Jesús Torres-Bacete

Optimization of 6-aminopenicillanic acid (6-APA) production by using a new immobilized penicillin acylase.

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 23 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 °C, whereas its soluble counterpart showed the same optimal pH value and a lower optimal temperature of 50 °C.

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

ABSTRACT Aculeacin A acylase from Actinoplanes utahensis produced by Streptomyces lividans revealed acylase activities that are able to hydrolyze penicillin V and several natural aliphatic penicillins. Penicillin K was the best substrate, showing a catalytic efficiency of 34.79 mM−1 s−1. Furthermore, aculeacin A acylase was highly thermostable, with a midpoint transition temperature of 81.5°C.

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

ABSTRACT The 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 (α-subunit) and 60.09 kDa (β-subunit). Based on sequence alignments and the three-dimensional model of SlPVA, the enzyme contains a hydrophobic pocket involved in catalytic activity, including Serβ1, Hisβ23, Valβ70, and Asnβ272, 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.

Optimization of culture medium and conditions for penicillin acylase production by Streptomyces lavendulae ATCC 13664

The culture medium for Streptomyces lavendulae ATCC 13664 was optimized on a shake-flask scale by using a statistical factorial design for enhanced production of penicillin acylalse. This extracellularenzyme recently has been reported to bea penicillin Kacylase, presenting also high hydrolytic activity against penicillin V and other natural aliphatic penicillins such as penicillin K, penicillin F, and penicillin dihydroF,. The factorial design indicated that the main factors that positively affect penicillin acylase production by S. lavendulae were the concentration of yeast extract and the presence of oligoelements in the fermentation medium, whereas the presence of olive oil in the medium had no effect on enzyme production. An initial concentration of 2.5% (w/v) yeast extract and 3 μg/mL of CuSO4·5H2O was found to be best for acylase production. In such optimized culture medium, fermentation, of the microorganism yielded 289 IU/L of enzyme in 72 h when employing a volume medium/volume flask ratio of 0.4 and a 300-rpm shaking speed. The presence of copper, alone and in combination with other metals, stimulated biomass as well as penicillin acylase production. The time course of penicillin acylase production was also studied in the optimized medium and conditions. Enzyme production showed catabolite repression by different carbon sources such as glucose, lactose, citrate, glycerol, and glycine.

The Kinetic Mechanism of Penicillin V Acylase from Streptomyces Lavendulae

Penicillin Vacylase isolated from Streptomyces lavendulae is an extracellular enzyme with interesting properties for the industrial production of 6-APA. The enzyme hydrolysed penicillin V and synthetic analogues such as 2-nitro-5-(phenoxyacetarnido)-benzoic acid (NTPOAB). The corresponding kinetic parameters were KM = 3 mM and V max = 92 μmol min-1 mg-1 for penicillin V; KM = 15.3 mM and V max = 14.5 μmol. min-1 mg-1 for NIPOAB. Studies of inhibition by products, alternative products and dead-end inhibition supported an ordered uni bi mechanism that could involve an acyl-enzyme intermediate as has been described for penicillin G acylases and other amidases.

Chemical mechanism of penicillin V acylase from Streptomyces lavendulae: pH-dependence of kinetic parameters

Abstract The variation of kinetic parameters of penicillin V acylase from Streptomyces lavendulae with pH was used to gain information about the chemical mechanism of the hydrolysis of penicillin V catalyzed by this enzyme. The pH-dependence of V max showed that a group with a p K value of 6.45 (p K 1 ) must be unprotonated for activity. The pH-dependence of V max / K m showed that a group with a p K value of 7.1 (p K 1 ) must be unprotonated and a group with a p K of 10.83 (p K 2 ) must be protonated for activity. The lower p K value corresponded to a group in the enzyme involved in catalysis and whose protonation state also affects binding. The higher p K value was only involved in binding. Results from chemical modification studies showed the importance of serine residues in the catalytic mechanism of the enzyme and pointed to the identity of the groups responsible for p K 1 and p K 2 as the α-amino nitrogen of the N-terminal residue and a lysine residue, respectively.

Kinetic mechanism of penicillin V acylase activation byshort-chain alcohols

Alcohol-induced activation of penicillin V acylase from Streptomyces lavendulae by some water-miscible primary alcohols such as methanol, ethanol, 1-propanol and 1-butanol has been extensively studied. The effects of various concentrations of these alcohols on the maximum velocity of penicillin V hydrolysis (Vmax) and the apparent Michaelis-Menten constant (Km) of the enzyme are reported. Lineweaver-Burk analyses of the kinetic data revealed a phenomenon of nonessential activation in the presence of short-chain primary alcohols. In the concentration range where activation was observed, conventional secondary replots of 1/Δ slope or 1/Δ intercept versus 1/[alcohol] yielded perfect straight lines, demonstrating the binding of a single molecule of alcohol to an activator site in penicillin V acylase. Furthermore, the enzyme displayed a propensity to bind the longer chain alcohols, as reflected by the dissociation constant KA values of 493, 369, 228 and 11.8 mM for methanol, ethanol, 1-propanol and 1-butanol, respectively. Therefore, we have proposed a high affinity activator site for alcohols in the enzyme. Very hydrophobic alcohols such as 1-butanol bind more efficiently to this activator site and convert the enzyme form one conformation to another one, which is catalytically more effective than the alcohol-free form of the penicillin acylase. Further addition of alcohol results in a competitive inhibition of the enzyme in its activated state. An inverse relationship between the chain length of the alcohols and the breakpoints in the biphasic normalized vmax/Km ratio versus alcohol concentration plots was observed.

Chromogenic analogues of penicillin dihydroF and penicillin K for the continuous spectrophotometric determination of aliphatic penicillin acylase activity

The synthesis of 2-nitro-5-[(hexanoyl)-amino]-benzoic acid and 2-nitro-5-[(octanoyl)-amino]-benzoic acid as chromogenic substrates for the determination of aliphatic penicillin acylase activity is described. During enzymatic hydrolysis, the released chromophore, 2-nitro-5-amino-benzoic acid, was detected at 405 nm. Penicillin acylase from Streptomyces lavendulae had an appreciable activity towards these substrates, which can then be used to detect penicillin acylases able to cleave hexanoyl and octanoyl residues off synthetic amides as well as penicillin dihydroF and penicillin K, their natural analogues.