catalyzes the hydrolysis of penicillins into 6-aminopenicillanic acid (6-APA) and the corresponding organic acid. The classification of PAs is based on their substrate specificity, i.e., penicillin G acylases (PGA) or penicillin V acylases (PVA), that preferentially cleave phenylacetyl penicillin (penicillin G [PG]) or phenoxymethyl penicillin (penicillin V [PV]), respectively (1, 2). The relevance of these enzymes lies in the fact that semisynthetic penicillins currently are industrially produced by the enzymatic hydrolysis of PG or PV.PVA is widely distributed among several microorganisms, being intra-and extracellularly produced (2-6). PVA from Streptomyces lavendulae ATCC 13664 (SlPVA) is an extracellular enzyme which has been exhaustively characterized (7-10) and immobilized (11, 12) due to its ability to hydrolyze very efficiently PV and other natural aliphatic penicillins that contaminate PV and usually reduce 6-APA yield at the end of the process. The broad substrate specificity of SlPVA allows this enzyme to hydrolyze several penicillins with aliphatic acyl chains, e.g., 3-hexenoyl-penicillin (penicillin F [PF]), hexanoyl-penicillin (penicillin dihydro-F [PdF]), and octanoyl-penicillin (penicillin K [PK]), as the catalytic constant for PK was even higher than that for PV (13). These observations indicate SlPVA is an effective industrial enzyme, provided that it can be obtained in large amounts.Here, we describe the heterologous overproduction of SlPVA in Streptomyces lividans and the characterization of its catalytic residues by site-directed mutagenesis. MATERIALS AND METHODS Materials.Penicillin V (potassium salt), penicillin G (potassium salt), phenoxyacetic acid, phenylacetic acid, aculeacin A, and fluorescamine were from Sigma-Aldrich (St. Louis, MO). 6-AP...

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A new immobilized penicillin acylase (ECPVA) was obtained by covalent binding of penicillin acylase from Streptomyces lavendulae on Eupergit C. Enzymic hydrolysis of penicillin V catalysed by ECPVA was optimized using a 2(3) factorial design of experiments, and the selected parameters for this study were pH, temperature and substrate concentration. The immobilized enzyme showed an optimal pH value of 9.5-10.5, and an optimal temperature of 60 degrees C, whereas its soluble counterpart showed the same optimal pH value and a lower optimal temperature of 50 degrees C.