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Covalent and Metal‐Chelate Immobilization of a Modified 2‐Haloacid Dehalogenase for the Enzymatic Resolution of Optically Active Chloropropionic Acid
Author(s) -
Ordaz E.,
GarridoPertierra A.,
Gallego M.,
Puyet A.
Publication year - 2000
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp990134k
Subject(s) - chelation , sepharose , chemistry , covalent bond , enzyme , dehalogenase , immobilized enzyme , affinity chromatography , chromatography , stereospecificity , peptide , enzyme assay , biochemistry , organic chemistry , catalysis
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 °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.