4. DIRECTED EVOLUTION OF METHYL PARATHION HYDROLASE FOR
4.4.5. Ideas to improve the thermostability evolution and future directed evolution of MPH
From the various observations noted during the library screening and enzyme characterisation experiments, several ideas to improve the thermostability and other future directed evolution experiments on MPH were conceived.
From the metal content of WT MPH without metal supplementation and other reports on metalloenzymes in the literature, it is obvious that overexpression of metalloenzymes in E. coli strains requires metal supplementation during expression and purification to obtain enzymes with fully occupied metal centre. The results obtained in MPH characterisation implied that the active site metal is important both catalytically and structurally. The evolution experiment performed in the absence of additional metals seemed to favour the stabilisation of the metal free enzyme, following a different pathway in enzyme stabilisation. Therefore, it is crucial in future thermostability and other directed evolution experiments of MPH that Zn2 ion are supplemented to the growth media and assays buffers to maintain a fully occupied metal centre. The purification buffers should also be supplemented with Zn2 ions for the same reason. This will ensure that the evolution will not select for metal free enzymes that are more stable.
In this thermostability evolution experiment, only two mutations were found to be responsible for thermostability enhancement. The difficulty in obtaining new mutations that confer substantial thermostability improvement might suggest that the pathway to attain stability enhancement was restricted by the criteria imposed during processing of selected enhanced variants. Giver and co-workers, in their work in evolving a thermostable esterase, found that the evolutionary pathway to better thermostability was restricted by selecting only highly active mutants at ambient temperature as parental template for subsequent evolution. 12 They suggested that by relaxing the requirement for high activity at ambient temperature, more evolution pathways to thermostability would be available and more mutants with upward shift in optimum temperature would be found. Gumulya and Reetz in evolving epoxide hydrolase, found that the best variants were obtained from parents consisting of neutral or inferior mutants and suggested using parents consisting of unimproved mutants as a technique to escape from local minima (“dead ends”) in evolution.42 In this experiment,
mutants with less than 60% WT MPH initial activity were removed from consideration. Relaxing this requirement in future thermostability evolution experiments might open up more evolutionary pathways and allow the selection of mutants with upward shift in activity profile.
Another possible improvement to the thermostability evolution experiment would be to adopt a high throughput activity-independent biophysical screen. One such method is the Hot-CoFi agar screening method developed by Asial and co-workers.4’ The method allows for the isolation of better performing mutants at the colony level. This method worked on the premise that only soluble enzymes will be able to pass though a filter membrane and insoluble aggregate would be eliminated. By exposing the colonies to a temperature range, a varying degree of enzyme aggregation can be obtained to construct a biophysical thermo stab ilty profile. A secondary activity screen can then be performed on isolated mutant to ensure that the mutants are still active and not dead because of mutations that confer enhanced stability at the expense of activity.
4.5. Summary
This chapter described the directed evolution of MPH for better thermostability. Four rounds of directed evolution and variants with enhanced thermostability were selected. A single variant, R413D4, containing L256Q and K316E double mutations gave the most thermostability improvement. Unexpectedly, the stability of the purified R413D4 was much lower than anticipated. Although there was an enhancement, the mutant showed only two-fold stability improvement, unlike the marked improvement seen during screening. Various possibilities for the issue were discussed and the most likely explanation is the increase of non-specific interaction between R413D4 and some cellular component introduced by the mutations, thus stabilising the enzyme. During the course of investigating the reason behind the discrepancy, it was also discovered that MPH is expressed in E. coli without additional metal had a lower than expected Zn2 ' content, leading to compromised stability. Therefore, it is very plausible that we had generated a mutant that was more stable in its “metal free” form and stability enhancement seen in this work was metal independent. The mutant, expressed and purified without metal supplementation was more stable than the WT variant, expressed and purified without metal supplementation.
Investigation on the effect of additives on MPH had exposed its susceptibility toward organic solvents. This revelation is important, as this can potentially be a major weakness of the enzyme when deployed in the field for bioremediation. Either protein engineering or protein entrapment methods can be used to improve and protect the protein against solvent-induced denaturation.
The experiment was terminated at this point due to time constraint. However, suggestions on how the experiment could be improved had been given and discussed. The uncovering of the metal requirement during growth and expression subsequently contributed to a successful evolution experiment in altering the substrate specificity of MPH to various organophosphates (see Chapter 5).
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