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

Abstract

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...

A Novel Quorum-Quenching N -Acylhomoserine Lactone Acylase from Acidovorax sp. Strain MR-S7 Mediates Antibiotic Resistance

“…This accords with the result of SDS-PAGE analysis of MacQ, implicating that MacQ might be synthesized as a precursor polypeptide and modified into two enzymatically active subunits by posttranslational modification ( Fig. 2 ), as other known acylases ( 19 , 25 ). Intriguingly, three residues (Ile-282 and Ser-289 in AHL acylase [AiiD] and Trp-443 in Kc PGA) associated with the substrate specificity ( 28 , 29 ) were not fully conserved in MacQ, and we identified alternative residues (i.e., Phe-283, Gln-290, and Trp-401 in MacQ) with the same alignment positions ( Fig.…”

“…The first 24 residues of MacQ are predicted to be a signal sequence by SignalP ( 24 ), implying that MacQ is an extracellular enzyme, as well as the known Ntn hydrolase proteins ( 19 , 25 ). Indeed, amino acid residues (Ser-234 and Asn-235; Fig.…”

Quorum Sensing versus Quenching Bacterial Isolates Obtained from MBR Plants Treating Leachates from Municipal Solid Waste

“…Penicillin acylases bear the ability to cleave penicillin, and, due to wide substrate specificity, also bacterial AHLs [ 60 , 64 ] Penicillin V acylase (PVA) hydrolyzes penicillin V producing 6-aminopenicillanic acid (6-APA) and phenoxyacetic acid [ 61 , 65 , 66 ], and this mechanism has been used in the present study to determine the presence of acylase activities in the four selected bacterial isolates. As can be seen in Figure 4 , all strains that were produced low levels of PVA when grown in LB medium, and, according to the results presented in the same figure, there were no important differences in PVA activity among the selected strains and controls, suggesting that the QQ activity that was ascribed to these strains were probably not due to an acylase activity.…”

Isolation And Identification Of Penicillin Acylase Producing Bacteria Originated From Cow Intestine

“…Isolation of penicillin acylase producing bacteria has been done, namely Acinetobacter sp. AP24 originating from Lake Loktak, India (Philem et al, 2015) and Streptomyces lavendulae (Torres-Bacete et al, 2015). The difference of the clear zone produced by the three bacterial isolates is closely related to the type and habitat of the bacteria.…”

“…6-APA is the result of penicillin hydrolysis by the activity of penicillin acylase. According to Torres-Bacete et al, (2015) penicillin acylase is an enzyme that catalyzes the hydrolysis of penicillin into 6-amino penicillanic acid (6-APA). Penicillin acylase is produced by various microorganisms, such as fungi and bacteria (Nandy et al, 2014).…”