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 wa s 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 Zn2+-chelating sepharose. The activity of the mo dified protein was tested after immobilization on Zn2+-chelating sepharose and on covalently bound acrylic polymer. Both immobilization systems were u sed 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 acryl ic polymer significantly extended the operational temperature and pH range of the enzyme: up to 60% of activity was recovered at either 80 degrees C o r pH 11, whereas no activity could be detected under these conditions in th e soluble or chelate-immobilized enzyme. Both forms of immobilization exten ded the enzyme effective storage periods, and after 10 cycles of reutilizat ion, 70% and 20% of the initial activity was recovered in the covalent- and chelate-immobilized enzyme, respectively.