In vivo screening of haloalkane dehalogenase mutants

Haloalkane dehalogenase (DhlA) from Xanthobacter autotrophicus GJ10 catalyz es the dehalogenation of short chain primary alkyl halides. Due to the high K-m and low turnover, wild type DhlA is not optimal for applications in bi oremediation. We have developed an in vivo screen, based on a colorimetric pH indicator, to identify DhlA mutant with improved catalytic activity. aft er screening 50,000 colonies, we identified a DhlA mutant with a lower pH o ptimum. Sequence analysis of the mutant revealed a single substitution, ala nine 149 to threonine, which is located close to the active site of DhlA. R eplacement of alanine 149 via site-directed mutagenesis with threonine, ser ine or cysteine retained the mutant phenotype. Other substitutions at posit ion 149 show little or no activity. (C) 1999 Elsevier Science Ltd. All righ ts reserved.