The first step in the utilization of the xenobiotic chlorinated hydrocarbon 1,2-dichloroethane by Xanthobacter autotrophicus is catalyzed by haloalkane dehalogenase (Dh1A). The enzyme hydrolyses 1-haloalkanes to the corresponding alcohols. This allows the organism to grow also on short-chain (C2-C4) 1-chloro-n-alkanes. We have expressed Dh1A in a strain of Pseudomonas that grows on long-chain alcohols and have selected 12 independent mutants that utilize 1-chlorohexane. Six different mutant

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... mes with improved Km or Vmax values with 1-chlorohexane were obtained. The sequences of the mutated dh1A genes showed that several mutants had the same 11-amino acid deletion, two mutants carried a different point mutation, and three mutants had different tandem repeats. All mutations occurred in a region encoding the N-terminal part of the cap domain of Dh1A, and it is concluded that this part of the protein is involved in the evolution of activity toward xenobiotic substrates.