Enhanced production of penicillin V acylase from Streptomyces lavendulae

Torres, R.; Ramón, Fernando; Mata, Isabel de la; Acebal, Carmen; Castillón, M.P.

doi:10.1007/s002530051618

Cited by 29 publications

(23 citation statements)

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“…The highest rSlPVA production was reached after 96 h of incubation, in contrast to the 275 h required by the native S. lavendulae strain (7). The recombinant strain produced 950 IU/liter of rSlPVA, which is a 5-fold higher yield than that found under the best production conditions of S. lavendulae (7). As expected, rSlPVA is secreted in S. lividans as a homogeneous heterodimeric form (Fig.…”

Section: Resultssupporting

confidence: 60%

“…The bacterial strains, plasmids, and oligonucleotides used are listed in Table 1. S. lavendulae ATCC 13664 was used as a PVA producer and DNA source (7,8). Escherichia coli DH5␣ was used as the host for subcloning experiments, and S. lividans 1326 was used as the host for gene expression.…”

Section: Methodsmentioning

confidence: 99%

“…SlPVA from S. lavendulae ATCC 13664 was produced as previously described (7,8) and purified with slight modifications, using hydroxyapatite for the last step instead of Ultrogel AcA44. Protein concentration was determined according to Bradford (21).…”

Section: Methodsmentioning

confidence: 99%

“…To determine substrate specificity, pure recombinant enzyme (0.5 g) was incubated with increasing concentrations of different penicillins (e.g., PV, PK, PF, PdF, and PG) in 100 mM potassium phosphate buffer, pH 8.0, at 40°C for 20 min in a final volume of 100 l. The reaction was stopped by addition of 400 l of 0.5 M sodium acetate. After centrifugation of the samples, the released 6-APA was monitored with fluorescamine (7,29). The reaction was linear under these assay conditions.…”

Section: Methodsmentioning

confidence: 99%

“…PVA from Streptomyces lavendulae ATCC 13664 (SlPVA) is an extracellular enzyme which has been exhaustively characterized (7)(8)(9)(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).…”

mentioning

confidence: 99%

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Overexpression of Penicillin V Acylase from Streptomyces lavendulae and Elucidation of Its Catalytic Residues

Torres‐Bacete

Hormigo

Torres‐Guzmán

et al. 2015

Appl Environ Microbiol

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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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Section: Resultssupporting

confidence: 60%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Overexpression of Penicillin V Acylase from Streptomyces lavendulae and Elucidation of Its Catalytic Residues

Torres‐Bacete

Hormigo

Torres‐Guzmán

et al. 2015

Appl Environ Microbiol

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Stabilization of penicillin V acylase from Streptomyces lavendulae by covalent immobilization

Torres‐Bacete

Arroyo

Torres‐Guzmán

et al. 2001

J of Chemical Tech & Biotech

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Penicillin V acylase from the actinomycete Streptomyces lavendulae ATCC 13664 has been immobilized to epoxy-activated acrylic beads (Eupergit C 1 ) by covalent binding. Further linkage of bovine serum albumin after enzyme immobilization was carried out in order to remove the remaining oxirane groups of the support. The obtained immobilized biocatalyst displayed double exponential deactivation kinetics at temperatures below 55°C, while the native enzyme followed single exponential decay at the same temperatures. We concluded that soluble penicillin acylase was deactivated in one step, whereas the immobilized enzyme showed an enzymatic intermediate state which is highly thermostable. As a consequence of the immobilization process, the enzyme displayed a 10-fold increase in its half-life at 40°C. At this temperature, the enzymatic intermediate state was progressively destabilized as the pH of the medium was increased. Thus, the optimum pH range for the immobilized enzyme preparation was established as being from 7.0 to 8.0. Higher pH values led to quicker enzyme deactivation.

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