{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,28]],"date-time":"2025-10-28T14:51:26Z","timestamp":1761663086835},"reference-count":41,"publisher":"Springer Science and Business Media LLC","issue":"1","content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Microb Cell Fact"],"published-print":{"date-parts":[[2012,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n          <jats:sec>\n            <jats:title>Background<\/jats:title>\n            <jats:p>Penicillin acylases (PACs) are enzymes of industrial relevance in the manufacture of \u03b2-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 <jats:italic>Escherichia coli<\/jats:italic> PAC was found in the genome of the thermophilic bacterium <jats:italic>Thermus thermophilus<\/jats:italic> (<jats:italic>Tth<\/jats:italic>) 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 <jats:italic>Tth<\/jats:italic> HB27 overexpressed in <jats:italic>Escherichia coli.<\/jats:italic>\n            <\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>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 <jats:italic>Tth<\/jats:italic> cells. In order to overexpress this PAC in <jats:italic>E. coli<\/jats:italic> 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<jats:sup>-1<\/jats:sup>.seg<jats:sup>-1<\/jats:sup>). The optimum pH was aprox. 4 and the optimum temperature was 75 \u00b0C. The half-life of the enzyme at this temperature was 9.2 h.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusions<\/jats:title>\n            <jats:p>This is the first report concerning the heterologous expression of a <jats:italic>pac<\/jats:italic> gene from a thermophilic microorganism in the mesophilic host <jats:italic>E. coli<\/jats:italic>. The recombinant protein was identified as a penicillin K-deacylating thermozyme.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/1475-2859-11-105","type":"journal-article","created":{"date-parts":[[2012,8,9]],"date-time":"2012-08-09T12:14:22Z","timestamp":1344514462000},"update-policy":"http:\/\/dx.doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Functional expression of a penicillin acylase from the extreme thermophile Thermus thermophilus HB27 in Escherichia coli"],"prefix":"10.1186","volume":"11","author":[{"given":"Leticia L","family":"Torres","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Eloy R","family":"Ferreras","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"\u00c1ngel","family":"Cantero","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aurelio","family":"Hidalgo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jos\u00e9","family":"Berenguer","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2012,8,9]]},"reference":[{"key":"704_CR1","doi-asserted-by":"publisher","first-page":"341","DOI":"10.1074\/jbc.M309281200","volume":"279","author":"J Yoon","year":"2004","unstructured":"Yoon J, Oh B, Kim K, Park J, Han D, Kim KK, Cha S-S, Lee D, Kim Y: A Bound Water Molecule Is Crucial in Initiating Autocatalytic Precursor Activation in an N-terminal Hydrolase. 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