A pH-indicator-based screen for hydrolytic haloalkane dehalogenase.

1. Introduction Microbial hydrolytic haloalkane dehalogenases catalyze the cleavage of halogen-carbon bonds of a variety of aliphatic halogenated compounds, including a broad range of chlorinated (C 2 –C 6) and brominated (C 2 –C 8) alkanes, with water as the sole co-substrate, resulting in the production of halide ions, protons, and alcohols (1,2). Based primarily on substrate specificity and sequence homology, these enzymes have been classified into two general classes that are represented by the enzymes from Xanthobacter autotrophicus GJ10 and Rhodococcus rhodochrous (3). The study of these enzymes has been motivated largely by their potential use in waste treatment, bioremediation and industrial biocatalysis (4,5). A substantial amount of mechanistic and structural information is available for these enzymes (6–9). The haloalkane dehalogenase from Rhodococcus rhodochrous (RrDHL) is of particular interest because it is capable of selectively converting several industrially important commodity chemicals, including 1,2-dichloropropane (DCP), 1,2,3-trichloropropane (TCP), and 1,2-dichlorobutane (DCB), into more valuable chlorohydrins. Traditional chemical catalysts cannot do this. These chemicals are generated at a scale of several hundred million pounds per year as side products in the existing manufacturing processes of propylene oxide, epichlorohydrin, and butylene oxide, and are presently incinerated at a cost. Thus, developing RrDHL-based biocatalytic processes to recover value from these chloroalkanes represents an important environmental and process opportunity (4). Unfortunately, as a practical biocatalyst, this RrDHL enzyme suffers from low activity and low stability under the industrial process conditions.