Substrate Specificity of Penicillin Acylase from Streptomyces lavendulae

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

“…Furthermore, the kinetic parameters for the rSlPVA hydrolysis of different natural ␤-lactam antibiotics, i.e., PV, PK, PF, PdF, and PG, have been determined (Table 3). The values were similar to that reported for the original SlPVA (13), showing the highest bimolecular constant (specificity constant) value, k cat /K m ϭ 244.11 mM Ϫ1 s Ϫ1 , with penicillin K as the substrate, indicating that the recombinant enzyme is processed and matured as in S. lavendulae. Remarkably, rSlPVA is able to hydrolyze aculeacin A, showing a specific activity of 13 mol/min/ mg. To date, no other PA enzyme described in the literature displays such hydrolytic capability, opening new and interesting bio- Active site of SlPVA.…”

“…S2 in the supplemental material). Interestingly, these five enzymes show similar substrate specificity with respect to the acyl moiety, because they catalyze the deacylation of substrates with long hydrophobic acyl moieties (4,13,48,49,60,61). In this sense, we have shown in this work that SlPVA is able to hydrolyze not only penicillins but also aculeacin A; in addition, rSlPVA hydrolyzes AHLs (data not shown), raising new questions about the genuine role of SlPVA in nature.…”

“…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.…”

“…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). These observations indicate SlPVA is an effective industrial enzyme, provided that it can be obtained in large amounts.…”

Newly Discovered Penicillin Acylase Activity of Aculeacin A Acylase from Actinoplanes utahensis

“…Remarkably, although this acylase shares sequence similarity with ␤-lactam acylases (8,11), it has not been reported to show penicillin acylase activity. Preliminary analyses carried out in our laboratory showed that AuAAC was very similar to the ␤-lactam acylase from Streptomyces lavendulae (AY611030), which shows a preference for penicillins with long hydrophobic acyl moieties (21). On the other hand, although AuAAC has been purified (8,9,18), few data on its structure function relationships have been provided so far and many biochemical properties of these proteins remain to be analyzed.…”