Abstract
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.
Highlights
Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of β-lactam antibiotics
TthPAC is produced and processed in T. thermophilus In the first place and in order to be able to study TthPAC production and maturation, we generated antibodies against the α- and β-subunits of the PAC codified in the Thermus thermophilus (Tth) HB27 genome, separately produced in E. coli cells
When analyzing TthPAC activity in the same conditions we observed that both specific and volumetric activities in the culture without chaperone co-expression were similar to those observed in cultures with DnaK/J and GrpE or trigger factor (TF) and GroEL/ES co-expression (Figure 8b). These results indicate that the production of mature TthPAC in E. coli cells can be increased through the co-expression of appropriate chaperone(s), but the fraction of TthPAC protein that is enzymatically active cannot be assumed from the total amount of TthPAC that is produced
Summary
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. Penicillin acylase (PAC, EC 3.5.1.11) is one of the most relevant enzymes in the pharmaceutical industry. It is used in the production of 6-amino penicillanic acid (6-APA), which is subsequently used in the chemical synthesis of new lactams with greater effectiveness. PACs. Industrially, the penicillin G acylase (PGA) from Escherichia coli is the enzyme of choice, whether recombinant or native. Operational stability of the catalytic process could be strongly improved by the use of enzymes with a longer half-life under the reaction conditions regularly used
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
