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Hydrolysis of penicillins and related compounds by the cell-bound penicillin acylase of Escherichia coli.

ABSTRACT: 1. A method is given for the preparation of penicillin acylase by using Escherichia coli N.C.I.B. 8743 and a strain selected for higher yield. The enzyme is associated with the bacterial cells and removes the side chains of penicillins to give 6-amino-penicillanic acid and a carboxylic acid. 2. The rates of penicillin deacylation indicated that p-hydroxybenzylpenicillin was the best substrate, followed in diminishing order by benzyl-, dl-alpha-hydroxybenzyl-, 2-furylmethyl-, 2-thienylmethyl-, d-alpha-aminobenzyl-, n-propoxymethyl- and isobutoxymethyl-penicillin. Phenylpenicillin and dl-alpha-carboxybenzylpenicillin were not substrates and phenoxymethyl-penicillin was very poor. 3. Amides and esters of the above penicillins were also substrates for the deacylation reaction, as were cephalosporins with a thienylmethyl side chain. 4. For the deacylation of 2-furylmethylpenicillin at 21 degrees the optimum pH was 8.2. The optimum temperature was 60 degrees at pH7. 5. By using selection A of N.C.I.B. 8743 and determining reaction velocities by assaying yields of 6-amino-penicillanic acid in a 10min. reaction at 50 degrees and pH8.2, the K(m) for benzylpenicillin was found to be about 30mm and the K(m) for 2-furylmethylpenicillin, about 10mm. The V(max.) values were 0.6 and 0.24mumole/min./mg. of bacterial cells respectively.

SUBMITTER: Cole M

PROVIDER: S-EPMC1185200 | biostudies-other | 1969 Dec

REPOSITORIES: biostudies-other

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Project description:BackgroundPenicillin 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.ResultsFusions 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.ConclusionsThis 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.

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Hydrolysis of penicillins and related compounds by the cell-bound penicillin acylase of Escherichia coli.

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