Methyl Parathion Hydrolase Research Articles

A Dual-Focus Workflow for Simultaneously Engineering High Activity and Thermal Stability in Methyl Parathion Hydrolase.

Industrial fermentation applications typically require enzymes that exhibit high stability and activity at high temperatures. However, efforts to simultaneously improve these properties are usually limited by a trade-off between stability and activity. This report describes a computational strategy to enhance both activity and thermal stability of the mesophilic organophosphate-degrading enzyme, methyl parathion hydrolase (MPH). To predict hotspot mutation sites, we assembled a library of features associated with the target properties for each residue and then prioritized candidate sites by hierarchical clustering. Subsequent in silico screening with multiple algorithms to simulate selective pressures yielded a subset of 23 candidate mutations. Iterative parallel screening of mutations that improved thermal stability and activity yielded, MPHase-m5b, which exhibited 13.3 °C higher Tm and 4.2 times higher catalytic activity than wild-type (WT) MPH over a wide temperature range. Systematic analysis of crystal structures, molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations revealed a wider entrance to the active site that increased substrate access with an extensive network of interactions outside the active site that reinforced αβ/βα sandwich architecture to improve thermal stability. This study thus provides an advanced, rational design framework to improve efficiency in engineering highly active, thermostable biocatalysts for industrial applications.

A Dual‐Focus Workflow for Simultaneously Engineering High Activity and Thermal Stability in Methyl Parathion Hydrolase

AbstractIndustrial fermentation applications typically require enzymes that exhibit high stability and activity at high temperatures. However, efforts to simultaneously improve these properties are usually limited by a trade‐off between stability and activity. This report describes a computational strategy to enhance both activity and thermal stability of the mesophilic organophosphate‐degrading enzyme, methyl parathion hydrolase (MPH). To predict hotspot mutation sites, we assembled a library of features associated with the target properties for each residue and then prioritized candidate sites by hierarchical clustering. Subsequent in silico screening with multiple algorithms to simulate selective pressures yielded a subset of 23 candidate mutations. Iterative parallel screening of mutations that improved thermal stability and activity yielded, MPHase‐m5b, which exhibited 13.3 °C higher Tm and 4.2 times higher catalytic activity than wild‐type (WT) MPH over a wide temperature range. Systematic analysis of crystal structures, molecular dynamics (MD) simulations, and quantum mechanics/molecular mechanics (QM/MM) calculations revealed a wider entrance to the active site that increased substrate access with an extensive network of interactions outside the active site that reinforced αβ/βα sandwich architecture to improve thermal stability. This study thus provides an advanced, rational design framework to improve efficiency in engineering highly active, thermostable biocatalysts for industrial applications.

Coupling Peptide-Based Encapsulation of Enzymes with Bacteria for Paraoxon Bioremediation.

The catalytic efficiency of enzymes can be harnessed as an environmentally friendly solution for decontaminating various xenobiotics and toxins. However, for some xenobiotics, several enzymatic steps are needed to obtain nontoxic products. Another challenge is the low durability and stability of many native enzymes in their purified form. Herein, we coupled peptide-based encapsulation of bacterial phosphotriesterase with soil-originated bacteria, Arthrobacter sp. 4Hβ as an efficient system capable of biodegradation of paraoxon, a neurotoxin pesticide. Specifically, recombinantly expressed and purified methyl parathion hydrolase (MPH), with high hydrolytic activity toward paraoxon, was encapsulated within peptide nanofibrils, resulting in increased shelf life and retaining ∼50% activity after 132 days since purification. Next, the addition of Arthrobacter sp. 4Hβ, capable of degrading para-nitrophenol (PNP), the hydrolysis product of paraoxon, which is still toxic, resulted in nondetectable levels of PNP. These results present an efficient one-pot system that can be further developed as an environmentally friendly solution, coupling purified enzymes and native bacteria, for pesticide bioremediation. We further suggest that this system can be tailored for different xenobiotics by encapsulating the rate-limiting key enzymes followed by their combination with environmental bacteria that can use the enzymatic step products for full degradation without the need to engineer synthetic bacteria.

Elucidating the Enzymatic Mechanism of Dihydrocoumarin Degradation: Insight into the Functional Evolution of Methyl-Parathion Hydrolase from QM/MM and MM MD Simulations.

Methyl-parathion hydrolase (MPH), which evolved from dihydrocoumarin hydrolase, offers one of the most efficient enzymes for the hydrolysis of methyl-parathion. Interestingly, the substrate preference of MPH shifts from the methyl-parathion to the lactone dihydrocoumarin (DHC) after its mutation of five specific residues (R72L, L273F, L258H, T271I, and S193Δ, m5-MPH). Here, extensive QM/MM calculations and MM MD simulations have been used to delve into the structure-function relationship of MPH enzymes and plausible mechanisms for the chemical and nonchemical steps, including the transportation and binding of the substrate DHC to the active site, the hydrolysis reaction, and the product release. The results reveal that the five mutations remodel the active pocket and reposition DHC within the active site, leading to stronger enzyme-substrate interactions. The MM/GBSA-estimated binding free energies are about -20.7 kcal/mol for m5-MPH and -17.1 kcal/mol for wild-type MPH. Furthermore, this conformational adjustment of the protein may facilitate the chemical step of DHC hydrolysis and the product release, although there is a certain influence on the substrate transport. The hydrolytic reaction begins with the nucleophilic attack of the bridging OH- with the energy barriers of 22.0 and 18.0 kcal/mol for the wild-type and m5-MPH enzymes, respectively, which is rate-determining for the entire process. Unraveling these mechanistic intricacies may help in the understanding of the natural evolution of enzymes for diverse substrates and establish the enzyme structure-function relationship.

Unveiling Eco-Friendly Solutions: A Comprehensive Review on Leveraging Mechanism-Based Pathways for Sustainable Mitigation of Pesticide Metabolite Contamination and Enhanced Public Health

The harmful consequences of several pesticides, such as Benzene Hexachloride (BHC), chlorpyrifos, cypermethrin and atrazine on the environment and human health are examined in this study. Through a variety of exposure pathways, these pesticides carry concerns including neurotoxicity, carcinogenicity, and reproductive problems. Even though traditional degrading methods like fire and hydrolysis exist, their usefulness is limited and they may produce hazardous materials. Alternative methods, such as bacteria-mediated remediation, have the potential to efficiently and sustainably break down pesticides. Pesticides can be converted into non-toxic chemicals by the enzymes and metabolic pathways found in bacterial species such as Sphingomonas sp., Clostridium sp. and Pseudomonas sp. BHC degradation is aided by enzymes like LinA and LinB, whereas the breakdown of chlorpyrifos is mediated by organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH). Pseudomonas alcaligenes and Bacillus thuringiensis SG4 are effective at breaking down cypermethrin and break down atrazine using enzymes such AtzA, AtzB, and AtzC. This work highlights the potential of bioremediation to mitigate pesticide pollution and provides ecologically friendly alternatives for sustainable farming practices and ecosystem preservation by providing an understanding of bacterial mechanisms.

Improving thermostability, solvent tolerance, and reusability of methyl parathion hydrolase through immobilization into the metal-organic framework ZIF-8

The metal-organic framework (MOF) ZIF-8 has found extensive application in the medicinal and agricultural fields. This study aimed to immobilize the methyl parathion hydrolase (MPH) enzyme from Azohydromonas australica (Azau-MPH) within ZIF-8 using an in-situ synthesis method. Interestingly, the immobilized Azau-MPH (abbreviated as Azau-MPH@ZIF-8) exhibited enhanced thermostability and tolerability towards organic solvents compared to the free enzyme. Moreover, Azau-MPH@ZIF-8 retained more than 50% of its initial activity after six cycles, demonstrating its desirable reusability. To further improve the recyclability and catalytic activity of the immobilized enzyme, we introduced magnetic Fe3O4 nanoparticles into the immobilization carrier. Notably, the resulting Azau-MPH@(Fe3O4@ZIF-8) biocomposite displayed superior catalytic activity and stability when compared to Azau-MPH@ZIF-8. In practical applications, we employed Azau-MPH@ZIF-8 for cyclic degradation of methyl parathion (20 mg/L) in cherries, and it exhibited exceptional degradation ability. Hence, both Azau-MPH@ZIF-8 and Azau-MPH@(Fe3O4@ZIF-8) hold great promise as valuable biodegradation tools against MP contamination.

Molecular binding of different classes of organophosphates to methyl parathion hydrolase from Ochrobactrum species.

Methyl parathion hydrolase (MPH) is an enzyme of the metallo-β-lactamase superfamily, which hydrolyses a wide range of organophosphates (OPs). Recently, MPH has attracted attention as a promising enzymatic bioremediator. The crystal structure of MPH enzyme shows a dimeric form, with each subunit containing a binuclear metal ion center. MPH also demonstrates metal ion-dependent selectivity patterns. The origins of these patterns remain unclear but are linked to open questions about the more general role of metal ions in functional evolution and divergence within enzyme superfamilies. We aimed to investigate and compare the binding of different OP pesticides to MPH with cobalt(II) metal ions. In this study, MPH was modeled from Ochrobactrum sp. with different OP pesticides bound, including methyl paraoxon and dichlorvos and profenofos. The docked structures for each substrate optimized by DFT calculation were selected and subjected to atomistic molecular dynamics simulations for 500 ns. It was found that alpha metal ions did not coordinate with all the pesticides. Rather, the pesticides coordinated with less buried beta metal ions. It was also observed that the coordination of beta metal ions was perturbed to accommodate the pesticides. The binding free energy calculations and structure-based pharmacophore model revealed that all the three substrates could bind well at the active site. However, profenofos exhibit a stronger binding affinity to MPH in comparison to the other two substrates. Therefore, our findings provide molecular insight on the binding of different OP pesticides which could help us design the enzyme for OP pesticides degradation.

Development of a whole-cell biosensor for detecting organophosphorus pesticide methyl parathion in the farmland soil

The evaluation of the bioavailability of pollutants in soil is crucial to accurately assess the pollution risk, and whole-cell biosensor is one of the important tools for such evaluation. This study aimed to develop a novel whole-cell biosensor for the detection of methyl parathion in soil using. First, a whole-cell biosensor was constructed by the screened methyl parathion hydrolase mpd gene, the existing specific induction element pobR, and the pUC19 plasmid skeleton. Then, the detection method of methyl parathion in soil extracts was established using 96-well microtiter plate as carrier and five whole-cell biosensors as indicator. The method was applied in the detection of methyl parathion in tested and field soil extracts. Taking E. coli DH5α/pMP-AmilCP with the best detection performance as an example, this biosensor had a detection limit of 6.21-6.66 µg/L and a linear range of 10-10 000 µg/L for methyl parathion in four soil extracts. E. coli DH5α/pMP-RFP and E. coli DH5α/pMP-AmilCP methods have good detection performance for the analysis of methyl parathion in soil extract samples. This biosensor method can help to quickly assess the bioavailability of methyl parathion in soil, and thus help to understand the risk of soil pollution caused by organophosphorus pesticide methyl parathion.

A transposon system for random insertion of a gene expression cassette into the chromosome of Bacillus subtilis

Bacillus subtilis is a robust industrial workhorse for the production of heterologous proteins. Chromosomal integration-based protein production has advantages over plasmid-based methods. Considering that the expression level of a gene is affected by its location in the chromosome, it is important to find an optimal integration site for the gene to be expressed. This work establishes a method for random insertion of a gene expression cassette into chromosomes, enabling the screening of optimal integration sites for high-level protein production. Specifically, a gene expression cassette and a chloromycetin-resistance marker are assembled into a transposon. This transposon is inserted between the promoter and the ribosomal binding site of the zeocin-resistance marker in the chromosome, which blocks the transcription of the zeocin-resistance gene. Transposase Himar1-mediated transposition of this transposon activates the zeocin-resistance marker, which can be selected on plates containing both chloromycetin and zeocin. The transposition frequency was over 10−5. This method was used to select proper insertion sites for the expression cassette of methyl parathion hydrolase (MPH). Compared with the common integration site amyE, the expression level of MPH was increased up to 50 % at the yjbH site.

Controlled co-immobilisation of proteins via 4′-phosphopantetheine-mediated site-selective covalent linkage

In Escherichia coli, acyl carrier protein (ACP) is posttranslationally converted into its active holo-ACP form via covalent linkage of 4′-phosphopantetheine (4′-PP) to residue serine-36. We found that the long flexible 4′-PP arm could react chemoselectively with the iodoacetyl group introduced on solid supports with high efficiency under mild conditions. Based on this finding, we developed site-selective immobilisation of proteins via the active holo-ACP fusion tag, independently of the physicochemical properties of the protein of interest. Furthermore, the molecular ratios of co-immobilised proteins can be manipulated because the tethering process is predominantly directed by the molar concentrations of diverse holo-ACP fusions during co-immobilisation. Conveniently tuning the molecular ratios of co-immobilised proteins allows their cooperation, leading to a highly productive multi-protein co-immobilisation system. Kinetic studies of enzymes demonstrated that α-amylase (Amy) and methyl parathion hydrolase (MPH) immobilised via active tag holo-ACP had higher catalytic efficiency (kcat/Km) in comparison with their corresponding counterparts immobilised via the sulfhydryl groups (-SH) of these proteins. The immobilised holo-ACP-Amy also presented higher thermostability compared with free Amy. The enhanced α-amylase thermostability upon immobilisation via holo-ACP renders it more suitable for industrial application.

Improving Catalytic Activity and Thermal Stability of Methyl-Parathion Hydrolase for Degrading the Pesticide of Methyl-Parathion

Pesticides are indispensable in today’s agriculture. Methyl-parathion hydrolase (MPH, E.C.3.1.8.1) could hydrolyze organophosphorus pesticides, including methyl-parathion. MPH could rehabilitate soil and water resources contaminated by organophosphorus pesticides. However, natural MPHs generally exhibited a low tolerance to high temperatures and low catalytic efficiency. In this study, we improved the catalytic efficiency toward methyl-parathion and the thermal stability of the MPH from Pseudomonas sp. WBC-3 through saturation mutagenesis and fusion with self-assembling amphipathic peptides (SAP). The experimental characterization showed that compared to the wild-type enzyme, the kcat/Km of the mutant T271S yielded by saturation mutagenesis was increased by 224.3% compared to the wild-type MPH. T50 and Tm of SAP3-MPH with an SAP fused at the N-terminus were increased by 6.2°C and 6.0°C, respectively. Compared to the wild-type enzyme, T271S fused with SAP3 at the N-terminus (SAP3-T271S) exhibited a 2.1-fold increase in kcat/Km without significantly affecting the thermal stability. The simultaneous improvement of the catalytic efficiency and thermal stability of MPH would be beneficial for its application in the degradation and detection of organophosphorus pesticides. Furthermore, our study provides a potential combination strategy for the design of the other enzyme preparations of pollutant degradation.

Environmental Distribution, Metabolic Fate, and Degradation Mechanism of Chlorpyrifos: Recent and Future Perspectives.

Pesticides play a significant role in crop production and have become an inevitable part of the modern environment due to their extensive distribution throughout the soil ecosystem. Prophylactic applications of chlorpyrifos (CP) affect soil fertility, modify soil microbial community structure, and pose potential health risks to the nontarget organisms. Bioremediation through microbial metabolism is found to be an ecofriendly and cheaper process for CP removal from the environment. So far, various bacterial and fungal communities have been reported for CP and its metabolites degradation. Organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH) are crucial bacterial enzymes for CP degradation as they efficiently hydrolyze the unbreakable P-O and P = S bond. This review discusses the prospects of toxicity level, persistency, and harmful effects of CP on the environment. CP degradation mechanisms, metabolic pathways, and key enzymes, along with their structural details, are also featured. The highlights on molecular docking with OPH and MPH enzyme for CP show the best binding affinity with OPH; hence, it is an essential part of CP degradation. Simultaneously, metagenomic analysis of soil from contaminated agricultural lands and wastewater was analyzed with the goal to identify the dominant CP degraders and enzymes. The identification of potent degraders, key enzymes, and evaluation of microbial community dynamics upon pesticide exposure can be used as a warning for its dissemination and biomagnification into the food chain.

The adaptive landscape of a metallo-enzyme is shaped by environment-dependent epistasis

Enzymes can evolve new catalytic activity when environmental changes present them with novel substrates. Despite this seemingly straightforward relationship, factors other than the direct catalytic target can also impact adaptation. Here, we characterize the catalytic activity of a recently evolved bacterial methyl-parathion hydrolase for all possible combinations of the five functionally relevant mutations under eight different laboratory conditions (in which an alternative divalent metal is supplemented). The resultant adaptive landscapes across this historical evolutionary transition vary in terms of both the number of “fitness peaks” as well as the genotype(s) at which they are found as a result of genotype-by-environment interactions and environment-dependent epistasis. This suggests that adaptive landscapes may be fluid and molecular adaptation is highly contingent not only on obvious factors (such as catalytic targets), but also on less obvious secondary environmental factors that can direct it towards distinct outcomes.

CityApps: A bioinformatics tool for predicting the key residues of enzymes weakly interacting with monovalent metal ions

Monovalent metal ions play an essential role in some enzyme reactions. Due to the loose interactions, identifying the critical residues interacting with monovalent metal ions based on a molecular dynamic (MD) analysis is still a challenge. In this study, we first defined the enrichment factor (Eaa) and the dwell time (td) to describe the interactions with metal ions in each enzyme residue during MD analysis. A "sandwich-like" solvent box for MD analysis was then constructed to avoid the interference of the initial position of metal ions. Finally, a bioinformatics tool, CityApps, was developed to facilitate the automatization of MD analysis and the calculation of the interaction parameter. Using the tool, the key residues (D191, Q272, and N329) responsible for K+-mediated activation of a methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3 were identified based on Eaa, td, and RMSF differences. Ala mutation showed that the mutant D191A/Q272A/N329A was difficult to be activated by 1 M K+. In addition, Q272A showed a 176.19 % increase in specific activity compared to the wild MPH type. Our results indicate that CityApps developed here could be an effective tool for identifying enzyme residues interacting with monovalent metal ions and those residues that are critical for catalytic reaction.

High-level extracellular production and immobilisation of methyl parathion hydrolase from Plesiomonas sp. M6 expressed in Pichia pastoris

Methyl parathion hydrolase (MPH) hydrolyses methyl parathion efficiently and specifically. Herein, we produced MPH from Plesiomonas sp. M6 using a Pichia pastoris multi-copy expression system. The original signal peptide sequence of the target gene was removed, and a modified coding sequence was synthesised. Multi-copy expression plasmids containing MPH were constructed using pHBM905BDM, and used to generate recombinant strains containing 1, 2, 3 or 4 copies of the MPH gene. The results showed that a higher target gene copy number increased the production of recombinant MPH (MPH-R), as anticipated. The expression level of the recombinant strain containing four copies of the MPH gene was increased to 1.9 U/ml using 500 ml shake flasks, and the specific activity was 15.8 U/mg. High-density fermentation further increased the target protein yield to 18.4 U/ml. Several metal ions were tested as additives, and Ni2+, Co2+ and Mg2+ at a concentration of 1 mM enhanced MPH-R activity by 196%, 201% and 154%, respectively. Enzyme immobilisation was then applied to overcome the difficulties in recovery, recycling and long-term stability associated with the free enzyme. Immobilised MPH-R exhibited significantly enhanced thermal and long-term stability, as well as broad pH adaptability. In the presence of inhibitors and chelating agents such as sodium dodecyl sulphate (SDS), immobilised MPH-R displayed 2-fold higher activity than free MPH-R, demonstrating its potential for industrial application.

MPH-GST sensing microplate for easy detection of organophosphate insecticides.

To develop a convenient and efficient means for organophosphate (OP) insecticide detection, a simple, cost-effective, and easy-to-use absorbance-based sensing device was generated using methyl parathion hydrolase fused with glutathione-S-transferase (MPH-GST) covalently immobilized onto a chitosan film-coated microplate. With methyl parathion (MP) as a representative substrate, this MPH-GST sensing microplate had the detection limit of 0.1µM and the linear range of 0.1-50µM. Despite its highest stability at 4°C, it was considerably stable at 25°C with high activity for 30 days. It was also most stable at pH 8.0 and could be efficiently reused up to 100 rounds. The device revealed a high percentage of recovery for tap water spiked with a known concentration of MP, which was also comparable to the result obtained from gas chromatography-mass spectrometry. It also showed a high recovery of 82-100% with MP spiked agricultural products and satisfactory results with non-spiked samples. This immobilized enzyme sensing system was more sensitive and efficient than the whole cell system from our previous work. All of the advantages of the MPH-GST sensing microplate developed have rendered it suitable for rapid and convenient OP screening, and for being a bio-element for fabricating a potential optical biosensor in the future.

In-depth biochemical identification of a novel methyl parathion hydrolase from Azohydromonas australica and its high effectiveness in the degradation of various organophosphorus pesticides

Organophosphorus pesticides are highly toxic phosphate compounds with the general structure of O = P(OR)3 and threaten human health seriously. Methyl parathion hydrolase from microbial is an important enzyme to degrade organophosphorus pesticides (OPs) into less toxic or nontoxic compounds like.p-nitrophenol and diethyl phosphate. Here, a gene encoding methyl parathion hydrolase from Azohydromonas australica was firstly cloned and expressed in Escherichia coli. The recombinant hydrolase showed its optimal pH and temperature at pH 9.5 and 50 °C. Leveraging 1 mM Mn2+, the enzyme activity was significantly enhanced by 29.3-fold, and the thermostability at 40 and 50 °C was also improved. The recombinant MPH showed the specific activity of 4.94 and 16.0 U/mg towards methyl parathion and paraoxon, respectively. Moreover, A. australica MPH could effectively degrade various of OPs pesticides including methyl parathion, paraoxon, dichlorvos and chlorpyrifos in a few minutes, suggesting a great potential in the bioremediation of OPs pesticides.

Random Mutagenesis by Insertion of Error-Prone PCR Products to the Chromosome of Bacillus subtilis.

Bacillus subtilis is an attractive host for the directed evolution of the enzymes whose substrates cannot be transported across cell membrane. However, the generation of a mutant library in B. subtilis suffers problems of small library size, plasmid instability, and heterozygosity. Here, a large library of random mutant was created by inserting error-prone PCR (epPCR) products to the chromosome of B. subtilis. Specifically, the epPCR product was fused with flanking regions and antibiotic resistant marker using a PCR-based multimerization method, generating insertion construct. The epPCR product was integrated into the chromosome via homologous recombination after the insertion construct was transformed into the supercompetent cells of B. subtilis strain SCK6. The transformation efficiency of the insertion construct was improved through co-expressing homologous recombination-promoting protein NgAgo, raising the number of competent cells, and increasing the length of flanking regions. A library containing 5.31 × 105 random mutants was constructed using per μg insertion construct, which is sufficient for directed evolution. The library generation process was accomplished within 1 day. The effectiveness of this method was confirmed by improving the activity of Methyl Parathion Hydrolase (MPH) toward chlorpyrifos and by enhancing the secretion level of MPH in B. subtilis. Taken together, the present work provides a fast and efficient method to integrate epPCR products into the chromosome of B. subtilis, facilitating directed evolution and expression optimization of target proteins.

Organophosphorus Nerve Agents: Types, Toxicity, and Treatments.

Organophosphorus compounds are extensively used worldwide as pesticides which cause great hazards to human health. Nerve agents, a subcategory of the organophosphorus compounds, have been produced and used during wars, and they have also been used in terrorist activities. These compounds possess physiological threats by interacting and inhibiting acetylcholinesterase enzyme which leads to the cholinergic crisis. After a general introduction, this review elucidates the mechanisms underlying cholinergic and noncholinergic effects of organophosphorus compounds. The conceivable treatment strategies for organophosphate poisoning are different types of bioscavengers which include stoichiometric, catalytic, and pseudocatalytic. The current research on the promising treatments specifically the catalytic bioscavengers including several wild-type organophosphate hydrolases such as paraoxonase and phosphotriesterase, phosphotriesterase-like lactonase, methyl parathion hydrolase, organophosphate acid anhydrolase, diisopropyl fluorophosphatase, human triphosphate nucleotidohydrolase, and senescence marker protein has been widely discussed. Organophosphorus compounds are reported to be the nonphysiological substrate for many mammalian organophosphate hydrolysing enzymes; therefore, the efficiency of these enzymes toward these compounds is inadequate. Hence, studies have been conducted to create mutants with an enhanced rate of hydrolysis and high specificity. Several mutants have been created by applying directed molecular evolution and/or targeted mutagenesis, and catalytic efficiency has been characterized. Generally, organophosphorus compounds are chiral in nature. The development of mutant enzymes for providing superior stereoselective degradation of toxic organophosphorus compounds has also been widely accounted for in this review. Existing enzymes have shown limited efficiency; hence, more effective treatment strategies have also been critically analyzed.

Supreme Catalytic Properties of Enzyme Nanoparticles Based on Ferritin Self-Assembly

In this study, we aimed to address if enzymes self-assembled on the biological nanosphere matrix would have some nanoscale effects and then improve the catalytic ability and other characteristics especially for the resistance to adverse conditions. Initially, we evaluated two different forms of ferritins, DNA-binding protein from starved cells (Dps) and recombinant heavy chain of human ferritin (rHF), fused with methyl parathion hydrolase (MPH) to be self-assembled as the dodecamer and 24-mer enzyme nanoparticles (enzyme-NPs), Dps-MPH and MPH-rHF. Both self-assembled enzyme-NPs showed superior catalytic activity and kcat drastically increased, but MPH-rHF showed better performance than Dps-MPH. Next, Escherichia coli alkaline phosphatase (EAP) and the canonical mutant EAP(D101S) were chosen to confirm if rHF-based self-assembly generally improves the catalysis of enzymes with the different catalytic abilities. Additionally, restoration of the enzyme native forms on the rHF shell by increasing the flexibility of the polylinker between the catalytic units and the nanoscaffold further improved the catalytic activities of enzyme-NPs. Some of the nanoparticles exhibited better residual activities under extreme conditions. Conclusively, the enzyme-NPs based on rHF self-assembly provided an effective strategy for enzyme engineering.