Covalent and metal-chelate immobilization of a modified 2-haloacid dehalogenase for the enzymatic resolution of optically active chloropropionic acid.
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.
Ordaz
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E
Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040 Madrid, Spain.
Garrido-Pertierra
A
A
Gallego
M
M
Puyet
A
A
D002614:Chelating Agents / Q000737:chemistry*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D006843:Hydrocarbons, Chlorinated; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D011422:Propionates / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000737:chemistry*
D002614:Chelating Agents; D004800:Enzymes, Immobilized; D006843:Hydrocarbons, Chlorinated; D011422:Propionates; D011993:Recombinant Fusion Proteins; D006639:Histidine; C021974:2-chloropropionic acid; D006867:Hydrolases; C034507:2-haloacid dehalogenase; D015032:Zinc