Functional screening of enzymes and bacteria for the dechlorination of hexachlorocyclohexane by a high-throughput colorimetric assay
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.
KeywordsColorimetric assay HCH Dehydrochlorinase (LinA) Haloalkane dehalogenase (LinB) Metagenomic libraries
This work was supported by grants under the Indo-Australian Biotechnology Fund from the Department of Education Science and Technology (DEST), Australia, the Department of Biotechnology (DBT), India, Indo Swiss Joint Research Programme (ISJRP) and Department of Science and Technology (DST). PS, SJ, KK, KB, NN and JK also acknowledge CSIR-UGC (Council for Scientific and Industrial Research-University Grants Commission), Government of India and DBT for providing their research fellowships. We also thank two anonymous reviewers for their excellent suggestions for improving the manuscript.
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