Purification and characterization of penicillin V acylase from Streptomyces lavendulae

Torres, R.; Mata, Isabel de la; Castillón, M.P.; Arroyo, Μ.; Torres, José Carlos Bravo; Acebal, Carmen

doi:10.1016/s0921-0423(98)80109-3

Cited by 10 publications

(14 citation statements)

References 9 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It could be argued that the recognized SlPVA activity on natural penicillins is due to the partial contamination of the original enzyme preparation, but we have found that the rSlPVA behaves as the native purified acylase (7,8,13). In this sense, the enzyme showed the highest activity at 55°C and pH 9.5 to 10.0 when PV was used as the substrate.…”

Section: Resultsmentioning

confidence: 69%

“…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%

“…The acylase activity was routinely assayed using PV as the substrate. The acylase activity present in the culture supernatants of S. lavendulae ATCC 13664 and the recombinant S. lividans strains was determined according to the method described previously (8). Crude protein samples were prepared by growing the cells in the appropriate production media.…”

Section: Methodsmentioning

confidence: 99%

“…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.…”

mentioning

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%

See 4 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

View full text Add to dashboard Cite

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...

show abstract

Section: Resultsmentioning

confidence: 69%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

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

View full text Add to dashboard Cite

show abstract

Stabilization of penicillin V acylase from Streptomyces lavendulae by covalent immobilization

Torres‐Bacete

Arroyo

Torres‐Guzmán

et al. 2001

J of Chemical Tech & Biotech

View full text Add to dashboard Cite

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.

show abstract

Characterization of smallest active monomeric penicillin V acylase from new source: A yeast, Rhodotorula aurantiaca (NCIM 3425)

Kumar

Prabhune

Suresh

et al. 2008

Process Biochemistry

View full text Add to dashboard Cite

Purification and characterization of penicillin V acylase from Streptomyces lavendulae

Cited by 10 publications

References 9 publications

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

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

Stabilization of penicillin V acylase from Streptomyces lavendulae by covalent immobilization

Characterization of smallest active monomeric penicillin V acylase from new source: A yeast, Rhodotorula aurantiaca (NCIM 3425)

Contact Info

Product

Resources

About