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

Background: Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of ß-lactamantibiotics. 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 homolo...

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Detalles Bibliográficos
Autores: Torres, Leticia L., Ferreras, Eloy, Cantero, Ángel, Hidalgo, Aurelio, Berenguer, José
Tipo de recurso: artículo
Fecha de publicación:2012
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/113121
Acceso en línea:http://hdl.handle.net/10261/113121
Access Level:acceso abierto
Palabra clave:Thermozyme
Thermophile
Autoprocessing
Pre-pro-protein
Thermus thermophilus
Penicillin acylase
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spelling Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coliTorres, Leticia L.Ferreras, EloyCantero, ÁngelHidalgo, AurelioBerenguer, JoséThermozymeThermophileAutoprocessingPre-pro-proteinThermus thermophilusPenicillin acylaseBackground: Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of ß-lactamantibiotics. 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.Spanish Ministry of Science; Fundación Ramon ArecesPeer ReviewedBioMed CentralMinisterio de Ciencia y Tecnología (España)Fundación Ramón Areces2015201520122015info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501http://hdl.handle.net/10261/113121reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglésinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1131212026-05-22T06:33:51Z
dc.title.none.fl_str_mv Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli
title Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli
spellingShingle Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli
Torres, Leticia L.
Thermozyme
Thermophile
Autoprocessing
Pre-pro-protein
Thermus thermophilus
Penicillin acylase
title_short Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli
title_full Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli
title_fullStr Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli
title_full_unstemmed Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli
title_sort Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli
dc.creator.none.fl_str_mv Torres, Leticia L.
Ferreras, Eloy
Cantero, Ángel
Hidalgo, Aurelio
Berenguer, José
author Torres, Leticia L.
author_facet Torres, Leticia L.
Ferreras, Eloy
Cantero, Ángel
Hidalgo, Aurelio
Berenguer, José
author_role author
author2 Ferreras, Eloy
Cantero, Ángel
Hidalgo, Aurelio
Berenguer, José
author2_role author
author
author
author
dc.contributor.none.fl_str_mv Ministerio de Ciencia y Tecnología (España)
Fundación Ramón Areces
dc.subject.none.fl_str_mv Thermozyme
Thermophile
Autoprocessing
Pre-pro-protein
Thermus thermophilus
Penicillin acylase
topic Thermozyme
Thermophile
Autoprocessing
Pre-pro-protein
Thermus thermophilus
Penicillin acylase
description Background: Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of ß-lactamantibiotics. 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.
publishDate 2012
dc.date.none.fl_str_mv 2012
2015
2015
2015
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/113121
url http://hdl.handle.net/10261/113121
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv BioMed Central
publisher.none.fl_str_mv BioMed Central
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15.228081

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