Directed evolution of an organophosphate hydrolase : methyl parathion hydrolase

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

Altering the substrate specificity of methyl parathion hydrolase with directed evolution

Abstract Many organophosphates (OPs) are used as pesticides in agriculture. They pose a severe health hazard due to their inhibitory effect on acetylcholinesterase. Therefore, detoxification of water and soil contaminated by OPs is important. Metalloenzymes such as methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3 hold great promise as bioremediators as they are able to hydrolyze a wide range of OPs. MPH is highly efficient towards methyl parathion (1 × 106 s-1 M-1), but its activity towards other OPs is more modest. Thus, site saturation mutagenesis (SSM) and DNA shuffling were performed to find mutants with improved activities on ethyl paraxon (6.1 × 103 s-1 M-1). SSM was performed on nine residues lining the active site. Several mutants with modest activity enhancement towards ethyl paraoxon were isolated and used as templates for DNA shuffling. Ultimately, 14 multiple-site mutants with enhanced activity were isolated. One mutant, R2F3, exhibited a nearly 100-fold increase in the kcat/Km value for ethyl paraoxon (5.9 × 105 s-1 M-1). These studies highlight the 'plasticity' of the MPH active site that facilitates the fine-tuning of its active site towards specific substrates with only minor changes required. MPH is thus an ideal candidate for the development of an enzyme-based bioremediation system

Altering the substrate specificity of methyl parathion hydrolase with directed evolution

Many organophosphates (OPs) are used as pesticides in agriculture. They pose a severe health hazard due to their inhibitory effect on acetylcholinesterase. Therefore, detoxification of water and soil contaminated by OPs is important. Metalloenzymes such as methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3 hold great promise as bioremediators as they are able to hydrolyze a wide range of OPs. MPH is highly efficient towards methyl parathion (1 x 10(6) s(-1) M-1), but its activity towards other OPs is more modest. Thus, site saturation mutagenesis (SSM) and DNA shuffling were performed to find mutants with improved activities on ethyl paraxon (6.1 x 10(3) s(-1) M-1). SSM was performed on nine residues lining the active site. Several mutants with modest activity enhancement towards ethyl paraoxon were isolated and used as templates for DNA shuffling. Ultimately, 14 multiple-site mutants with enhanced activity were isolated. One mutant, R2F3, exhibited a nearly 100-fold increase in the k(cat)/K-m value for ethyl paraoxon (5.9 x 10(5) s(-1) M-1). These studies highlight the 'plasticity' of the MPH active site that facilitates the finetuning of its active site towards specific substrates with only minor changes required. MPH is thus an ideal candidate for the development of an enzyme-based bioremediation system. (C) 2015 Elsevier Inc. All rights reserved

Engineering Yarrowia lipolytica towards food waste bioremediation: Production of fatty acid ethyl esters from vegetable cooking oil

10.1016/j.jbiosc.2019.06.009Journal of Bioscience and Bioengineering129131-4

Rewriting the Metabolic Blueprint: Advances in Pathway Diversification in Microorganisms

Living organisms have evolved over millions of years to fine tune their metabolism to create efficient pathways for producing metabolites necessary for their survival. Advancement in the field of synthetic biology has enabled the exploitation of these metabolic pathways for the production of desired compounds by creating microbial cell factories through metabolic engineering, thus providing sustainable routes to obtain value-added chemicals. Following the past success in metabolic engineering, there is increasing interest in diversifying natural metabolic pathways to construct non-natural biosynthesis routes, thereby creating possibilities for producing novel valuable compounds that are non-natural or without elucidated biosynthesis pathways. Thus, the range of chemicals that can be produced by biological systems can be expanded to meet the demands of industries for compounds such as plastic precursors and new antibiotics, most of which can only be obtained through chemical synthesis currently. Herein, we review and discuss novel strategies that have been developed to rewrite natural metabolic blueprints in a bid to broaden the chemical repertoire achievable in microorganisms. This review aims to provide insights on recent approaches taken to open new avenues for achieving biochemical production that are beyond currently available inventions

Rewriting the metabolic blueprint: advances in pathway diversification in microorganisms

10.3389/fmicb.2018.00155Frontiers in Microbiology9FEB155completedcomplete

Organophosphate-degrading metallohydrolases: structure and function of potent catalysts for applications in bioremediation

Organophosphate compounds (OPs) have been employed in the agricultural industry as pesticides and insecticides for several decades. Many of the methods used currently for the detoxification of OPs are harmful and possess serious environmental consequences. Therefore, utilizing enzymes for the detection and decontamination of OPs is gaining increasing attention as an efficient and clean bioremediation strategy. Microbial enzymes, such as OP hydrolases, OP acid anhydrolases or methyl parathion hydrolase (MPH), are potent agents for OP decontamination. Their biochemical properties and biotechnological applications are discussed in this review, including a discussion on methods that may be employed to immobilize such enzymes, and essential steps to generate reusable and affordable biocatalytic systems for use in bioremediation and biorestoration