Stabilization of Hexa-Histidine-Containing Organophosphorus Hydrolase

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1		Olga Senko	--	4252	2023-02-13 10:52:02	\|
2	layout	Camila Xu	Meta information modification	4252	2023-02-14 02:28:42	\|

This entry is adapted from the peer-reviewed paper 10.3390/polym15030591

Organophosphorus hydrolase, containing a genetically introduced hexahistidine sequence (His₆-OPH) can hydrolyze various substrates, such as organophosphorus pesticides and chemical warfare agents, mycotoxins, and N-acyl homoserine lactones. The application of various carrier materials (metal-organic frameworks, polypeptides, bacterial cellulose, polyhydroxybutyrate, succinylated gelatin, etc.) for the immobilization and stabilization of His₆-OPH by various methods, enables creation of biocatalysts with various properties and potential uses, in particular, as antidotes, recognition elements of biosensors, in fibers with chemical and biological protection, dressings with antimicrobial properties, highly porous sorbents for the degradation of toxicants, including in flow systems, etc. Immobilized variants of His6-OPH are characterized by increased stability, and the hydrolytic process of destruction of many substrates can be carried out in wider temperature and pH ranges than when using a free form of the enzyme. The variation in methods and carriers for the immobilization of His6-OPH makes it possible to create a wide palette of biocatalysts, significantly expanding the boundaries of enzyme use.

organophosphorus hydrolase hexa-histidine tag immobilization

1. Introduction

Interest in the enzyme aryldialkylphosphatase (EC 3.1.8.1)—which is now known in the scientific literature under two names that are actively used in various studies, organophosphorus hydrolase (OPH) and phosphotriesterase (PTE)—increases due to a number of objective reasons. On the one hand, among these reasons are problems associated with the widespread use of organophosphorus compounds (OPCs) as agricultural pesticides ^[1], their toxic properties, and their ability to accumulate in various biological objects, environmental objects (aquatic environments and soils), as well as organs and tissues ^[2]. On the other hand, this is dictated by the need to degrade these substances in the most favorable conditions for ecosystems and their components, including animals and humans, and to carry out bioremediation measures aimed at reducing pollution of environmental objects (soil, water, and air), food, and agricultural raw materials by various OPCs ^[3]. In addition, there is a demand for highly effective antidotes in the event of serious cases of human intoxication with such compounds ^[2], which may be the result of their penetration into the human body through oral, inhalation, and/or transdermal routes.

Today, the possibility of using enzymes as catalysts for the destruction of OPCs is characterized by a fairly high action efficiency and a wide range of different substrates—which, as it turns out, may include not only the OPCs and not only pesticides, but also chemical warfare agents (CWAs) as well as products of their chemical and biocatalytic degradation ^[3]^[4]^[5]^[6].

Among the main advantages of using enzymes for the decomposition of OPCs, especially in natural objects, are their high level of catalytic activity, chemical and stereoselectivity, minimal amounts of byproducts, and compatibility with different biological systems ^[7]. The latter is extremely important when the enzyme is used as a part of a hydrolytic antidote or a long-circulating preventive nanoscavenger acting against the OPCs in vivo ^[8]^[9] (Figure 1). In addition, the enzymatic hydrolytic degradation of OPCs can be used for the biosensing determination of these substances ^[10]^[11].

Figure 1. Various methods of immobilization (red) and applications (blue) of hexa-histidine-containing OPH.

It should be noted that the search for new sources of OPH, the genetic modification of which is carried out through the use of various plasmids encoding the synthesis of an enzyme with a polyhistidine sequence mainly at the N-terminus of a protein molecule, is constantly being conducted in different laboratories around the world ^[5]^[12]^[13]^[14]. A study of the properties of such genetically modified OPH variants has shown that the presence of a hexahistidine tag in the enzyme molecule (His₆-OPH) gives it new properties: stability of action in the presence of various surfactants ^[15], action at increased temperatures, and changed pH of the medium ^[16]. The improved biocatalytic characteristics and altered stereospecificity of the catalytic action were also revealed for the His₆-OPH as compared to OPH ^[17]. The latter property can be achieved by combining the genetic introduction of a polyhistidine sequence into the enzyme molecule with site-directed mutations in the active center of the enzyme and in the amino acid residues around it or in the substrate-binding domain ^[11]^[18].

For a number of enzymes, the term PTE-like lactonases (PLLs) has been introduced, since they had a fairly high homology with PTE/OPH but low activity in OPC hydrolysis reactions compared to OPH. These enzymes had greater hydrolytic activity against N-acyl homoserine lactones (AHLs), which are signaling molecules of the quorum of Gram-negative bacteria. Verification of similar lactonase activity in His₆-OPH led to the conclusion that this enzyme may be of practical interest not only for solving the issues of destruction and detection of OPCs but also for serving as the basis for such a process as Quorum Quenching (QQ), since the lactonase activity of His₆-OPH in reactions with different AHLs was comparable with that of PLLs ^[19] (Figure 1).

2. Stabilization of Hexa-Histidine-Containing OPH for OPC Hydrolysis

When discussing the possibilities of using various approaches to stabilize hexahistidine-containing OPH, it should be noted, first of all, the expediency of using carriers for immobilized metal ion affinity chromatography is intended for the interaction through which His₆-tag is generically introduced into the enzyme molecule. It is on this interaction of the affine sequence and metals in the composition of chelating groups (which, as a rule, modify various polymer carriers (agarose, cryogels of synthetic and natural polymers, etc.)) is based that fast and highly effective immobilization, and then isolation and purification of the enzyme itself occurs. The emerging coordination bonds between metal atoms in the carrier and nitrogen atoms in the imidazole rings of the polyhistidine sequence in the protein molecule make it possible to anchor the enzyme on the carrier quite firmly ^[2]^[20]^[21] and use it for a long time, in such an immobilized form, for the destruction of OPCs (Table 1, ^[21]^[22]^[23]^[24]^[25]^[26]^[27]^[28]^[29]^[30]^[31]^[32]^[33]^[34]^[35]^[36]^[37]^[38]^[39]^[40]^[41]^[42]^[43]^[44]^[45]). Of particular interest are such immobilization variants for the stabilization and repeated use of the enzyme in the degradation of OPC-polluting water systems ^[20]^[21]. In fact, another variant of carriers that can be used for the ion-chelating immobilization of His₆-OPH is metal–organic frameworks (MOFs), which are usually used as porous functional materials ^[46]. They are formed through the self-assembly of organic ligands containing oxygen or nitrogen atoms and metal atoms (or metal clusters) as the inorganic part of MOFs. By varying organic ligands and metals, it is possible to control the pore size of materials, the specificity and functional modification of their surface area, and their interaction with various proteins. Owing to these capabilities, the MOFs are widely used in the study of catalytic processes and biosensors, generally. MOFs used for the immobilization of enzymes usually provide significantly improved stability and recyclability of biocatalysis in diverse applications ^[47]^[48]^[49]^[50]^[51]^[52].

Table 1. Samples of immobilized and stabilized forms of His₆-OPH for reactions with OPCs.

Variants of His-Tagged OPH or PTE [Reference]	Immobilization Technique and Carrier	Properties	Application
Use of metalorganic interactions and frameworks
OpdA@Ni-NTA-VMSN ^[21]	Ni²⁺-nitrilotriacetic acid (NTA)-modified, virus-like, mesoporous silica nanoparticles	The affine capacity of substrates became higher for the enzyme after its oriented immobilization	Hydrolysis of methyl parathion in a plug-flow reactor
OpdA@MIL-88A ^[22]	MOF synthesized on the basis of fumaric acid and FeCl₃	The catalytic activity is 5 times higher as compared to the free form of the enzyme. The enzyme possessed improved organic detergent and solvent tolerance and thermal and storage stability	Degradation of organophosphorus pesticides on grapes and cucumbers
BSA-Cu@CaPs-OPH ^[23]	A hybrid organic–inorganic, calcium phosphate-containing nanocrystal, based on the use of bovine serum albumin modified by Cu²⁺ ions	Improved multiple usages with up to 56% retainment of activity after 10 working cycles; advanced thermal and pH stability	Hydrolysis of methyl parathion on fruits or vegetables
OPH^6His/UiO-66-NH₂ ^[24]	MOF, synthesized using amino-terephthalic acid and zirconium chloride with further pre-activation by N,N′-dicyclohexylcarbodiimide, was covalently bonded with the enzyme over the surface via UiO-66-NH₂	Improved storage stability (up to 60 days), reusability (in 9 working cycles), and 37% improved catalytic activity	Degradation of methyl parathion
OPH^6His@Tb-BTC ^[25]	Encapsulation of the enzyme in MOF, formed by terbium nitrate and 1,2,4-benzenetricarboxylic acid (BTC)	Improved storage stability and a 30% increase in activity	Sensing of methyl parathion
OpdA@Co/C@SiO₂@Ni/C ^[26]	An enzyme created multiple affine coordination bonds with Co and Ni introduced into carbonized hybrid nanocomposite ZIF-67@ SiO₂ with a yolk-shell structure	Increased pH, thermal and storage stability, and SDS resistance; long reusability (in 7 working cycles) with 60% of residual activity	Chemo-enzymatic cascade degradation of methyl parathion
Co/MnHF@PTE ^[27]	An enzyme was involved in the multimetallic nanoflower structure during its formation in a mixture of metal salt solutions and protein: Co-PTE (CoHF@PTE) and Mn-PTE (MnHF@PTE) hybrid	The improved catalytic activity of 4 times due to the use of two metals (Co and Mn) in the frame of the nanoflower-like structure of MOF	Hydrolysis of methyl parathion, VX and soman
OPH@H-Au-TiO₂ ^[28]	An enzyme was adsorbed on the hollow-structured nanoparticles, Au-TiO₂	Increased reusability in the destruction of OPCs and very stable activity due to the synergistic combination of photo- and enzymatic catalysis	Degradation of methyl parathion
OPH@0.8CoZIF ^[29]	An enzyme was encapsulated to Zn-doped Co-based ZIF via biomimetic mineralization	The enzyme is involved in a cascade of chemical reactions where the 4-aminophenol is the last product	Conversion of methyl parathion
YT-PTE on FE ^[30]	A mutant enzyme was immobilized by sorption on the Fuller’s Earth (FE)	The temperature and storage stability were improved, but there were no notable changes in the activity of the immobilized enzyme compared to its free form; several bivalent ions (Co, Ni, Cu, Fe, Zn) exhibited significantly higher increases in the activity of the immobilized enzyme	Hydrolysis of paraoxon
Use of fusion proteins and bioconjugations
[scGFP-arPTE][S⁻] ^[31]	Fusion proteins, containing green fluorescent protein (GFP) and PTE, were involved in an electrolytic interaction with monomeric S⁻ with further drying and cross-linking to obtain a catalytically active porous film or cross-linking in the presence of cotton fiber to obtain composite textiles	Improved thermal stability and higher catalytic constant (k_cat)	Introduction into cotton fibers for the creation of personalized protective composite material useful for the recyclable hydrolysis of paraoxon
[arPTE][S⁺][S⁻] ^[32]	Obtainment of bioconjugates through the subsequent introduction of the enzyme into contact with cationic (Ethoquad) and anionic (oxidized IGEPAL) polymer surfactants (S⁺ or S⁻), resulting in the formation of such a structure as corona encapsulating the enzyme	Enhancement of the efficiency of catalytic action by 3 times	Hydrolysis of paraoxon
[arPTE][S⁺][S⁻] –ABC, [arPTE][S⁺][S⁻] –PCL ^[33]	An enzyme, in the presence of cationic or anionic polymer surfactants (S⁺ or S⁻), was lyophilized with further melting of the obtained powder and used in co-dispersion with curtain polymers (ABC, acrylonitrile butadiene styrene; PCL, polycaprolactone) for 3D-printing on the surface of stainless steel rings	Improved stability of enzyme action	Hydrolysis of paraoxon-ethyl by composite enzyme plastics developed for use in self-decontaminating surfaces
PoOPH_M9-CLEPC ^[34]	An enzyme containing 10 residues of genetically introduced phenylalanine was conjugated with Pluronic F127 to form a cross-linked enzyme–polymer conjugate (CLEPC)	Increased optimal temperature (50 °C) and pH stability in the range of 7–11	Hydrolysis of malathion
OPH@pID-MSN ^[35]	An enzyme was covalently immobilized on mesoporous silica nanoparticles (MSNs) coated with a zwitterionic polymer containing short hydrophobic chains, which is a product of the ring-opening reaction between poly (isobutylene-alt-maleic anhydride) and N,N-dimethylethylenediamine (pID)	Significantly improved stability	Hydrolysis of methyl parathion
Sorption and covalent immobilization on natural carriers
OPH/PCD ^[36]	An enzyme was adsorbed onto the microparticles of poly-β-cyclodextrin (PCD) with further freeze-drying	Increased sorption capacity of the substrate and product of the enzymatic hydrolysis; possible regeneration of activity via new enzyme sorption and the self-decontamination of the biocatalyst	Hydrolysis of methyl paraoxon
OpdA-PHB ^[37]	An enzyme was immobilized on non-porous poly(hydroxyl butyrate) (PHB) microspheres	The carrier increased the sorption of the hydrophobic substrate from the medium	Hydrolysis of coumaphos
SpOpdA-SPS-S ^[38]	An enzyme was immobilized via the covalent binding of His-tag as SpyTag (Sp) to such fusion proteins as SpyCatcher (SPS)-coated poly(hydroxy alkanoates) (PHAs) spheres (S)	Improved catalytic activity and stability	Hydrolysis of coumaphos
Conjugation using DNA molecules
QD-DNA-PTE ^[39]	An enzyme was attached, via the DNA-containing linker, to PEGylated quantum dots (QDs)	Enhanced efficiency and rate of catalytic reaction (k_cat)	Hydrolysis of paraoxon
DNA cage-QD-PTE ^[40]	Molecules of DNA were conjugated into a cage by His₅-peptide and modified by quantum dots (QDs) of ZeS with further immobilization of PTE	Enhanced catalytic activity by 12.5 times	Hydrolysis of paraoxon
PTE^pAzF -DNA ^[41]	An enzyme was conjugated to a DNA scaffold modified by dibenzocyclooctyl via a site-specific incorporated azido-group to the enzyme molecule structure	Improved catalytic constants	Hydrolysis of paraoxon
Formation of complexes
OPT−PIMs ^[42]	An enzyme (OPT) was involved in the formation of pollen-inspired microparticles (PIMs), prepared based on complexation between CaCO₃, gelatin, and the enzyme	Improved stability at 50 °C and low pH 4.8 (citric acid/sodium citrate buffer)	Detoxification of pollen contaminated by paraoxon or malathion
His₆-OPH/PLE_50, His₆-OPH/PLD₅₀ ^[43]	Enzyme was involved in polyelectrolyte complexes with poly-l-glutamic acid (PLE₅₀) or poly-l-aspartic acid (PLD₅₀)	Increased stability of catalytic action in the soil	Destruction of chlorpyrifos in different types of soil
His₆OPH/PEG₁₁₃PLE₁₀, His₆OPH/PEG₁₁₃PLE₅₀, His₆OPH/PEG₁₁₃PLE₁₀₀, His₆OPH/PEG₁₁₃PLD₅₀, His₆OPH/PEG₂₂PLE₅₀, His₆OPH/PLE₅₀PEG₁₁₃PLE₅₀ His₆OPH/Hydroxyethyl starch His₆OPH/Succinylated gelatin ^[44]	An enzyme was involved in polyelectrolyte complexes with PEGylated poly-l-glutamic acid (PLE_10-100), poly-l-aspartic acid (PLD₅₀) of a different polymerization degree, hydroxyethyl starch, or succinylated gelatin	20–40% increased catalytic efficiency of enzyme action	Hydrolysis of methyl parathion and paraoxon
PCL-RHP-OPH ^[45]	An enzyme was immobilized through electrospinning in poly-ε-caprolactone fibrous mats in the form of a lyophilized complex dissolved in toluene with random heteropolymers (RHPs)	Enhanced stability and reusability (40% residual activity after everyday use for 3 months)	Hydrolysis of methyl parathion and paraoxon

The interest in such carriers is additionally motivated by the possible detoxifying effects of self-MOFs in relation to toxins, including OPCs (pesticides and CWA) ^[50]^[51]^[52]. Since then, MOFs as supports look very attractive for the obtainment and use of immobilized forms of His₆-OPH ^[22]^[23]^[24]^[25]^[26]^[27].

Combinations of MOFs with enzymes catalyzing the hydrolysis of OPCs are promising since they allow for the combination of the potential of chemical and biological catalysts. An example of such a combination obtained on the basis of His₆-OPH is the development of a conjugate of this enzyme with UiO-66-NH₂-containing MOFs ^[24]. This variant of the enzyme combination with MOFs guarantees the production of a catalyst, which is characterized by several improved properties at once (activity, stability, substrate specificity of action), allowing for the use of such an immobilized enzyme both for the decomposition of methyl parathion and for its detection in fairly low concentrations (10 ng/mL) and, at the same time, in a fairly wide range of concentrations (10–10⁶ ng/mL) ^[24].

The use of MOF modified by Terbium-BTC, where BTC is 1,2,4-benzenetricarboxylic acid, for the encapsulation of His-tagged organophosphorus hydrolase (OPH^6His) allowed the researchers of the development to improve the stability of the enzyme and increase the activity of the resulting biocatalyst by 30% in comparison with the original enzyme ^[25]. The sample of the modified enzyme was obtained by mixing terbium nitrate pentahydrate (Tb(NO₃)₃ × 5H₂O) and an aqueous solution of the enzyme and BTC in ethanol. It was successfully applied for providing a highly sensitive determination of methyl parathion in grapes and tomatoes with a low enough limit of detection (2.6 nM).

An interesting solution of coordinating the immobilization of His-tagged organophosphohydrolase (OpdA) via the metal chelating interactions was realized using the yolk-shell structured Co/C@SiO₂@Ni/C nanocomposites ^[26]. It was obtained through the carbonization treatment of the polyhedral ZIF-67@SiO₂ composite. The stabilization of the structure of the carbonized nanocomposite was completed through the creation of SiO₂-layer protection. Further, the Ni-nanoparticles were introduced to the surface of the formed structure and further enzymes were successfully immobilized. This example of the combined variant of the chemo-enzymatic nanocatalyst was capable of hydrolyzing OPCs and detecting methyl-parathion with a low enough limit of detection (300 nM). Comparisons of the activity and stability of the immobilized biocatalyst against the initial enzyme demonstrated their improvement.

Recently developed and reviewed methods of obtaining new hybrid nanostructures, named “nanoflowers” ^[53]^[54]^[55], were applied for the immobilization of His-tagged PTE ^[27]. Usually, chlorides or phosphates of metals are used in the mixture with proteins for the creation of organic–inorganic structures of nanoflowers. In this investigation, the combination of the concurrent introduction of both Co and Mn provided the best results with the enzyme.

The use of a carrier enzyme (hollow-structured nanoparticles, Au-TiO₂) for immobilization, which, due to its composition, provides the possibility of the photocatalytic decomposition of OPCs, makes it possible to obtain a material with new functions combining chemical and biological catalysis. This makes the process of the degradation of toxins more resistant to the variation in external factors, and the material itself, with the synergistic effect of its constituent components, allows for more long-term use ^[28]. It should be noted that the immobilization itself leads to a noticeable decrease in the hydrolytic activity of the enzyme, but the possibility of photocatalysis compensates for this decrease during the decomposition of methyl parathion.

The use of hollow-structured nanoparticles, Au-TiO₂, as a carrier for enzyme immobilization (which, due to its composition, provides the possibility of photocatalytic decomposition of OPCs) makes it possible to obtain a material with new functions combining chemical and biological catalysis, which makes the process of degradation of organophosphorus toxins more resistant to the variations in external factors. The material itself, with the synergistic effect of its constituent components, allows for more long-term usage ^[28]. It should be noted that the immobilization itself leads to a noticeable decrease in the hydrolytic activity of the enzyme, but the opportunity of photocatalysis compensates for this decrease during the decomposition of methyl parathion.

Polyhistidine-containing OPH was immobilized on a carrier known as Fuller’s Earth, which, in addition to hydrated aluminum-magnesium silicate, contained various metal ions (Mg²⁺ and Ca²) as well as montmorillonite, kaolinite, and attapulgite/bentonite ^[30]. The carrier used is known for its high sorption capacity with respect to proteins as well as to various chemical agents and toxins, and this possibility provided the enzyme with the presence of a substrate in excess, simulating catalytic (non-limiting) reaction conditions.

A fundamentally different approach, based on the use of polyelectrolyte interactions of the surface of the His-tagged enzyme with variously charged surfactants, was used by researchers for the creation of various bioconjugates to improve the stability and catalytic characteristics of the enzyme ^[31]^[32]^[33]. At the same time, the expediency of using such coatings for the enzyme, including forming a capsule in the form of a so-called corona, was noted. Such structures made it possible to avoid the direct influence of various unfavorable factors on the enzyme itself. They simultaneously retained the possibility for the covalent modification of surfactants, the possible addition of various polymers to them, and their introduction into diverse plastics, fabric materials, and 3D printing systems, allowing the enzyme to be applied to metal surfaces as a part of the created conjugates. This creates scientific and practical foundations for the development of personal protective equipment against the toxic effects of OPCs. Additional genetic modifications of the enzyme itself—particularly due to the connection with the fusion protein (GFP) ^[31] before its encapsulation in the layers of surfactants—create the possibility of additional control of the effect of immobilization conditions on the properties of the main biocatalyst through the properties of the selected fusion partner (fluorescence intensity of GFP).

The traditional approach to the immobilization of His₆-tagged OPH continued to be developed in recent years, and the cross-linking conjugation of the enzyme modified by the genetic introduction of 10 residues of phenylalanine was demonstrated with Pluronic F127 ^[34]. This work and another study based on the application of mesoporous silica nanoparticles coated with a zwitterionic polymer ^[35] were good illustrations for the successful covalent binding of the enzyme to a polymeric molecule to retain and improve its stability. The results obtained ^[34] revealed better catalytic activity of enzyme conjugates as compared to samples prepared by the covalent immobilization of the same enzyme into well-known, cross-linked enzyme aggregates (CLEA) by using glutaraldehyde.

An example of how His₆-tagged OPH can be immobilized by sorption and covalent immobilization on natural carriers, varying the properties of the carriers themselves, are studies based on poly-β-cyclodextrin ^[36] and polyhydroxyalkanoate) (PHA) microspheres ^[37]^[38]. Interestingly, multiple modifications of these microparticles with the introduction of various spacers and fusion proteins onto their surface had virtually no effect on the fact that, in the end, the activity of the enzyme under study exceeded the same parameter known for the soluble enzyme.

The solution arrived at by the researchers of the development, in which His₆-tagged OPH was connected directly to the shells of PEGylated quantum dots (QD) through a linker, which was comprised of DNA connected to nanoparticles, looks unusual and promising ^[39]^[40]. Firstly, the DNA appeared to be a good anchor for the enzyme, since it provided the availability of its active center for catalysis and ensured its successful conformation, which contributed to the increase in the OPH catalytic constant in paraoxon hydrolysis. Secondly, the modification of DNA molecules—on the one hand by an enzyme, which can potentially be used as an antidote, and on the other hand, the possible attachment of DNA to nanoparticles, which can have targeted delivery to certain cells and tissues—can be continued in the form of the development of new nanobiosensors.

It is interesting to note that an increase in the DNA concentration relative to QD-OPH and the creation of dense composites led to an increase in the enzymatic reaction rate by 12.5 times ^[41]. It should be noted that, in principle, the use of DNA molecules modified for their use as bioscaffolds for the enzyme under discussion has proven to be very good in terms of an effective means of improving the catalytic characteristics of the obtained biocatalysts even in the absence of QD ^[42]. It turned out that, depending on how the site-specific introduction of functional groups into the enzyme molecule for its subsequent conjugation with DNA occurs, K_M, k_cat, and, accordingly, their ratio (k_cat/K_M) can be directionally changed.

The method of immobilization and stabilization of His₆-tagged OPH, due to the formation of various polyelectrolyte complexes, turned out to be very simple in implementation and very promising according to the results of its application ^[8]^[9]^[42]^[43]^[44]^[45]. At the same time, proteins (gelatin), poly(carboxybetaine), poly(amino acids), and their copolymers with poly(ethylene glycol), turned out to be partners in such enzyme–polyelectrolyte complexes. All these developments have proved to be highly successful not only in terms of the simplicity of the formation of stable forms of the enzyme (characterized by significantly improved resistance in the manifestation of catalytic activity with a significant decrease in pH and a temperature increase) but also in terms of their possible applications—not only to solve problems of OPC destruction in aqueous media, in particular samples of contaminated soils ^[43], but also in the blood ^[8]^[9] and gastrointestinal tracts of animals ^[44]. Thus, the preparation of OPT-PIMs enzyme complexes allowed the researchers to develop a sample of an enzyme included in a polyelectrolyte complex, whose microparticles can be consumed by bees (together with plant pollen particles) and catalyze the destruction of several organophosphorus pesticides while maintaining activity at pH 4.8 in the insect digestive system.

The creation of non-covalent complexes based on His₆-OPH and the PEG₁₁₃-PLE₅₀ block copolymer ^[8] showed that the enzyme acquires improved catalytic characteristics and increased stability during storage compared to the original enzyme. The introduction of such non-covalent complexes in vivo showed a decrease in immune and inflammatory reactions to His₆-OPH. The pharmacokinetic parameters of such complexes were also improved compared to the soluble enzyme, which ensured their longer circulation in the bloodstream after intravenous (iv) administration in rats. In addition, the enzyme remained bioavailable after intraperitoneal (ip), intramuscular (im), and even transbuccal (tb) administration and had the ability to protect animals from exposure to OPCs (paraoxon, VX) in vivo.

The development of similar biocatalysts, by applying a thin, ultra-hydrophilic, semi-permeable polymer gel layer poly(carboxybetaine) (PCB) to the surface of the enzyme ^[9], showed that the resulting gel-coating layer of the zwitterionic polymer also provides long-term circulation and minimal immunogenicity of the resulting form of the enzyme biocatalyst. The study of the properties of such an enzyme in vivo in models of different animals before or after exposure to OPCs has demonstrated the protective and antidote effectiveness of their action. In guinea pig models, a single prophylactic administration of such a biocatalyst effectively prevented the mortality of animals after repeated exposure to sarin for 1 week. The study showed an increase in catalytic characteristics and the stabilization of the enzymatic activity of His₆-tagged OPH. The results obtained show that the profile application of such a biocatalyst can be effective in preventing the toxic effects of OPCs (paraoxon, sarin) in vivo.

It should be noted that the variants of the immobilization of His₆-tagged OPH for the hydrolysis of OPCs described here have been developed by researchers only in recent years, but even their diversity is very wide. It should be noted that the use of the enzyme under discussion is the most well-studied precisely for the degradation of OPCs. However, the spectrum of the substrates of this enzyme is not limited to OPCs ^[2]^[19]^[56], and therefore, the options for its stabilization and immobilization for use in other hydrolytic reactions remain extremely interesting. A lot of parameters of biocatalytic systems can be predicted in silico: the interaction of the enzyme with novel substrates ^[4]^[6], the interaction of the enzyme microenvironment/partner with substrates ^[41], the binding of enzymes with individual polymers ^[44], etc. Prospectively, computer modeling may allow the designing of the necessary catalytic system on a turnkey basis.

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Subjects: Materials Science, Biomaterials

Contributors MDPI registered users' name will be linked to their SciProfiles pages. To register with us, please refer to https://encyclopedia.pub/register :

Elena Efremenko

Ilya Lyagin

Aysel Aslanli

Nikolay Stepanov

Olga Maslova

Olga Senko

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Update Date: 14 Feb 2023

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1. Introduction

2. Stabilization of Hexa-Histidine-Containing OPH for OPC Hydrolysis

References