Fahrul Huyop, Ng Hong Jing and Ronald A Cooper
Keywords: Dehalogenase D, D,L-2-chloropropionic acid, Rhizobium sp.
Abstract: Halogenated organic compounds are found widely throughout the environment and microbial catabolism can lead to their biodegradation. The action generally involves enzyme-catalysed carbon-halogen bond cleavage. Dehalogenase D enzyme act only on D-2-chloropropionic acid and D,L-2-chloropropionic acid. Other substrate for the enzyme included monochloroacetic acid and monobromoacetic acid but not 2,2-dichloropropionic acid. From 4 g (wet weight) of cells, 2.8 mg protein (4.3 U enzyme) was efficiently purified using Fast Protein Liquid Chromatography (FPLC). The cell-free extract was prepared in 0.01M Tris-acetate buffer pH7.6 and was applied to a FPLC Mono Q HR 5/5 anion-exchange column equilibrated with 5 mM sodium phosphate, 1 mM EDTA, 1 mM dithiothreitol (DTT), 10%(mass/vol.) glycerol buffer, pH7.6 and eluted with sodium phosphate gradient to 100 mM. The dehD gene consisted of 798 bp which encoded a 266 amino acid protein with a predicted molecular mass of 29 386 Da. This value corresponds to the value of 29 000 Da estimated by SDS/PAGE. DehD protein has some similarity to the previously published sequence of several stereospecific dehalogenases with 59% identity to Rhizobium sp. NHG3 and 23% identity to Pseudomonas putida strain AJ1.
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