In vivo screening of haloalkane dehalogenase mutants Original Research Article

Haloalkane dehalogenase (DhlA) from Xanthobacter autotrophicus GJ10 catalyzes the dehalogenation of short chain primary alkyl halides. Due to the high Km and low turnover, wild type DhlA is not optimal for applications in bioremediation. We have developed an in vivo screen, based on a colorimetric pH indicator, to identify DhlA mutant with improved catalytic activity. After screening 50,000 colonies, we identified a DhlA mutant with a lower pH optimum. Sequence analysis of the mutant revealed a single substitution, alanine 149 to threonine, which is located close to the active site of DhlA. Replacement of alanine 149 via site-directed mutagenesis with threonine, serine or cysteine retained the mutant phenotype. Other substitutions at position 149 show little or no activity.