title: Cloning, expression, purification and three-dimensional structure prediction of haloalkane dehalogenase from a recently isolated Ancylobacter aquaticus strain UV5.
abstract: 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 35kDa) exhibited optimum activity at temperature 37°C and pH 9.0. The enzyme retained approximately 50% of its activity after 1h of incubation at 50°C, and showed moderate stability against denaturing agent urea. The DhlA displayed a Km value of 842μM and kcat/Km ratio of 168mM(-)(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.
authors.affiliation: Discipline of Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal (Westville Campus), Private Bag X54001, Durban 4000, South Africa.
authors.forename: Ajit
authors.initials: A
authors.lastname: Kumar
authors.affiliation: Discipline of Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal (Westville Campus), Private Bag X54001, Durban 4000, South Africa.
authors.forename: Balakrishna
authors.initials: B
authors.lastname: Pillay
authors.affiliation: Discipline of Microbiology, School of Life Sciences, College of Agriculture, Engineering and Science, University of KwaZulu-Natal (Westville Campus), Private Bag X54001, Durban 4000, South Africa. Electronic address: olanirana@ukzn.ac.za.
authors.forename: Ademola O
authors.initials: AO
authors.lastname: Olaniran
mesh_terms: D020561:Alphaproteobacteria / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005025:Ethylene Dichlorides / Q000378:metabolism; D006867:Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D016415:Sequence Alignment
chemical_list: D005025:Ethylene Dichlorides; C024565:ethylene dichloride; D006867:Hydrolases; C046137:haloalkane dehalogenase
keywords: 1,2-Dichloroethane; Ancylobacter aquaticus UV5; Haloalkane dehalogenase (DhlA)