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1.
Appl Environ Microbiol ; 79(5): 1555-62, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23263966

ABSTRACT

A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position α188, α189, or ß24 improved the K(m) for penicillin G between 9- and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.


Subject(s)
Penicillin Amidase/genetics , Penicillin Amidase/metabolism , Protein Engineering , Thermus thermophilus/enzymology , Amino Acid Substitution , Catalytic Domain , Escherichia coli/enzymology , Escherichia coli/genetics , Models, Molecular , Penicillin Amidase/isolation & purification , Penicillin G/metabolism , Penicillins/metabolism , Protein Conformation , Substrate Specificity , Thermus thermophilus/genetics
2.
Microb Cell Fact ; 11: 105, 2012 Aug 09.
Article in English | MEDLINE | ID: mdl-22876915

ABSTRACT

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


Subject(s)
Bacterial Proteins/genetics , Escherichia coli/genetics , Gene Expression , Penicillin Amidase/genetics , Thermus thermophilus/enzymology , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Enzyme Stability , Escherichia coli/metabolism , Hot Temperature , Penicillin Amidase/chemistry , Penicillin Amidase/metabolism , Penicillins/chemistry , Penicillins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate Specificity
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