Covalent and metal-chelate immobilization of a modified 2-haloacid dehalogenase for the enzymatic resolution of optically active chloropropionic acid |
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Authors: | Ordaz E Garrido-Pertierra A Gallego M Puyet A |
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Institution: | Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain. |
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Abstract: | The stereospecific L-2-haloacid dehalogenase DehCI from Pseudomonas CBS3 was tagged with a peptide tail containing six histidines and overexpressed in Escherichia coli. The His-tagged protein was purified after a single-step affinity chromatography on Zn(2+)-chelating sepharose. The activity of the modified protein was tested after immobilization on Zn(2+)-chelating sepharose and on covalently bound acrylic polymer. Both immobilization systems were used for the transformation of racemic 2-chloropropionic acid into D-lactate and D-chloropropionic acid. Although immobilization on chelating sepharose produced a limited increase in stability, covalent immobilization on acrylic polymer significantly extended the operational temperature and pH range of the enzyme: up to 60% of activity was recovered at either 80 degrees C or pH 11, whereas no activity could be detected under these conditions in the soluble or chelate-immobilized enzyme. Both forms of immobilization extended the enzyme effective storage periods, and after 10 cycles of reutilization, 70% and 20% of the initial activity was recovered in the covalent- and chelate-immobilized enzyme, respectively. |
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