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.

Fragments of pro-peptide activate mature penicillin amidase of Alcaligenes faecalis.

Abstract: Penicillin amidase from Alcaligenes faecalis is a recently identified N-terminal nucleophile hydrolase, which possesses the highest specificity constant (kcat/Km) for the hydrolysis of benzylpenicillin compared with penicillin amidases from other sources. Similar to the Escherichia coli penicillin amidase, the A. faecalis penicillin amidase is maturated in vivo from an inactive precursor into the catalytically active enzyme, containing one tightly bound Ca2+ ion, via a complex post-translational autocatalytic processing with a multi-step excision of a small internal pro-peptide. The function of the pro-region is so far unknown. In vitro addition of chemically synthesized fragments of the pro-peptide to purified mature A. faecalis penicillin amidase increased its specific activity up to 2.3-fold. Mutations were used to block various steps in the proteolytic processing of the pro-peptide to obtain stable mutants with covalently attached fragments of the pro-region to their A-chains. These extensions of the A-chain raised the activity up to 2.3-fold and increased the specificity constants for benzylpenicillin hydrolysis mainly by an increase of the turnover number (kcat).

Kinetics and reaction engineering of penicillin G hydrolysis

Abstract: A general diffusion reaction model for immobilised biocatalysis has been developed. The model has been used to study the deacylation of penicillin G to 6-aminopenicillanic acid using two commercially available immobilised Penicillin acylases. The values of D e /R 2 for the enzyme pellets have been estimated using data on uptake of 6-aminopenicillanic acid and phenylacetic acid by the enzyme pellets. The kinetic parameters of the model were individually estimated from a suitably designed set of experiments. The values of the Thiele modulus from the kinetic parameters so calculated have been found to be in the range 1.67 to 9.8 for the two enzymes studied, implying that diffusional effects cannot be ignored. The effect of such diffusional limitations on the overall rates and hence on the utilisation of the intrinsic kinetic ability of the enzyme has been demonstrated. This paper also reports on the implementation of the fed-batch strategy for this system. The proposed strategy, which involves maintaining the substrate concentration at the optimum value for a large part of the conversion, results in higher product concentrations than in batch operation, thereby reducing downstream procesing costs. The productivity was also shown to be considerably higher than for batch operation. Further, the ease of implementation of this mode of operation has been demonstrated.

3D‐QSAR Applied to the Quantitative Prediction of Penicillin G Amidase Selectivity

Abstract: A new approach for predicting the selectivity of penicillin G amidase (PGA) - expressed as k cat / K M - is here described. Regression models were constructed correlating the experimentally determined k cat /K M of a limited number of substrates to molecular descriptors calculated by using methods generally employed in drug discovery for quantitative structure-activity relationship (3D-QSAR methods). Two different methods for the calculation of molecular descriptors have been tested, namely GRIND and Volsurf. The real predictions, made on molecules not used for constructing the models, had an accuracy sufficient for being useful in the experimental practice. Both approaches led to models able to predict substrate selectivity even without modelling the enzyme-substrate complex, whereas the prediction of enantioselectivity was feasible only by combining the GRIND approach with the conformational analysis of the substrates inside the enzyme's active site. The present approach represents an actual alternative to screening procedures since it allows one to develop a whole predicting model in a few hours, once a small set of experimental data is made available.

Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli.

Abstract: Background: Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of β-lactam antibiotics. Development of a PAC with a longer half-life under the reaction conditions used is essential for the improvement of the operational stability of the process. A gene encoding a homologue to Escherichia coli PAC was found in the genome of the thermophilic bacterium Thermus thermophilus (Tth) HB27. Because of the nature of this PAC and its complex maturation that is crucial to reach its functional heterodimeric final conformation, the overexpression of this enzyme in a heterologous mesophilic host was a challenge. Here we describe the purification and characterization of the PAC protein from Tth HB27 overexpressed in Escherichia coli. Results: Fusions to a superfolder green fluorescent protein and differential membrane solubilization assays indicated that the native enzyme remains attached through its amino-terminal end to the outer side of the cytoplasmic membrane of Tth cells. In order to overexpress this PAC in E. coli cells, a variant of the protein devoid of its membrane anchoring segment was constructed. The effect of the co-expression of chaperones and calcium supplementation of the culture medium was investigated. The total production of PAC was enhanced by the presence of DnaK/J and GrpE and even more by trigger factor and GroEL/ES. In addition, 10 mM calcium markedly improved both PAC specific and volumetric activities. Recombinant PAC was affinity-purified and proper maturation of the protein was confirmed by SDS-PAGE and MALDI-TOF analysis of the subunits. The recombinant protein was tested for activity towards several penicillins, cephalosporins and homoserine lactones. Hydrophobic acyl-chain penicillins were preferred over the rest of the substrates. Penicillin K (octanoyl penicillin) was the best substrate, with the highest specificity constant value (16.12 mM -1 .seg -1 ). The optimum pH was aprox. 4 and the optimum temperature was 75 °C. The half-life of the enzyme at this temperature was 9.2 h. Conclusions: This is the first report concerning the heterologous expression of a pac gene from a thermophilic microorganism in the mesophilic host E. coli. The recombinant protein was identified as a penicillin K-deacylating thermozyme.