Cloning, expression, purification and three-dimensional structure prediction of haloalkane dehalogenase from a recently isolated Ancylobacter aquaticus strain UV5

• A haloalkane dehalogenase (DhlA) from Ancylobacter aquaticus UV5 was cloned.
• The clone was overexpressed in E. coli BL21 (DE3) cells and purified to homogeneity.
• Ultrafiltration, anion-exchange and gel-filtration techniques were applied.
• DhlA exhibited Mr 35 kDa with optimum activity at 37 °C and pH 9.0.
• DhlA showed a Km value of 842 μM and kcat/Km ratio of 168 mM−1 min−1 for 1,2-DCA.

Haloalkane dehalogenase (DhlA) converts 1,2-dichloroethane (1,2-DCA) to 2-chloroethane in the genus Ancylobacter and Xanthobacter autotrophicus GJ10 (XaDhlA) and allows these organisms to utilise 1,2-DCA and some other halogenated alkanes for growth. The DhlA encoding gene (dhlA) was PCR-amplified from the genomic DNA of a recently isolated Ancylobacter aquaticus UV5 strain, cloned and overexpressed in Escherichiacoli BL21 (DE3). The recombinant enzyme was purified by using Amicon ultra-15 centrifugal filter units, an anion-exchange QFF column followed by a gel-filtration column (Sephacryl HR100). Enzyme activity was determined by using 1,2-DCA as a substrate. Three-dimensional structure of the enzyme was predicted using SWISS-MODEL workspace and the biophysical properties were predicted by submitting the amino acid sequence of DhlA on ExPASy server. DhlA (Mr 35 kDa) exhibited optimum activity at temperature 37 °C and pH 9.0. The enzyme retained approximately 50% of its activity after 1 h of incubation at 50 °C, and showed moderate stability against denaturing agent urea. The DhlA displayed a Km value of 842 μM and kcat/Km ratio of 168 mM−1 min−1 for its substrate 1,2-DCA. This DhlA was found to belong to the α/β hydrolase family with a catalytic triad composed of Asp-His-Asp in its active site. This is the first study reporting on the characterisation and reaction kinetics of purified DhlA from A.aquaticus UV5 indigenous to contaminated site in Africa.