Enzyme based bioremediation

Details Enzyme based bioremediation Enzyme based bioremediation

1996-10-07

1998-12-01

Wax, Robert A.

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

435196, 4353201, 435 711, 435262, 4352625, 536 232, 530350, C12N 120, C12N 916, C12N 1200, B09B 300

Patent

active

058437584

DESCRIPTION:

BRIEF SUMMARY
This application is a 371 of PCT/AU94/00016 filed Jan. 1, 1995.
This invention relates to enzymes capable of hydrolysing organophosphate and/or carbamate pesticide residues. In particular, it relates to esterase enzymes purified from organophosphate resistant strains of Lucilia cuprina, and isolated DNA molecules encoding such enzymes.
Residues of organophosphates and carbamate pesticides are undesirable contaminants of the environment and a range of commodities. Areas of particular sensitivity include contamination of domestic water supplies, residues above permissible levels in meat and horticultural exports and contamination of health products like lanolin. Bioremediation strategies are therefore required for eliminating or reducing these pesticide residues. One proposed strategy involves the use of enzymes capable of immobilising or degrading the pesticide residues. Such enzymes may be employed, for example, in bioreactors through which contaminated water could be passed; in sheep or cattle dips to reduce problems with contaminated pasture and run off into water supplies; in the wool scour process to reduce contamination of liquid effluent, wool grease and lanolin; or in washing solutions after post harvest disinfestation of fruit and vegetables to reduce residue levels and withholding times. Suitable enzymes for degrading pesticide residues include esterases. It is desirable that the esterases be relatively specific and hydrolyse the pesticide residues at a rapid rate.
Esterases in insects have been implicated in reproductive behaviour, pheromone and hormone metabolism, digestion, neurotransmission, and in the action of, and resistance to, insecticides, particularly organophosphates (OPs). Three different mechanisms of OP resistance in insects involving esterases have been proposed. One such mechanism involves possible alterations to the structure of an esterase to increase its ability to degrade OPs. This mechanism has been proposed for the house fly, Musca domestica and Lucilia cuprina. The proposed structural changes are thought to have resulted in the loss of activity for synthetic substrates, such as .alpha.-naphthyl acetate and its related esters. For example, esterase E3 in L. cuprina has lost the ability to utilize .alpha.- and .beta.-naphthyl acetate as substrates in all the OP-resistant strains examined to date. However, concomittantly with this loss in ability to utilise .alpha.- and .beta.-naphthyl acetate substrates, it would appear that the OP-resistant E3 esterase becomes capable of hydrolysing OPs into non-toxic products whereas the susceptible E3 esterase cannot. Thus, the E3 esterase from OP-resistant strains of L. cuprina may be a suitable enzyme for development as a catalytic bioremediant for organophosphates and/or carbamates.
The present inventors have now developed a method for purifying the E3 esterase from L. cuprina. Kinetic data obtained from assays using homogenates suggests that the E3 esterase from OP-resistant strains of L. cuprina, hydrolyses OPs quickly even under suboptimal conditions (prevalent in most bioremediation applications). Further, the inventors have isolated the gene encoding the E3 esterase from an OP-susceptible and resistant strains of L. cuprina, and identified a homologue of this gene in Drosophila melanogaster.
Accordingly, in a first aspect the present invention consists in an E3 esterase from an organophosphate-resistant strain of Lucilia cuprina, in substantially pure form.
Preferably, the E3 esterase is from one of the isochromosomal OP-resistant L. cuprina strains selected from the group consisting of der-S, Inverell 22, Landillo 103 and Sunbury 5.2.
In a second aspect, the invention provides an isolated DNA molecule comprising a nucleotide sequence encoding a Lucilia cuprina E3 esterase or portion thereof capable of hydrolysing organophosphates and/or carbamate pesticide residues.
Preferably, the isolated DNA molecule comprises a nucleotide sequence encoding an E3 esterase or portion thereof, from an OP-resistant strain of L. cuprina (such as th

REFERENCES:
Merrin E. Spackman et al., "A Cluster of Esterase Genes on Chromosome 3R of Drosophila Melanogaster Includes Homologues of Esterase Genes Conferring Insecticide Resistance in Lucilia Cuprina"., Dec. 1993., pp. 39-62.
Steven Whyard et al., "Insecticide Resistance and malathion Carboxylesterase in the Sheep Blowfly, Lucilia Cuprina"., Oct. 1993., pp. 9-24.
Kerrie-Ann Smyth et al., "Genetics of Insecticide Resistance"., pp. 1-18.
Joel L. Sussman et al., "Atomic Structure of Acetylcholinesterase From Torpedo California: A Prototypic Acetylcholine-Binding Protein"., pp. 872-878.
Anthony G. Parker. et al., "Biochemistry and Physiology of Esterases in Organoposphate-Susceptible and Resistant Strains of the Australian Sheep Blowfly, Lucilia Cuprina."., 1991 pp. 305-318.
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John M. Chirgwin et al., "Isolation of Biologically Active Ribonucleic Acid From Sources Enriched in Ribonuclease." pp. 5294-5299.
Parker et al. (1991) Pesticide Biochemistry and Physiology 41, 305-318.

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