By Davidson, Sreve
During the late 1980s, CSIRO entomologists Dr John Oakeshott and Dr Robyn Russell were immersed in studies of the sheep blowfly and associated mysteries of insecticide resistance. Little did they guess their work would lead to the use of enzymes to combat toxic pesticide residues.
Oakeshott, Russell and their colleagues at CSIRO Entomology discovered that the sheep blowfly had evolved resistance to the insecticide used against it via a mutation that enabled the fly maggots to degrade the insecticide to non-toxic products.
The resistant flies had mutated the active site of a particular esterase enzyme, which meant that their metabolism could now break down the normally highly toxic insecticide (an organophosphate) and render it harmless. This work led the scientists to the idea of using a similar technique to clean up pesticide residues in the environment.
For the past few years, the CSIRO group, in partnership with Dr Hung Nguyen and his colleagues at Orica Australia, have been trying to mimic the success of the sheep blowfly by developing commercial enzyme technologies for bioremediation.
There is mounting pressure to reduce agricultural pesticide residues, as they can be a threat to both the environment and to the marketing of many of Australia's commodities. Grains, cotton, wool, meat, fruit and vegetables, nursery plants and cut flowers are all at risk.
Contaminated waters, such as irrigation wastes on rice and cotton farms, or spent animal dip liquors, are also environmental hazards and ones that the scientists believe are amenable to enzyme treatment.
The CSIRO Entomology team mostly looks for suitable enzymes in bacteria, rather than insects. This is because bacteria can degrade larger amounts of residue. For example, soil in cotton fields is full of bacteria that either use pesticide residues as a source of nutrients, or detoxify the residues to survive and multiply.
`What's amazing is that when you look in the right place, you find enzymes,' Russell says. `It depends where you go for your bit of dirt. Our best microbial enzyme came from a back yard in Brisbane. A lady regularly washed her dog there with an insecticide formulation and we found resistant bacteria in the soil.'
To obtain an enzyme from such a source, an enrichment culture of the bacteria is set up in the laboratory. Insecticide is added and left for a while, so selecting for still more resistant microbes. The culture is then shaken and divided into sub-cultures, which are tested for their ability to degrade pesticide. If such activity occurs, the culture is progressively diluted to isolate the organism responsible.
Once a promising strain of bacteria has been isolated, the gene responsible for producing the enzyme -- or several genes and enzymes -- has to be identified and cloned. An `expression system' in which to multiply the gene for commercial use must also be developed. This involves inserting the gene into a, usually circular, unit of bacterial DNA known as a plasmid, and allowing the bacteria to multiply. …