Penicillin amidase

About: Penicillin amidase is a research topic. Over the lifetime, 576 publications have been published within this topic receiving 15563 citations. The topic is also known as: penicillin amidohydrolase & ampicillin acylase.

Selectivity of the enzymatic synthesis of ampicillin by E. coli PGA in the presence of high concentrations of substrates

Abstract: Penicillin G acylase (PGA) catalyzes the synthesis/hydrolysis of acyl derivatives of phenylacetic acid through the formation of a covalent intermediate (the acyl–enzyme complex). When used for the kinetically controlled synthesis of β-lactam antibiotics, this enzyme promotes two undesired side reactions: the hydrolysis of the acyl side-chain precursor and of the antibiotic. Therefore, a high selectivity (synthesis/hydrolysis, S/H ratio) is very important for the process economics. Here, the enzymatic synthesis of ampicillin from d -phenylglycine methyl ester (PGME) and 6-aminopenicillic acid (6-APA), using PGA from Escherichia coli (EC 3.5.1.11) is studied. Kinetic assays provided S/H for high concentrations of substrates (up to 200 mM of 6-APA and 500 mM of PGME), using soluble PGA, at 25 °C, pH 6.5. S/H increased with 6-APA concentration, in accordance with the literature. However, when the concentration of 6-APA approached saturation, the rate of enzymatic hydrolysis tended towards zero (i.e., S/H tended to infinity). On the other hand, when the concentration of ester was augmented, S/H consistently decreased. This behavior, to the best of our knowledge still not reported, indicates that the acylation step may occur with 6-APA already positioned for the nucleophilic attack.

The production of 6‐aminopenicillanic acid by a multistage tubular reactor packed with immobilized penicillin amidase

Abstract: A theoretical model equation was derived to find the correlation between the conversion and the amount of immobilized penicillin amidase in column. The theoretical values of the conversion were predicted form this correlation and compared with experimental results. It was observed in a column reactor that the pH drop along the column path was linear versus the enzyme loading and that the enzyme activity was also linearly dependent on pH up to 8.0. In order to diminish the effect of pH drop, a continuous two-stage plug-flow reactor (PFR) with pH adjustment between the two columns was used was used in the experiments, and two- and three-stage PFRs were simulated by computer. In the case of the two-stage PFR, the maximum productivity was demonstrated experimentally and theoretically as well. when an equal amount of the immobilized enzyme was packed in both columns. It was also predicted in the tree-stage PFR system that the optimal distributions of enzyme loading in three columns were found to be 1:1:1. It was demonstrated that the increased number of reactors in series could enhance the level of the maximum productivity with a given amount of enzyme loading.

High-throughput strategies for penicillin G acylase production in rE. coli fed-batch cultivations

Abstract: Penicillin G acylase (PGA) is used industrially to catalyze the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid. In Escherichia coli, the most-studied microorganism for PGA production, this enzyme accumulates in the periplasmic cell space, and temperature plays an important role in the correct synthesis of its subunits. This work investigates the influence of medium composition, cultivation strategy, and temperature on PGA production by recombinant E. coli cells. Shake flask cultures carried out using induction temperatures ranging from 18 to 28°C revealed that the specific enzyme activity achieved at 20°C (3000 IU gDCW-1) was 6-fold higher than the value obtained at 28°C. Auto-induction and high cell density fed-batch bioreactor cultures were performed using the selected induction temperature, with both defined and complex media, and IPTG and lactose as inducers. Final biomass concentrations of 100 and 120 gDCW L-1, and maximum enzyme productivities of 7800 and 5556 IU L-1 h-1, were achieved for high cell density cultures using complex and defined media, respectively. To the best of our knowledge, the volumetric enzyme activity and productivity values achieved using the complex medium are the highest ever reported for PGA production using E. coli. Overall PGA recovery yields of 64 and 72% after purification were achieved for crude extracts obtained from cells cultivated in defined and complex media, respectively. The complex medium was the most cost-effective for PGA production, and could be used in both high cell density and straightforward auto-induction protocols.