Directed evolution of an organophosphate hydrolase : methyl parathion hydrolase

Abstract

Organophosphates (OPs) are the most common pesticides used in agriculture. Although they can be broken down in nature, OPs pose a severe health hazard to human due to their inhibitory effect on acetylcholine, a major enzyme in nervous transmission. Therefore, detoxification of water and soil contaminated by OPs is important. One way of achieving this is by bioremediation of sites with OP-degrading enzymes. One such enzyme is methyl parathion hydrolase (MPH) isolated from Pseudomonas sp. WBC-3. MPH is a highly efficient enzyme that is capable of hydrolysing methyl parathion at near diffusion-limited rate. While MPH can hydrolyse a wide range of OPs, the substrate specificity of the enzyme was not well characterised. In order to study MPH, the enzyme was expressed and purified. In the process of MPH protein purification, proteolytic degradation was observed. Various methods, including protein engineering and optimising the purification, were employed to investigate and overcome the degradation. A protocol that allowed rapid purification of MPH was developed so that the proteolysis can be minimised. Due to initial suspicion of autoproteolysis, nickel affinity chromatography was also used in further investigations and autoproteolysis was eventually ruled out. Stability is one of the most important characteristics that define an enzyme's practical use in the industry. For MPH to be an effective bioremediator, it needs to be thermally and chemically stable. Unfortunately, MPH does not have exceptional thermostability and could benefit from extra thermostability. To achieve this, MPH was subjected to directed evolution for enhanced thermostability. In the course of characterising the mutants isolated, it was discovered that MPH expressed in E. coli had lower than expected metal content. It was also found that Zn2+ supplementation prior to activity and stability assay drastically increased the activity and stability of WT MPH. Since the evolution was performed without metal supplementation and the isolated mutant did not have enhanced stability with Zn2+ supplementation, we hypothesised that the mutant isolated was stabilised "metal independently". Another desired characteristic for a bioremediator is the ability to hydrolyse various OPs efficiently. The substrate profile characterisation of WT MPH revealed that while MPH is highly efficient towards methyl parathion, its activity towards other OPs varies. To alter and broaden the substrate specificity of MPH, structure-guided site saturation mutagenesis (SSM) on active site lining residues was performed to obtain mutants with enhanced activity towards ethyl paraoxon. Mutants with modest improvements were isolated and two rounds of DNA shuffling were performed to compound the mutations. The best mutant towards ethyl paraxon exhibited 98-fold increase in kcat/Km. Several other mutants exhibited interesting and respectable changes in their substrate profiles. One mutant with selective activity towards chlorpyrifos class substrates was found. These results highlighted the 'plasticity' of MPH active site that allow efficient hydrolysis of other OPs with only minor changes. In short, progress had been made in purifying MPH and in evolving it to be more stable - although further work is required in this area. Considerable progress had been made in identifying mutations that alter the substrate specificity of MPH.