Pesticide Conversion Mechanisms in the Environment Enzymatic Conversion
The transformation of the different types of pesticides to various degradation products is brought about by physical, chemical, and biological agents (Coats, 1991). Microbial metabolism is however responsible for the degradation of a vast majority of pesticides in the environment.
Microbial degradation is the breakdown of pesticides by fungi, bacteria, and other microorganisms that use pesticides as a food source, and most microbial degradation of pesticides occurs in the soil.
According to the definition by the International Union of Pure and Applied Chemistry, the term biodegradation is “Breakdown of a substance catalyzed by enzymes in vitro or in vivo”.
The biodegradation of these pesticides, is often complex and involves a series of biochemical reactions. These reactions are catalyzed by enzymes, hence they are termed enzymatic conversion.
Enzymatic Conversion
The potential to use enzymatic treatment in biodegradation is a modern technology, which is currently receiving attention as a step ahead of the use of microorganisms for bioremediation.
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An array of enzymes and other natural substances have proved highly effective in the breakdown of pesticides and other xenobiotic compounds in the soil.
Enzymes are catalytic in nature and therefore speed up the rates of biochemical reactions by lowering the activation energy needed to drive these reactions. These enzymes can detoxify many times their own weight of agent within very short time.
The advantages of enzymatic treatment over microbial biodegradation include;
(1) No acclimation phase,
(2) Use over a wider range of environmental conditions (pH, moisture, temperature),
(3) Effectiveness at high and low pollutant concentrations,
(4) Movement of enzymes readily into soil micropores and their protection from inactivation, and
(5) Little effect of inhibitors of microbial metabolism on enzymes (Dec and Bollag 2001).
The limitations of enzymatic treatment in bioremediation include the high cost of isolation and storage, the difficulty in maintaining enzyme stability, the requirement for expensive cofactors, and the lack of xenobiotic mineralization (Dec and Bollag 2001).
Microbial enzymes with potential for pesticide metabolism include hydrolases, oxidoreductases, hydroxylases, amidases, and esterases.
However, enzymatic treatments are not ideal for complete xenobiotic mineralization because mineralization usually requires many enzymes and several cofactors such as NAD(P)H and FAD. Enzymatic treatment holds great promise in bioremediation of contaminated soil and water (Van Eerd et al., 2003).
Enzymes with activity against nerve agents were first discovered during World War II, which include numerous organophosphorus pesticides. Some others were researched into very recently. Below is a list of some of enzymes with potentials to detoxify pesticides in the environment:
Organo phosphorus Hydrolase
Organophosphorus Hydrolase (OPH) is a known Nerve Agent Detoxifying Enzymes which enhances activity of the Bacteria Pseudomonas diminutain degradation process.
OPH is an enzyme found in a number of bacterial isolates that has optimal activity against a variety of organophosphorus pesticides (originally called parathion hydrolase) in addition to its activity against nerve agents.
The gene for this enzyme has been cloned, sequenced, and expressed in a number of prokaryotic and eucaryotic host organisms. The three-dimensional crystal structure of OPH also has been determined revealing that the native enzyme is a homodimer and contains two Zn2+ ions per sub-unit. The Co2+ substituted enzyme has greater activity on nerve agents and substrates with P-F and P-S bonds.
Organo phosphorus Acid Anhydrolase
Organophosphorus Acid Anhydrolase (OPAA) is another Nerve Agent Detoxifying Enzymes which enhances activity of the Bacteria Alteromonassp. in degradation of hosphorus base pesticides.
OPAA was originally identified in the obligate halophilic bacterium Alteromonassp. that was isolated from Grantsville Warm Springs in Utah. Unlike OPH, OPAA has very little activity against pesticides.
The OPAA gene has been cloned, sequenced, and expressed at very high levels in Escherichia coli (up to 50% of cell protein). The enzyme can be freeze- dried and survive for many years at room temperature with no loss of activity.
From the amino acid sequence of OPAA and functional studies on a variety of dipeptides, it was identified as an X- Pro dipeptidase (or prolidase) having nothing at all to do with phosphorus metabolism.
Through serendipity, it is ideally positioned for hydrolytic attack on the phosphorus atom. This class of enzymes can be found throughout nature in organisms as primitive and diverse as Archea and bacteria all the way up to humans.
The gene for squid enzyme Diisopropylfluorophosphatase (DFPase) also a Nerve Agent Detoxifying Enzymes which aid the microbe Loligovulgaris has been cloned, sequenced, and expressed in both E. coli and the yeast Pichia pastoris.
The squid-type DFPase has only been found in cephalopods, requires Ca2+ for activity and stability, and hydrolyzes DFP five times faster than soman. Its chemical and biological properties are completely different from those of all other types of DFPases as well as OPH and OPAA.
Phenoloxidases (peroxidases and laccases)
Phenoloxidases are produced by microbial activity in biobeds with straw-degrading fungi being the driving force.
Here the straw is the main substrate for pesticide degradation and microbial activity, especially from lignin-degrading fungi such as white rot fungi, which produce phenoloxidases (peroxidases and laccases).
The broad specificity of these enzymes makes them suitable for degradation of mixtures of pesticides. The degradation of individual pesticides by white rot fungi/peroxidases has been demonstrated in several studies.
Moreover in laboratory scale biobeds, the dissipation of most of the pesticides in a mixture is correlated with phenoloxidase activity and/or basal respiration and both activities are correlated to the levels of straw.
Therefore, a high amount of straw in the biomixture is recommended, although in practice not more than 50 vol-% due to the requirement to achieve a homogeneous mixture.
The lignin-degrading system of many white rot fungi is nitrogen- regulated. At low nitrogen levels the fungi activate the production of phenoloxidases, while higher nitrogen levels can enhance growth but inhibit the production of enzymes. Therefore, addition of nitrogen to biomixtures is not recommended.
Esterases
The esterases involved are somewhat characterized, but relatively little is known about which ones modify xenobiotics. Many of these enzymes are non-specific and reside in cuticles and cell walls.
Herbicides applied as esters are fairly lipophilic and mobile in the cuticle; however, de- esterification is required prior to entry of the herbicide (now an acid) into the phloem via ion trapping.
De-esterification will also increase or maintain the concentration gradient because the ester is converted into an acid and therefore, the gradient is steeper for entry of additional ester.
De-esterification can be viewed as a form of bioactivation because the herbicide will not be translocated as readily in the ester form. In some cases, the de-esterified form of the herbicide is more toxic as well (i.e. fenoxyprop is more toxic to grassy weeds than fenoxyprop-ethyl).
Oxido reductases
Oxidoreductases, such as laccase, tyrosinase, and horseradish peroxidase, can be used to decontaminate soil and water. These enzymes oxidize the substrate to free radicals, which are susceptible to chemical coupling, forming oligomers.
For example, oligomer formation reactions can take place between humic acid and xenobiotics, resulting in the polymerization of the substrate to soil, as was observed with 2, 4- dichlorophenol. In another experiment, horseradish root tissue and hydrogen peroxide (an electron acceptor) decontaminated water containing 850 ppm of 2, 4- dichlorophenol and other chlorinated phenols.
Depending on the concentration of hydrogen peroxide, up to 100% of the contaminants were removed by polymerization. Furthermore, horseradish root tissue contributed to the irreversible binding of 2,4-dichlorophenol to soil.
Enzyme immobilization
For enzymatic treatment to be effective in bioremediation, the enzymes must be stabilized. The most effective way to stabilize enzymes is by immobilization. Immobilization can be accomplished by enzyme linkage to organic or inorganic solid supports by adsorption on solid surfaces such as glass, entrapment in polymeric gels, encapsulation, or intermolecular cross-linking.
Although preparing supports can be time- consuming and expensive, the support can generally be reused. The use of immobilized enzymes to metabolize pesticides is not new.
For example, enzymes from crude Pseudomonas cell extracts immobilized on glass beads hydrolyzed 95% of parathion (10 to 250 ppm) from wastewater.
The same enzyme preparation hydrolyzed parathion at 2,500 ppm in soil and was also effective in hydrolyzing other organophosphate insecticides (triazophos, diazinon, and fenitrothion).
In conclusion, from the above, it is obvious that though the cost of pesticide conversion with enzymes may be quite prohibitive, it is an efficient and eco-friendly technology for pesticide cleanup in the environment.
The prospects of this technology in pesticide breakdown is also vast because more classes of enzymes can be explored for future use in bioremediation of pesticide contaminated environment.
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Pesticide conversion in the environment is mainly by microorganisms of which enzymatic breakdown forms a major aspect. The use of enzymes as biodegradation agents presents many advantages.
However, enzymatic treatments are not ideal for complete xenobiotic mineralization because of certain limitations, which include high cost of enzyme isolation and storage, difficulty in maintaining enzyme stability, and the requirement of many enzymes and several cofactors such as NAD(P)H and FAD to achieve mineralization.
