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Genetic engineering of halohydrolase for the treatment of chloroacetate toxic waste

Objective

To develop a biotechnological process for the production of large amounts of monochloroacetate (MCA) dehalogenase for use in the detoxification of wastes that arise as a result of MCA manufacture and use.

MCA wastes include residual herbicide that remains in the emptied container after use, the washings from spraying equipment used to apply the compound and waste waters contaminated at point of manufacture. The enzyme will be immobilized for the most cost effective treatment of dilute aqueous waste.

A range of bacteria capable of growth on 50 mM MCA as sole carbon and energy source will be isolated.

To identify the most suitable enzyme for the process, a selection of the bacteria will be grown on MCA liquid media and cell free extracts prepared by ultrasonication. The dehalogenase activity will be assayed by measuring chloride ion release by means of a chloride ion electrode attached to a millivolt meter. The dehalogenase enzyme in the various crude extracts will be assessed for the properties related to operational use. The dehalogenase which best meets the desired requirements will be selected and a biotechnological procedure developed for production of large amounts of the enzyme. For this the gene encoding the enzyme will be cloned and its over expression engineered.

To purify the dehalogenase enzyme using fast protein liquid chromatography (FPLC) procedures such that the amino terminal amino acid sequence can be obtained using an automatic gas phase protein sequencer. A synthetic oligonucleotide corresponding to an appropriate region of the sequence will then be synthesized on an automatic deoxyribonucleic acid(DNA) synthesizer. The oligonucleotide, terminally labelled with phosphorous-32, will be used in colony hybridizations to identify the complementary DNA. Once a clone has been obtained the dehalogenase gene will be localized by restriction mapping and subcloning. The nucleotide sequence of the gene will be determined to provide information that will permit polymerise chain reaction methodology to be used in the direct linking of the dehalogenase gene to the tac promoter.

The culture conditions, in terms of type of medium and extent of cell growth, will be investigated to identify conditions for optimal production of the enzyme from the cloned gene.

Coordinator

UNIVERSIDAD COMPLUTENSE DE MADRID
Address
Ciudad Universitaria S/n
Madrid
Spain

Participants (1)

UNIVERSITY OF LEICESTER
United Kingdom
Address
The Adrian Building, University Road
LE1 7RH Leicester