title: Functional screening of enzymes and bacteria for the dechlorination of hexachlorocyclohexane by a high-throughput colorimetric assay.
abstract: Two distinct microbial dehalogenases are involved in the first steps of degradation of hexachlorocyclohexane (HCH) isomers. The enzymes, LinA and LinB, catalyze dehydrochlorination and dechlorination reactions of HCH respectively, each with distinct isomer specificities. The two enzymes hold great promise for use in the bioremediation of HCH residues in contaminated soils, although their kinetics and isomer specificities are currently limiting. Here we report the functional screening of a library of 700 LinA and LinB clones generated from soil DNA for improved dechlorination activity by means of a high throughput colorimetric assay. The assay relies upon visual colour change of phenol red in an aqueous medium, due to the pH drop associated with the dechlorination reactions. The assay is performed in a microplate format using intact cells, making it quick and simple to perform and it has high sensitivity, dynamic range and reproducibility. The method has been validated with quantitative gas chromatographic analysis of promising clones, revealing some novel variants of both enzymes with superior HCH degrading activities. Some sphingomonad isolates with potentially superior activities were also identified.
authors.affiliation: Molecular Biology Laboratory, Department of Zoology, University of Delhi, Delhi, 110007, India.
authors.forename: Pooja
authors.initials: P
authors.lastname: Sharma
authors.forename: Swati
authors.initials: S
authors.lastname: Jindal
authors.forename: Kiran
authors.initials: K
authors.lastname: Bala
authors.forename: Kirti
authors.initials: K
authors.lastname: Kumari
authors.forename: Neha
authors.initials: N
authors.lastname: Niharika
authors.forename: Jasvinder
authors.initials: J
authors.lastname: Kaur
authors.forename: Gunjan
authors.initials: G
authors.lastname: Pandey
authors.forename: Rinku
authors.initials: R
authors.lastname: Pandey
authors.forename: Robyn J
authors.initials: RJ
authors.lastname: Russell
authors.forename: John G
authors.initials: JG
authors.lastname: Oakeshott
authors.forename: Rup
authors.initials: R
authors.lastname: Lal
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000737:chemistry* / Q000201:enzymology* / Q000302:isolation & purification / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003124:Colorimetry / Q000379:methods*; D054879:Halogenation; D001556:Hexachlorocyclohexane / Q000737:chemistry / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007536:Isomerism; D007700:Kinetics; D008190:Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D001556:Hexachlorocyclohexane; D006867:Hydrolases; C046137:haloalkane dehalogenase; D008190:Lyases; C019994:dehydrochlorinases