title: Detection of halogenated organic compounds using immobilized thermophilic dehalogenase.
abstract: Environmental pollutants containing halogenated organic compounds can cause a plethora of health problems. Detection, quantification, and eventual remediation of halogenated pollutants in the environment are important to human well-being. Toward this end, we previously identified a haloacid dehalogenase, L-HAD(ST), from the thermophile Sulfolobus tokodaii. This thermophilic enzyme is extremely stable and catalyzes, stereospecifically, the dehalogenation of L-2-haloacids. In the current study, we covalently linked L-HAD(ST) to an N-hydroxysuccinimidyl Sepharose resin to construct a highly specific sensor with long shelf life for the detection of L-2-haloacids. The enzyme-modified resin was packed into disposable columns. Samples containing L-2-haloacids were first incubated in the column, and were then collected to quantify the chloride produced through the breakdown of the substrate. The optimum pH of the immobilized enzyme is around 9.5, similar to that of the soluble protein. Its catalytic activity increased with temperature up to the highest temperature measured (50 degrees C). The resin could be fully regenerated after multiple reaction cycles and retained 70% of the initial activity after being stored at 4 degrees C for 6 months. The L-HAD(ST)-modified resin could be used to breakdown and quantify L-2-haloacids spiked in the simulated environmental samples, indicating dehalogenases from extremophiles can potentially be employed in the detection and decontamination of L-2-haloacids.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington, KY 40506, USA.
authors.forename: Philip G
authors.initials: PG
authors.lastname: Bachas-Daunert
authors.forename: Zachariah P
authors.initials: ZP
authors.lastname: Sellers
authors.forename: Yinan
authors.initials: Y
authors.lastname: Wei
mesh_terms: D002384:Catalysis; D004785:Environmental Pollutants / Q000032:analysis*; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D006846:Hydrocarbons, Halogenated / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism*; D016944:Sulfolobus / Q000201:enzymology; D013696:Temperature
chemical_list: D004785:Environmental Pollutants; D004800:Enzymes, Immobilized; D006846:Hydrocarbons, Halogenated; D006867:Hydrolases; C046137:haloalkane dehalogenase