RESUMEN
A textile-based reaction system for new peroxidase reactions in non-native media was implemented. The epoxidation of cyclohexene by the commercial peroxidase MaxiBright® was realized with the textile-immobilized enzyme in an adapted liquid-liquid two-phase reactor. A commercially available polyester felt was used as low-price carrier and functionalized with polyvinyl amine. The covalent immobilization with glutardialdehyde lead to an enzyme loading of 0.10 genzyme/gtextile. The textile-based peroxidase shows a high activity retention in the presence of organic media. This catalyst is shown to enable the epoxidation of cyclohexene in various solvents as well as under neat conditions. A model reactor was produced by 3D printing which places the textile catalyst at the interphase between the liquid reaction phase and the product extracting solvent.
Asunto(s)
Ciclohexenos/metabolismo , Enzimas Inmovilizadas/metabolismo , Peroxidasas/metabolismo , Textiles , Biocatálisis , Colorantes , Glutaral/metabolismo , Oxidación-Reducción , Solventes/metabolismoRESUMEN
TehATehB is a tellurite (TeO(2-)(3)) resistance determinant found on the Escherichia coli chromosome. Normally silent, it specifies a minimal inhibitory concentration (MIC) of 2 microg K(2)TeO(3)/ml unless upregulated or present on a multicopy plasmid which results in an MIC of 128 microg/ml. Both TehA and TehB have three cysteine residues. Oligonucleotide site-directed mutagenesis was carried out to systematically replace all six cysteine residues by alaninies. The results showed that cysteine residues in both TehA and TehB play a role in tellurite resistance: A single cysteine change had no effect, however increasing combinations of two or three cysteine substitutions demonstrated strong phenotypic effects with minimal inhibitory concentrations ranging from 16-64 microg K(2)TeO(3)/ml. A cysteine-free mutant in which all six cysteine residues were replaced by alanines maintained a MIC of 16 microg/ml. Further investigations on the role of cysteines in resistance were studied using thiol reactive reagents on the soluble subunit TehB. These studies confirmed that TehB is a dimer and undergoes a conformational change with tellurite and S-adenosyl-l-methionine binding. Studies using native and SDS denaturing PAGE under reducing and oxidizing conditions suggested that a cysteine in TehB is involved in binding tellurite.
Asunto(s)
Proteínas Bacterianas/genética , Cisteína/química , Cisteína/fisiología , Resistencia a Medicamentos , Proteínas de Escherichia coli , Telurio/farmacología , Alanina/química , Alquilantes/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Ácido Ditionitrobenzoico/farmacología , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , Escherichia coli/metabolismo , Etilmaleimida/farmacología , Yodoacetamida/farmacología , Yodoacetatos/farmacología , Ligandos , Pruebas de Sensibilidad Microbiana , Mutagénesis Sitio-Dirigida , Fenotipo , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica , Desnaturalización Proteica , S-Adenosilmetionina/química , S-Adenosilmetionina/metabolismo , Reactivos de Sulfhidrilo/farmacología , Regulación hacia ArribaRESUMEN
The Escherichia coli chromosomal determinant for tellurite resistance consists of two genes (tehA and tehB) which, when expressed on a multicopy plasmid, confer resistance to K(2)TeO(3) at 128 microg/ml, compared to the MIC of 2 microg/ml for the wild type. TehB is a cytoplasmic protein which possesses three conserved motifs (I, II, and III) found in S-adenosyl-L-methionine (SAM)-dependent non-nucleic acid methyltransferases. Replacement of the conserved aspartate residue in motif I by asparagine or alanine, or of the conserved phenylalanine in motif II by tyrosine or alanine, decreased resistance to background levels. Our results are consistent with motifs I and II in TehB being involved in SAM binding. Additionally, conformational changes in TehB are observed upon binding of both tellurite and SAM. The hydrodynamic radius of TehB measured by dynamic light scattering showed a approximately 20% decrease upon binding of both tellurite and SAM. These data suggest that TehB utilizes a methyltransferase activity in the detoxification of tellurite.