WO2015145222A2 - Recombinant and stable ssopox enzymes, method of generation thereof and reusable nanobiocatalyst of the same - Google Patents

Recombinant and stable ssopox enzymes, method of generation thereof and reusable nanobiocatalyst of the same Download PDF

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WO2015145222A2
WO2015145222A2 PCT/IB2014/064616 IB2014064616W WO2015145222A2 WO 2015145222 A2 WO2015145222 A2 WO 2015145222A2 IB 2014064616 W IB2014064616 W IB 2014064616W WO 2015145222 A2 WO2015145222 A2 WO 2015145222A2
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rssopox
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polypeptides
enzyme
recombinant
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French (fr)
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WO2015145222A3 (en
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Abhay Hariram PANDE
Priyanka BAJAJ
Rajan Kumar TRIPATHY
Ankita JADHAV
Gaurav Shriharishankar CHANDAK
Harsh Dineshchandra PARIKH
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National Institute Of Pharmaceutical Education And Research (Niper)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
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    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
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    • C12P21/00—Preparation of peptides or proteins
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    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/08—Phosphoric triester hydrolases (3.1.8)
    • C12Y301/08001—Aryldialkylphosphatase (3.1.8.1), i.e. paraoxonase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2305/00—Use of specific compounds during water treatment
    • C02F2305/08—Nanoparticles or nanotubes

Definitions

  • the present disclosure relates generally to the field of biotechnology. More specifically it relates to novel nucleotides which encode recombinant SsoPox (rSsoPox) enzymes, a novel method of production of rSsoPox enzymes, their stable compositions, and a reusable nanobiocatalyst of rSsoPox enzyme.
  • rSsoPox recombinant SsoPox
  • Organophosphate (OP)-compounds are highly toxic chemicals that exert their harmful effects by inhibiting the biological functions of enzymes required for the transmission of nerve message and proper functioning of the nervous system (Bloch-Shilderman., et al., 2008, Toxicol. Appl. Pharmacol. 231, 17). OP-compounds are easy to manufacture and are widely used as pesticides, fungicides, insecticides, herbicides and petroleum additives in agriculture and other industries. Certain OP-compounds developed by armies as chemical warfare nerve agents (CWNAs) are much more dangerous and have become important terrorist chemical weapons in today's world.
  • CWNAs chemical warfare nerve agents
  • OP-compounds contaminate terrestrial and aquatic ecosystems throughout the world, and are responsible for large number of OP-associated poisoning cases world-wide.
  • Current treatments of OP- poisoning include a combined administration of a cholinesterase reactivator (oxime), a muscarinic receptor antagonist (atropine), and an anticonvulsant (diazepam).
  • oxime cholinesterase reactivator
  • atropine muscarinic receptor antagonist
  • diazepam anticonvulsant
  • biofilms are commonly formed on important surfaces viz., surgical instruments and machineries and equipment's used in various industries, e.g., food-, paper-, power generating-, agriculture- and aquaculture-industries and water filtration and distribution system, and this is recognized as a big problem.
  • Some bacteria use quorum sensor molecules (acyl homoserine lactones; AHLs) to regulate biofilm formation. By inhibiting the quorum sensing signaling (by degrading AHLs) it is possible to control biofilm-forming bacterial contamination and infections and there is a need to develop agents which can inhibit quorum sensing and prevent biofilm formation.
  • SsoPox is a ⁇ 35 kDa, metal-dependent enzyme from thermophilic organism Sulfolobus solfataricus.
  • SsoPox belongs to an enzyme family called as phosphotriesterase-like lactonase (PLLs) and hydrolyzes and inactivates many OP-compounds as well as different type of AHLs.
  • PLLs phosphotriesterase-like lactonase
  • the enzyme is exceptionally stable under harsh conditions viz, high temperature, in the presence of organic solvents and detergents (Hiblot, J., et al., 2012, Sci. Rep., 2, 779).
  • SsoPox is a strong candidate for the development of agent not only for the therapeutic intervention of OP-poisoning and certain bacterial infections in humans and other animals, but also for the development of safe, effective, and environmental-friendly means for decontamination (clean-up) of OP-contaminated objects and areas and biofilm inhibitor in the industrial and environmental settings.
  • rSsoPox suffers from certain limitations associated with the use of recombinant protein pharmaceuticals, viz., immunogenicity, low circulating half-life due to kidney clearance or protease digestion, etc.
  • Chemical conjugation of recombinant protein pharmaceuticals with polyethylene glycol (PEG) (PEGylation) is a successful approach known in art to overcome some of these limitations.
  • Conjugation of recombinant proteins with PEG improve various properties of proteins, viz. increased solubility, enhanced circulating half-life, decreased renal clearance, lower immunogenicity and resistance to protease degradation.
  • Limitations and challenges regarding the use of SsoPox :
  • Non-availability of a cost effective method to produce SsoPox enzyme in pure and active form and in high yield The methods described in the art for the production of SsoPox enzymes are complex, expensive, and result in low yield of pure and active SsoPox enzymes.
  • Protease sensitivity of purified enzyme Sensitivity of SsoPox enzyme towards proteolytic degradation and potential immunogenicity is a major challenge for its therapeutic use.
  • Prior Art Limitations of prior art Novelty in the present documents invention 1) Merone, L., Prior arts 1 to 9 discloses use of E. coli and The present invention discloses et al, 2005, Pseudomonas putida expression systems for an E. coli expression system for
  • Elias, M., et yield of 0.89 mg/g of wet cell mass of E. coli The present invention provides al. 2008, J. Mol. 1 mg/1 of E. coli culture, and 3.6 mg/g of wet E. coli expression system for the
  • Prior art 9 discloses immobilization of high yield (300 - 450 mg / 3-4 g ) Hiblot, J., et purified rSsoPox on non-magnetic of wet cell mass of E. coli). al, 2012, Sci. nanoalumina-functionalized membrane and
  • ONE 8(9) variant of rSsoPox enzyme having enhanced a method of production of e75272 OP-hydrolyzing activity disclosed in the rSsoPox enzymes, by refolding ) Ng, F.S.W., current invention. the recombinant enzymes et al, 2010, expressed as inclusion bodies in
  • composition(s) for long-term storage • Novel improved variant: stabilization of purified SsoPox enzyme.
  • the invention discloses novel variant of r SsoPox enzyme
  • composition(s) the reaction mixture after their for long-term storage stabilization of purified use and stored and can be reused
  • Novel polynucleotide sequence encoding rSsoPox enzyme having enhanced hydrolytic activities towards at least one SsoPox- substrate
  • compositions for long-term storage stabilization of pure rSsoPox enzyme iv. N-terminal mono-PEGylated-r SsoPox conjugate having decreased protease sensitivity, and
  • a novel reusable nanobiocatalyst produced by covalently immobilizing pure rSsoPox enzyme on magnetic nanoparticles, which can be isolated from the reaction mixture after its use and stored and reused multiple times to degrade OP-compounds.
  • One object of the present invention is to provide a novel bacterial construct for the generation and production of rSsoPox enzymes.
  • Still another object of the present invention is to provide a reusable nanobiocatalyst generated by covalently immobilizing pure rSsoPox enzyme onto magnetic nanoparticles.
  • the reusable nanobiocatalyst can be recovered from the reaction mixture/environment after its use and can be stored and reused multiple times to degrade OP-compounds.
  • the invention discloses novel polynucleotide sequences encoding rSsoPox ( wt ) enzyme and its variant having enhanced hydrolytic activity toward at least one SsoPox- substrate, a novel method to produce r SsoPox enzymes in highly pure and active form in high yield by refolding the recombinant enzymes expressed as inclusion bodies in E.
  • the reusable nanobiocatalyst can be recovered from the reaction mixture/environment after its use and can be stored and reused multiple times to degrade OP- compounds.
  • One aspect of the present invention is to provide a recombinant polynucleotide (rSsoPox) and variant thereof comprising of SEQ ID NOs. 1-2.
  • Another aspect of the present invention is to provide a recombinant polynucleotide (rSsoPox) and variant, wherein the polynucleotide encodes at least one polypeptide chain comprising at least one amino acid sequence of SEQ ID NOs. 3-4, wherein the at least one polypeptide chain has increased hydrolytic activity toward at least one SsoPox- substrate.
  • rSsoPox recombinant polynucleotide
  • the SsoPox- substrate is selected from a group consisting of organophosphates, lactones and acylhomoserine lactones.
  • nucleic acid constructs comprising the isolated polynucleotides of SEQ ID NOs. 1-2.
  • nucleic acid constructs comprising the isolated polynucleotides encoding polypeptide of SEQ ID NOs. 3-4.
  • Still another aspect of present invention provides isolated host cell comprising the nucleic acid constructs comprising the isolated polynucleotides of SEQ ID NOs. 1-2.
  • compositions of rSsoPox polypeptide comprising rSsoPox polypeptide in combination with at least a buffering agent, a cofactor, a salt, a detergent, an amino acid, a sugar, or mixture thereof.
  • stabilized pharmaceutical composition comprises of recombinant polypeptide, wherein said recombinant polypeptide exhibits OP- hydrolyzing activity and can be rSsoPox ( wt ) or its variant.
  • the present invention provides a N-terminal mono-PEGylated-rSsoPox conjugate, wherein the PEGylated rSsoPox enzyme is mono-PEGylated-rSsoPox, wherein PEG is covalently linked to rSsoPox enzyme at its N-terminal end.
  • Another aspect of the present invention provide a mono-PEGylated-rSsoPox conjugate wherein the molecular weight of the conjugated PEG was selected among the molecular weights of 2 kDa, 5 kDa, 10 kDa, 20 kDa, preferably 5 kDa.
  • the PEG molecule is a linear molecule selected among the group of linear and branched PEGs.
  • the PEG molecule of is methoxy PEG aldehyde selected from a group of PEG-succinimidyl carbonate, PEG-pN0 2 phenyl carbonate, PEG- AA-NHS and PEG-carbonylimidazole.
  • Still another aspect of the present invention is to provide a reusable nanobiocatalyst produced by covalently immobilizing pure rSsoPox enzyme onto magnetic nanoparticles.
  • Another aspect of the present invention provides use of polynucleotides and polypeptides of the present invention for contacting/coating on wearables and devices; for treating bacterial infections; for sterilization; for water purification systems; for air filtration systems and for decontaminating OP-contaminated objects and surfaces.
  • Panel A is a schematic representation of a synthetic gene designed for the expression of rSsoPox ⁇ ) enzyme in E. coli.
  • Panel B shows a map of expression plasmid (construct) pET23a(+) containing the designed gene for rSsoPox ( wt ) cloned between Ndel and Xhol restriction sites.
  • Figure 2 shows the alignment of rSsoPox( Wt ) and native SsoPox gene sequences. Dashes and shaded area represents identical and dissimilar nucleotides, respectively.
  • Panels A and B show the representative images of Coomassie stained (4-20%) sodium dodecyl sulphate-polyacrylamide gel (SDS-PAGE) and western blot, respectively, of the fractions collected during a typical purification of inclusion bodies of rSsoPox enzyme. Anti-His antibody was used as a primary antibody in the western blot analysis.
  • Panel C shows a representative chromatogram illustrating separation of enzymatically active rSsoPox enzyme from the inactive enzyme present in the refolding mixture, by using ion-exchange chromatography.
  • (- ⁇ -) and (- .4 -) denote the absorbance at 280 nm and paraoxonase activity of the eluted fractions from the columns, respectively.
  • (»»» ⁇ ) represent increasing ionic strength (NaCl concentration) of the elution buffer.
  • Panel D is an image of silver stained (4-20%) sodium dodecyl sulphate-polyacrylamide gel of fractions obtained at different stages of rSsoPox enzyme separation by ion-exchange chromatography. Symbols: lane M: protein molecular weight markers; lane 1 : protein refolding mixture before loading onto the column; lane 2: unbound fraction; lane 3 : washes; lane 4: pooled fractions from peak PI (containing inactive enzyme); and lane 5: pooled fractions from peak P2 (containing active enzyme).
  • Figure 4 shows Lineweaver-Burk plot for paraoxon-hydrolysis by rSsoPox enzymes.
  • Purified enzyme 0.2 M final concentration
  • concentrations of paraoxon 0.2-2 mM
  • the hydrolysis data were fitted by Lineweaver-Burk equation and the R 2 values were 0.94- 0.99.
  • the kinetic parameters are given in the table. Figure 5.
  • Panel A shows a representative chromatogram illustrating separation of N-terminal mono-PEGylated-rSsoPox(variant-A) from the PEGylation reaction mixture, by using ion- exchange chromatography.
  • (- .4 -) and (-0-) denote the absorbance at 280 nm (xlO "2 ) and paraoxonase activity of the eluted fractions from the columns, respectively.
  • ( ⁇ ) represent increasing ionic strength (NaCl concentration) of the elution buffer.
  • Panel B shows the image of silver-stained sodium dodecyl sulphate-polyacrylamide gel.
  • lane M protein molecular weight markers
  • lane 1 unmodified rSsoPox( va riant-A)
  • lane 2 N-terminal mono- PEGylated-rSsoPox(variant-A)-
  • Panel C shows protease sensitivity of unmodified rSsoPox( va riant- A) (bars i and ii) and N-terminal mono-PEGylated-rSsoPox( va riant-A) (bars iii and iv) when the enzymes were incubated with trypsin for 4h at 37°C and the stability of proteins was determined by measuring the residual paraoxonase activity.
  • bar i unmodified rSsoPox(variant-A); bar ii: unmodified rSsoPox( va riant-A) + trypsin; bar iii : N-terminal mono- PEGylated-rSsoPox(variant-A); and bar iv: N-terminal mono-PEGylated-rSsoPox( va riant-A) + trypsin.
  • Panel A show a schematic representation of the chemistry employed for covalent immobilization of pure rSsoPox( va riant-A) enzyme onto magnetic nanoparticles.
  • Panels B and C shows paraoxon-hydrolyzing activity of (i) equal amount of free rSsoPox( va riant-A), ( ⁇ ) free rSsoPox(variant-A) present in the supernatant of immobilization reaction after centrifugation, and (iii) resuspended rSsoPox( va riant-A)-MNPs (nanobiocatalyst).
  • Panels D and E are the image depicting dispersion of rSsoPox( va riant-A)-immobilized nanobiocatalyst in TBS buffer (20 mM Tris-HCl, pH 8.0 containing 150 mM NaCl and 0.2 mM CoCl 2 ) in the absence (panel D) and the presence (panel E) of externally applied magnetic field.
  • Figure 7 shows a bar graph depicting paraoxon-hydrolyzing activity of rSsoPox( va riant-A)- immobilized nanobiocatalyst in aqueous TBS buffer.
  • the rSsoPox( va riant-A)-immobilized nanobiocatalyst was suspended in TBS containing 1 mM paraoxon and the sample was incubated for 6 h at 40°C. At the end of incubation the rSsoPox( va riant-A)-irnmobilized nanobiocatalyst was recovered from the reaction mixture by applying external magnetic field (i.e., a magnet) and the OD 40 5 of the reaction mixture was measured in a spectrophotometer to determine the amount of paraoxon hydrolyzed by the rSsoPox( va riant-A)-immobilized nanobiocatalyst.
  • external magnetic field i.e., a magnet
  • Biocatalyst refers to a substance, especially an enzyme that initiates or modifies the rate of conversion of one chemical compound (substrate) into another (product).
  • Chaotropic agent refers to a compound that in a suitable concentration in aqueous solution is capable of changing the spatial conformation of proteins so as to render the proteins soluble in an aqueous solution.
  • Denatured refers to a biologically inactive form of expressed recombinant protein obtained after dissolving the inclusion bodies with chaotropic agents.
  • Enzymatically active or functionally active refers to ability of enzyme to exert one or more activities known to be associated with SsoPox enzyme, such as ability to hydrolyze paraoxon.
  • Excipients or stabilizing agents refer to various chemicals whose presence alters the stability of proteins. The terms also refer to pharmacologically inactive substances used as a carrier for the active ingredients of a medication.
  • Error prone PCR refers to error prone polymerase chain reaction which introduces mutations into a target gene during the reaction.
  • Helper plasmid refers to plasmid that encodes tRNAs for the rare codons of the gene of heterologous recombinant proteins produced in bacterial expression systems, and includes plasmids like pRARE, pRIG, pACYC, pSClOl, etc.
  • Heterologous proteins refer to those proteins that are foreign to the host cells used for the production of such proteins, such as rSsoPox produced m E. coli.
  • Inclusion bodies refer to cytoplasmic aggregates of over-expressed, misfolded recombinant proteins expressed in transformed E. coli host cells and which may not be biologically active.
  • Magnetic nanoparticles refers to a class of nanoparticles which can be manipulated using magnetic field. Such particles commonly consist of magnetic elements such as iron, nickel and cobalt and their chemical compounds. The size of nanoparticles are smaller than 1 micrometer in diameter.
  • Misfolded and enzymatically inactive protein refer to protein molecules that do not possesses native three-dimensional conformation, are biologically inactive and in some instances have toxic functionality.
  • Mono-pegylation refers to attachment of single PEG molecule to one protein molecule.
  • Mono-PEGylated-rSsoPox conjugate refers to r SsoPox having one PEG attached to the enzyme.
  • Mutagenesis refers to any process of change in the base sequence of DNA that can be deletion, insertion, inversion or substitution such that information of a gene is changed or altered in a stable manner.
  • Nanobiocatalyst refers to nanoparticles coated with particular enzyme and which is capable of initiation or modification of the rate of conversion of one chemical compound (substrate) into another (product).
  • Nanoparticles refers to small objects whose size ranges from less than 100 nm to 1000 nm.
  • Native SsoPox enzyme refers to SsoPox enzyme from thermophilic organism Sulfolobus solfataricus (EC 3.1.8.1) that hydrolyzes and inactivates SsoPox-substrates.
  • Optimized polynucleotide sequence refers to a synthetic nucleic acid sequence optimized for high level expression of recombinant protein in E. coli.
  • Organophosphate (OP)-compounds refer to toxic chemicals that exert their harmful effects by inhibiting neurotransmitter-metabolizing enzymes required for the transmission of nerve messages and proper functioning of the nervous system.
  • PEGylation refers to the modification of biological molecules by covalent conjugation with polyethylene glycol (PEG).
  • Pharmaceutically accepted excipient refers to pharmacologically inactive substances used as a carrier for the active ingredients of a medication. Such substances do not interfere with the biological activity of the therapeutic proteins.
  • Polypeptide term 'polypeptide' in this invention refers to a rSsoPox enzyme or protein.
  • Immunogenicity refers to the ability of a particular substance, such as an antigen, to provoke an immune response in the body of a human or animal.
  • Proteolytic enzymes refers to a group of enzymes that break the long chain like molecules of proteins into shorter fragments (peptides), e.g., trypsin.
  • Protease resistance refers to ability of the proteins to resist the degradation by various proteolytic enzymes.
  • SsoPox-substrate refers to a synthetic or natural chemical selected from a group of but not limited to organophosphates or phosphotriesters; such as paraoxon, diazoxon, chlorpyrifosoxon, methyl-DEPCyC, bramophos-ethyl, chlorpyrifos, chlorfenvinphos, chlorothiophos, chlorpyrifos-methyl, coumaphos, crotoxyphos, crufomate, cyanophos, diazinon, dichlofenthion, dichlorvos, dursban, EPN, ethoprop, ethyl-parathion, etrimifos, famphur, fensulfothion, fenthion, fenthrothion, isofenphos, jodfenphos, leptophos-oxon, malathion, methyl-parathion, mevinphos, parathion, parathi
  • Random mutagenesis refers to process of introducing a permanent change in desired DNA sequence randomly and selecting the mutated sequence based on desired characteristics.
  • Random PEGylation refers to attachment of PEG molecules in a random manner to all the available H 2 groups in the protein molecule.
  • Recombinant SsoPox refers to a recombinantly produced SsoPox enzymes produced in foreign host cells like mammalian cell, bacterial cell, insect cell etc.
  • Refolding additives refers to various chemicals that are known to help in refolding of the denatured proteins.
  • Refolding buffer refers to a buffered solution containing various chemical additives that assist in the refolding of denatured proteins.
  • Refolding refers to a process of reintroducing secondary and tertiary structure to a protein that has had some or all of its native secondary or tertiary structure lost, either in vitro or in vivo, e.g., as a result of expression conditions or intentional denaturation and/or other modification.
  • the correctly refolded protein is biologically active and may possesses three- dimensional conformation similar to native protein (enzyme).
  • Reusable refer to conditions in which the nanobiocatalyst are recovered from the reaction mixture after its use, stored appropriately, and are reused multiple times to carry out a particular reaction (like hydrolysis of OP-compounds).
  • rSsoPox polypeptide or protein or enzyme the term 'rSsoPox polypeptide or rSsoPox protein or rSsoPox enzyme' collectively refers to rSsoPox ( wt ) and its variant of this invention that are produced in E. coli.
  • rSsoPox refers to a wild-type recombinant SsoPox enzyme produced in E. coli which is similar to the naturally occurring native SsoPox enzyme in terms of its enzymatic activity towards at least one SsoPox-substrate (MTCC accession # MTCC5840; GenBank accession # KF924249).
  • rSsoPox variant refer to mutant of rSsoPox ( wt ) enzyme that contain at least one gene mutation (e.g., insertion, deletion, substitution).
  • gene mutation e.g., insertion, deletion, substitution.
  • One variant of the enzyme is disclosed in the present invention and coding of the same has been done as below:
  • rSsoPoX( var iant-A)- refers to a rSsoPox enzyme containing two amino acid substitutions (Y40N and N297D) in rSsoPox (wt) .
  • MTCC accession # MTCC5841; GenBank accession # KF924250 refers to a rSsoPox enzyme containing two amino acid substitutions (Y40N and N297D) in rSsoPox (wt) .
  • Site-specific/N-terminal PEGylation refers to attachment of PEG molecule at N-terminal end of the protein molecule.
  • Stability of protein refers to the tendency of proteins to maintain their functional activity.
  • Transformed cells refer to host E. coli cells containing plasmid (construct) containing gene coding for target recombinant protein.
  • Unmodified rSsoPox refers to non-PEGylated-rSsoPox.
  • the present invention relates to novel polynucleotide sequences encoding rSsoPox( Wt ) enzyme and its variant having enhanced hydrolytic activity towards at least one SsoPox- substrate, a novel method to produce rSsoPox enzymes in highly pure and active form in high yield by refolding the recombinant enzymes expressed as inclusion bodies in E.
  • rSsoPox a recombinant polynucleotide (rSsoPox) and variant thereof comprising at least one nucleotide sequence selected from the group comprising SEQ ID NOs: 1-2.
  • polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 3-4.
  • polypeptide in another embodiment of the present invention is provided a recombinant polypeptide wherein the said polypeptide has hydrolytic activity towards at least one SsoPox- substrate and wherein at least one polypeptide chain has increased hydrolytic activity towards at least one SsoPox- substrate.
  • a recombinant polynucleotide and variant thereof comprising at least one nucleotide sequence selected from the group comprising SEQ ID NOs: 1-2, wherein the polynucleotide encodes at least one polypeptide chain comprising at least one amino acid sequence selected from the group comprising SEQ ID NOs: 3-4, wherein at least one polypeptide chain has increased hydrolytic activity towards at least one SsoPox- substrate.
  • a recombinant polypeptide chain selected from the group comprising SEQ ID NOs: 3-4, wherein the SsoPox-substrate is selected from the group comprising organophosphates (phosphotriesters), lactones, and acylho mo serine lactones.
  • a recombinant polypeptide chain selected from the group comprising SEQ ID NOs: 3-4, wherein the SsoPox-substrate is selected from the group comprising organophosphates (phosphotriesters), lactones, and acylho mo serine lactones, wherein the organophosphates (phosphotriesters) are selected from the group comprising paraoxon, diazoxon, chlorpyrifosoxon, methyl-DEPCyC, bramophos- ethyl, chlorpyrifos, chlorfenvinphos, chlorothiophos, chlorpyrifos-methyl, coumaphos, crotoxyphos, crufomate, cyanophos, diazinon, dichlofenthion, dichlorvos, dursban, EPN, ethoprop, ethyl-parathion, etrimifos, famphur, fensulfothion
  • a recombinant polypeptide chain selected from the group comprising SEQ ID NOs: 3-4, wherein the SsoPox-substrate is selected from the group comprising organophosphates (phosphotriesters), lactones, and acylho mo serine lactones, wherein the lactones are selected from the group comprising ⁇ - butyrolactone, ⁇ -caprolactone, ⁇ -octanoiclactone, ⁇ -nonanoiclactone, ⁇ -decaanoiclactone, ⁇ - nonanoic-5-lactone, undecanoic-5-lactone, dodecanoic-5-lactone, ⁇ -caprolactone, etc.
  • a recombinant polypeptide chain selected from the group comprising SEQ ID NOs: 3-4, wherein the SsoPox-substrate is selected from the group comprising organophosphates (phosphotriesters), lactones, and acylho mo serine lactones, wherein the acyl homoserine lactones are selected from the group comprising N-butrylhomoserine lactone, N-hexanoyal-DL-homoserinelactone, N-heptanoyl- DL-homoserine lactone, N-octanoyal-DL-homoserine lactone, N-decanoyal-DL-homoserine lactone, N-dodecanoyal-DL-homoserine lactone, N-tetradecanoyal-DL-homoserine lactone, N-oxodecanoyal-DL-homoserine lactone, N-oxodecanoyal
  • a recombinant polynucleotide and variant thereof comprising at least one nucleotide sequence selected from the group comprising SEQ ID NOs: 1-2, wherein the polynucleotide encodes at least one polypeptide chain comprising at least one amino acid sequence selected from the group comprising SEQ ID NOs: 3-4, wherein at least one polypeptide chain has increased hydrolytic activity towards at least one SsoPox-substrate, wherein at least one of the said polypeptide chain possesses increased hydrolytic activity (k cat /K m ratio) by at least 40-folds for paraoxon substrate with respect to the rSsoPox ⁇ ).
  • a method for production of recombinant polypeptide comprising amino acid sequence of rSsoPox and variant thereof by refolding inclusion bodies comprising the steps of- ⁇ designing rSsoPox gene and subjecting it to random-mutagenesis,
  • the host cell selected from the group of living cells but not limiting to bacterial cells, yeast cells, animal cells, plant cells, insect cells, and may be cell free expression system.
  • the host cell is E. coli.
  • the transformed E. coli cells are grown under the conditions favorable for high level expression of heterologous recombinant proteins.
  • One or more favorable experimental conditions may include effective media, temperature, pH, oxygen condition, shaking time and speed, and like.
  • various chemical or physical agents such as IPTG, lactose, low or high temperature change, and the like, are used to induce high level expression of rSsoPox enzymes iri E. coli cells.
  • the E. coli cells were lysed by using either physical or chemical methods such as sonication, French press, lysozyme- and detergent-treatment, and the like, to release the rSsoPox enzymes.
  • the purity of the isolated inclusion bodies varies from at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100%.
  • a method for production of recombinant polypeptide comprising amino acid sequence of rSsoPox and variant thereof by refolding inclusion bodies wherein the chaotropic agent is selected from the group comprising organic compounds, solvents, salts, detergents and is preferably urea.
  • the rSsoPox enzymes are refolded with the help of chemical assisted dilution refolding selected from a group of refolding procedures viz., simple batch dilution, continuous dilution, dialysis, chromatography-column based refolding, chaperone assisted refolding, and the like.
  • a method for production of recombinant polypeptide comprising amino acid sequence of rSsoPox ( wt ) and variant thereof by refolding inclusion bodies, wherein the refolding buffer comprises at least one buffering agent selected from the group of Tris-HCl, CHES, EPPS, HEPES, Glycine-NaOH, Phosphate, TAPS, MOPS, and MES.
  • a method for production of recombinant polypeptide comprising amino acid sequence of rSsoPox ( wt ) and variant thereof by refolding inclusion bodies, wherein the refolding buffer further comprises a mixture of at least one redox pair selected from a group of reduced glutathione/oxidized glutathione; oxidized nicotinamide dinucleotine / reduced nicotinamide dinucleotide and cystamine / cysteamine.
  • a method for production of recombinant polypeptide comprising amino acid sequence of rSsoPox ( wt ) and variant thereof by refolding inclusion bodies, wherein the refolding buffer further comprises at least one cofactor selected from the group of CaCl 2 , CoCl 2 , MgCl 2 , and ZnCl 2 .
  • a method for production of recombinant polypeptide comprising amino acid sequence of rSsoPox ( wt ) and variant thereof by refolding inclusion bodies, wherein the refolding buffer further comprises at least one refolding additives selected from the group of salts, sugars, polymers, polyols, detergents and surfactants.
  • a method for production of rSsoPox polypeptides wherein said polypeptide are derived from at least one nucleotide sequence selected from the group comprising SEQ ID NOs: 1-2, wherein said polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 3-4, wherein the method results into high level expression of rSsoPox polypeptides as enzymatically non-functional aggregated inclusion bodies, wherein said method comprises the step of inducing the host cell culture with 0.1-2.0 M IPTG concentration and growing the cultures for 4-24 h at 37°C.
  • a method of production of enzymatically active rSsoPox polypeptides from inclusion bodies wherein said polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 3-4, wherein said method comprises the steps of:
  • a method of production of enzymatically active rSsoPox polypeptides from inclusion bodies wherein said polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 3-4, wherein the refolding buffer comprises at least one buffering agent, at least one redox pair, at least one cofactor, at least one refolding additives, or mixture thereof;
  • the buffering agent is selected from the group of Tris-HCl, CHES, EPPS, HEPES, Glycine- NaOH, Phosphate, TAPS, MOPS, and MES
  • the redox pair is selected from a group of reduced glutathione/oxidized glutathione; oxidized nicotinamide dinucleotine / reduced nicotinamide dinucleotide and cystamine / cysteamine
  • the cofactor is selected from the group of CaCl 2 , CoCl 2 , MgCl 2 , and ZnC
  • a method of production of enzymatically active rSsoPox polypeptides from inclusion bodies wherein said polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 3-4, wherein the purity of enzymatically active rSsoPox polypeptides is at least 80%.
  • the final concentrations of rSsoPox enzymes used in the refolding reaction varies from less than 1 mg/ml including less than 300 ⁇ g/ml, such as less than 100 ⁇ g/ml, including less than 40 ⁇ g/ml.
  • the optimum time of refolding varies from 2 to 24 h depending upon the refolding condition and rSsoPox enzymes.
  • the yield of refolding varies from at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100% depending on different refolding conditions, purity of inclusion bodies and rSsoPox enzymes.
  • the method used for the separation/isolation is ion-exchange chromatography selected from a group of ion-exchange chromatography, gel filtration chromatography, adsorption chromatography, affinity chromatography, hydrophobic chromatography, reversed phase chromatography, and like.
  • the optimum refolding conditions can be same or different for rSsoPox enzymes.
  • the yield of enzymatically active rSsoPox enzymes is at least 10 mg, 100 mg, 1 g, 10 g, 100 g, 0.1 kg, 10 kg, or 100 kg. According to the still another embodiment of the present invention the yield of enzymatically active rSsoPox enzymes ranges from 300-450 mg / 3-4 g wet cell mass of E. coli.
  • compositions for long-term storage stability of rSsoPox enzymes in aqueous solution and freeze-dried form at 25°C wherein the rSsoPox can be rSsoPox(wt) or any of its variant and preferably is rSsoPox( va riant-A).
  • the storage-stable rSsoPox compositions of the present invention are fully solubilized in aqueous solution, i.e., in a water-based solution.
  • aqueous-based gels could also be used in the present invention, so long as such material permits the complete solubilization of rSsoPox enzyme contained therein.
  • the storage-stable rSsoPox compositions are stably stored in ready-to-use fluid form and in a freeze-dried form.
  • the temperature of the solution during storage is not particularly restricted, so long as the rSsoPox enzyme contained therein remains stable ⁇ i.e., retains functional activity).
  • the preferred temperature for storage of the rSsoPox(variant-A) enzyme compositions of the present invention ranges from -80°C to 90°C, depending upon the state of the solution (aqueous solution or freeze-dried form).
  • the composition of this invention contains pharmaceutically accepted excipients alone or in combination and have been chosen from a group of various buffers, sugars, polyols, amino acids, polymers, salts, non-ionic detergents, cofactors, proteins, and the like.
  • the composition of this invention contains at least one excipient or component.
  • concentration of such excipient(s) and/or component(s) may vary in the compositions, depending on the objective, and the concentration must be sufficient to allow such excipient(s) and/or component(s) to achieve their intended or stated purpose.
  • concentration of such excipient(s) and/or component(s) can be empirically determined by one of ordinary skill in the art by testing various concentrations and selecting that which is effective for the intended purpose and site of application.
  • the pharmaceutical excipients in the composition can be replaced by any of the similar excipients such as:
  • Tris-HCl can be replaced with HEPES, EPPS, TAPS and like.
  • proline can be replaced by any other amino acid like serine, threonine, alanine, and like.
  • sugars For e.g. maltose can be replaced by any other sugar like trehalose, sucrose, glucose and like.
  • Non-ionic surfactants For e.g. tween 20 can be replaced by any other non-ionic surfactant like tween 80, P-10, P-40, and like.
  • Salt For e.g. NaCl can be replaced by any other salt like KC1 and like.
  • compositions of present invention may include, besides stabilizers, isotonic reagents and buffering reagents, any other substances or materials known in the art within the range of not impairing the effects of the invention.
  • the percentage stability of rSsoPox enzyme varies from at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100%) which may varies for different compositions.
  • no antimicrobial agent is added to the storage stable composition of rSsoPox.
  • antimicrobial agent(s) can be added to avoid microbial contamination of the storage stable composition of rSsoPox enzyme over long-term storage.
  • the concentration of enzyme may vary from 1 nM to 1M.
  • the present invention provides a mono-PEGylated-rSsoPox, wherein the rSsoPox can be rSsoPox(wt) or any of its variant and preferably is rSsoPox( va riant-A).
  • the rSsoPox is modified with site specific PEGylation selected from a group of site specific and random PEGylation.
  • the PEGylated enzyme has a PEG attached at the N-terminus of protein molecule selected among the groups of C-terminus, cysteine specific and others.
  • the molecular weight of PEG attached is 5 kDa, selected among the molecular weights of 2 kDa, 5 kDa, 10 kDa, 20 kDa and more.
  • the PEG attached is a linear molecule selected among the group of linear and branched PEGs.
  • the PEG attached is methoxy PEG aldehyde selected from a group of PEG-succinimidyl carbonate, PEG-pN0 2 phenyl carbonate, PEG-AA-NHS and PEG-carbonylimidazole.
  • the N-terminal mono-PEGylated-rSsoPox has decreased protease sensitivity as compared to unmodified enzyme.
  • N-terminal mono-PEGylated-rSsoPox is particularly suitable for treatment of OP-poisoning in human and other live stocks.
  • nucleic acid constructs comprising the isolated polynucleotides of the present invention.
  • nucleic acid constructs comprising the isolated polynucleotides encoding polypeptides of the present invention.
  • an isolated host cell comprising the nucleic acid constructs of the present invention.
  • the rSsoPox enzyme having enhanced OP-hydrolyzing activity can be used for decontaminating OP-contaminated surfaces.
  • synthetic and biological surfaces contemplated according to embodiments of the invention include, but are not limited to, equipment's, laboratory hardware's, devices, fabrics, skin, eatables (organic food and vegetable's surfaces) and delicate membranes (e.g., biological), and the like.
  • the rSsoPox variant having enhanced OP-hydrolyzing activity can be used to treat or prevent OP-poisoning in OP- poisoned subjects.
  • use of polynucleotides and polypeptides of the present invention for treating bacterial infections; for sterilization; for water purification systems; for air filtration systems and for decontaminating OP- contaminated objects and surfaces.
  • a rSsoPox-immobilized magnetic nanobiocatalyst comprising a magnetic nanoparticles coated with rSsoPox, wherein the rSsoPox can be rSsoPox( Wt ) or any of its variant and preferably is rSsoPox( va riant-A), wherein
  • the immobilization surfaces can be carrier particles, nanomaterials, fabrics, membranes, discs, metals and more, preferably MNPs,
  • the immobilization can be done by either covalent binding, non-covalent binding and adsorption method, preferably by covalent binding, wherein rSsoPox enzyme immobilized on the surface of nanoparticles are used for degradation of OP-compounds.
  • a method for degrading OP-compounds in water system using the rSsoPox-immobilized magnetic nanobiocatalyst described above is provided.
  • the rSsoPox enzyme having enhanced OP-hydrolyzing activity can be used for making masks, tissue paper, soaps, foam, aerosols, air fresheners, zeolites, wipes, plastic bags, glass, wall papers, paints, hydrogels, spray, sponges, inhalers, silicates, manures, for coating various medical devices / equipments / tools, gloves, nanomaterials, fabrics, membranes, discs, metals and more, but essentially are MNPs.
  • the rSsoPox enzyme has been immobilized on the surface of carrier particles preferably MNPs. MNPs have many advantages over nonmagnetic nanoparticles like targeted delivery, easy recovery and reusability.
  • any method for immobilization of protein e.g. covalent linkage, entrapment and non-covalent interactions
  • the method of immobilization selected here for magnetic nanoparticles, is covalent linkage.
  • the magnetic particles chosen for enzyme coating essentially contain CMX as functional group among a group of amines, carboxylic acids, chitosan and the like.
  • the percentage binding of enzyme to MNPs vary from at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100%.
  • the activity retained by the immobilized enzyme varies from at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100%) with respect to activity of free enzyme.
  • the enzyme coated MNPs are reusable and the reusability varies to at least 1 cycle to 5, 10, 100 and 1000 cycles.
  • the immobilized-rSsoPox enzyme can be used in water purification system for decontaminating the drinking water, in air filtration system for decontaminating the air, in biosensors for sensing the various toxic OP contaminants in environmental samples, cleaning the accidental spillage of OP-compounds during manufacturing, transport and war scenarios, in making kits and cartridges containing enzyme for decontamination of various surfaces.
  • Example 1 Generation of nucleic acid construct (plasmid) containing novel polynucleotide sequence encoding rSsoPox(wt).
  • Nucleic acid sequence of the designed gene coding for rSsoPox(wt) was aligned with the nucleic acid sequence of naturally occurring SsoPox and the comparison is given in FIG. 2.
  • the designed gene for rSsoPox ⁇ exhibits only 34.6% similarity with the gene of naturally occurring SsoPox.
  • the rSsoPox(wt) protein contains 314 amino acids (Met ⁇ Ser 14 ) of naturally occurring native SsoPox enzyme followed by 2 extra amino acids (Leu and Glu) and a (His) 6 -tag at the C-terminal of the protein.
  • Example 2 Enhancement of OP-hydrolyzing activity of rSsoPox(wt) enzyme by random mutagenesis to produce rSsoPox( va riaiit-A>
  • the native SsoPox enzyme does not have sufficiently high OP-hydrolyzing activity and there was a need to develop improved variant(s) of SsoPox enzyme that exhibits enhanced OP- hydrolyzing activity.
  • the rSsoPox( Wt ) gene was subjected to random mutagenesis to generate variants having enhanced OP-hydrolyzing activity.
  • the gene for rSsoPox ⁇ ) (SEQ ID NO: 1) present in pET23a(+) plasmid was subjected to ep-PCR for error prone amplification using GeneMorph II random mutagenesis kit, by following the instructions given by the manufacturer (Stratagene, California, USA).
  • the forward and the reverse primers used in the ep-PCR experiment were designed using Primer X software (w ⁇ . w .b i nionnai i org pri merx/) and were T7 f-(5 -TAATACGACTC ACT AT AGGG-3 ⁇ ) and T7 r-(5 -GCTAGTTATTGCTCAGCGG-3 ).
  • the resulting randomly amplified rSsoPox ( wt ) gene (ep-PCR amplification product) was subjected to restriction digestion with Ndel and Xhol enzymes and the digested product was purified and ligated into pET23a(+) plasmid between Ndel and Xhol sites by following the standard molecular biology techniques known in the art.
  • the plasmid was then transformed into E. coli DH5a cells and the transformed cells were streaked on a Luria Bertani (LB)-agar plate containing 50 g/ml carbenicillin and the cells were allowed to grow at 37°C for 14-16 h.
  • the isolated plasmid was transformed into competent E. coli BL21DE3 cells and the transformed cells were grown on fresh LB-agar plates containing 50 g/ml carbenicillin at 37°C for 14-16 h. From these plates individual clones were hand-picked and inoculated separately into fresh LB-media supplemented with 50 g/ml carbenicillin. The cells were allowed to grow at 30°C for 3-5 h, 200 rpm till the OD 6 oo of the cultures reached to 0.6-0.8. At this point the cultures were induced with 0.5 mM isopropyl ⁇ -D-l-thiogalactopyranoside (IPTG) and 0.2 mM CoCl 2 was added to each culture tubes.
  • IPTG isopropyl ⁇ -D-l-thiogalactopyranoside
  • the cultures were further incubated for 48 h at 20°C and 200 rpm for expression of rSsoPox protein in soluble and active form. The cultures were then centrifuged and the cell pellets were collected. The cells were lysed by incubating with lysis buffer (TBS containing 1 mM PMSF, 0.5 mg/ml lysozyme and 0.2 % triton X-100). The samples were incubated for 1 h at 4°C and centrifuged to get clear cell supernatants (containing active rSsoPox protein). Equal amount of cell supernatants were used to check the paraoxon-hydrolyzing activity and the activity of variants (mutants) was compared by taking the activity of rSsoPox( Wt ) as 100%. Results:
  • rSsoPox( va riant-A) SEQ ID No 2 and 4 showing increased OP-hydrolyzing activity was selected for further characterization.
  • the DNA sequence of the generated variant was determined by bi-directional DNA sequencing (Eurofinn, India). Compared to rSsoPox( Wt ), the rSsoPox( va riant-A) was found to contain following two amino acid substitutions: Y40N and N297D.
  • Example 3 Production of rSsoPox enzymes.
  • the cell suspensions were gently stirred at room temperature for 1 h, passed through syringe with a needle and the cells were then disrupted by sonication.
  • the sonicated cell suspensions were immediately cooled on ice and treated with DNase and MgCl 2 for 1 h.
  • To the sample was then added 2V of buffer to make total volume 3 V.
  • the samples were vortexed and incubated at 4°C for 30-60 min with gentle shaking.
  • the samples were then centrifuged to separate clear cell lysates form insoluble fraction containing rSsoP ox-enriched inclusion bodies.
  • the inclusion bodies were then washed with inclusion bodies-washing buffer and centrifuged to remove the contaminants present in the inclusion bodies. Purified inclusion bodies were then collected and stored at -80°C till further use.
  • B. Refolding of rSsoPox enzymes from inclusion bodies Predicting the conditions that will promote the optimal refolding of any given protein and identifying the optimal refolding conditions is very difficult.
  • Different refolding additives that are known to facilitate in vitro refolding of recombinant proteins were selected and include: a buffering agent selected from the group of Tris-HCl, CHES, EPPS, HEPES, Glycine-NaOH, Phosphate, TAPS, MOPS, and MES, a cofactor selected from the group of CaCl 2 , CoCl 2 , MgCl 2 , and ZnCl 2 , a salt selected from the group of NaCl, KC1 and H 4 CI, a detergent selected from the group of tween-20, tween-20, NP-10, NP-40, triton X-100, CHAPS and Brij 35, an amino acid selected from the group of arginine, lysine, his
  • rSsoPox For screening of optimal refolding condition(s) of rSsoPox enzymes, purified inclusion bodies of rSsoPox were dissolved in freshly prepared saturated solution of urea (in water) and the denatured protein solutions was rapidly diluted into refolding buffers containing various combinations of refolding additives. Since cobalt is an essential cofactor for SsoPox, all refolding buffers contained 0.2 mM CoCl 2 . The refolding reactions were kept at 25°C for 4-24 h with gentle shaking. The extent of refolding was checked by monitoring the paraoxonase activity of the enzyme using paraoxon as substrate.
  • the refolding buffer comprised of 50 mM Tris-HCl, 9.6 mM NaCl, 0.4 mM KC1, 2 mM MgCl 2 , 2 mM CaCl 2 , 0.5 M sucrose, 0.05% PEG-3350, 0.5% Triton X-100, 1 mM GSH and 0.1 mM GSSH.
  • the overall temperature conditions varied from 4-37 °C and the time of refolding varied from 4-24 h at pH ranging from 6-10.
  • Results A representative chromatogram showing resolution of proteins present in the refolding mixture on ion-exchange column in a typical separation experiment is given in FIG. 3C. The results show that the enzymatically active rSsoPox enzyme (peak 2) is separated from the inactive enzyme (peak 1) present in the refolding mixture (FIG. 3). Purification yield of one of the rSsoPox enzyme is given in Table 2.
  • Table 2 Summary of purification of rSsoPox enzyme from 1 liter E. coli culture ( ⁇ 3-4 g wet cell mass)
  • Example 4 Characterization of OP- hydro lyzing activity of rSsoPox enzymes.
  • Enzymatic properties of purified rSsoPox(wt) and rSsoPox( va riant-A) were determined under in vitro conditions by comparing their OP-hydrolyzing (phosphotriesterase) activity using paraoxon as substrate.
  • Purified rSsoPox enzymes (0.2 M final concentration) were incubated at 25°C with a range of paraoxon concentrations in TBS containing 10 % acetonitrile and the product formation was monitored at 405 nm using Molecular Devices SPECTRA ⁇ PLUS Microplate spectrophotometer. Appropriate blank was included to correct for the spontaneous, non-enzymatic hydrolysis of substrate and was subtracted from the total rate of hydrolysis.
  • rSsoPox(variant-A) Compared to rSsoPox(wt), rSsoPox(variant-A) exhibited >40-folds higher k cat /K m value suggesting that the rSsoPox(variant-A) possesses enhanced OP-hydrolyzing activity than rSsoPox( Wt ).
  • Example 5 Compositions of rSsoPox enzyme for long-term storage in aqueous buffer and freeze-dried form at 25°C.
  • Purified r SsoPox enzyme was diluted in storage buffer (50 mM Tris-HCl, pH 8.0, and 0.2 mM CoCl 2 ) containing excipients and the samples were dispensed in vials and (a) stored at 25°C (as liquid formulation) and (b) lyophilized and stored at 25°C (as freeze-dried formulation), for different period of time.
  • storage buffer 50 mM Tris-HCl, pH 8.0, and 0.2 mM CoCl 2
  • the samples were dispensed in vials and (a) stored at 25°C (as liquid formulation) and (b) lyophilized and stored at 25°C (as freeze-dried formulation), for different period of time.
  • the samples were first frozen by incubating at -80°C for 10 h. The loosely capped vials were then placed into a freeze-drying flask that had been pre-cooled in liquid nitrogen.
  • the frozen samples were dried for 12 h on a BTK Bench Top K Manifold freeze dryer (with a condenser temperature pre-set at -112°C and vacuum ⁇ 10 mTorr). After the drying cycle was completed, the samples were removed from the flask, tightly capped and incubated at 25°C.
  • the storage stability of rSsoPox enzyme was determined by monitoring the paraoxon-hydrolyzing activity of the enzyme as a function of storage time. On indicated days, the vials were removed from the incubator and the paraoxonase activity was checked.
  • Stable compositions identified enable long-term storage stability of rSsoPox( va riaiit-A) enzyme and comprise of purified rSsoPox( va riaiit-A) enzyme in combination with at least a buffering agent, a cofactor, a salt, a detergent, an amino acid, a sugar, or mixture thereof, wherein the buffering agent is selected from the group of Tris-HCl, CHES, EPPS, HEPES, Glycine-NaOH, Phosphate, TAPS, MOPS, and MES, wherein the cofactor is selected from the group of CaCl 2 , CoCl 2 ,MgCl 2 , and ZnCl 2 , wherein the salt is selected from the group of NaCl, KC1 and NH 4 C1, wherein the detergent is selected from the group of tween-20, tween- 20, NP-10, NP-40, tritonX-100, CHAPS and Brij 35, wherein an
  • the PEGylation reaction mixture was applied onto a Q-Sepharose (ion- exchange) column pre-equilibrated with buffer containing 20 mM Hepes, pH 8.0 containing 1 mM CoCl 2 . After washing the column with same buffer, the bound protein was eluted using increasing concentrations of NaCl (0.1-1 M) in the same buffer. Eluted fractions were analyzed for both protein contents (OD 2 8o) and enzyme activity (using paraoxon as substrate) and fractions containing active enzyme were pooled, concentrated and stored at 4°C. Protein content in the samples was routinely determined using Bradford reagent using bovine serum albumin as standard. The purity of the isolated proteins was monitored by SDS-PAGE and the protein bands on the gel were detected by staining the gel with silver stain.
  • Protease sensitivity assay 0.1 mg of unmodified rSsoPox( va riant-A) and N-terminal mono- PEGylated-rSsoPox(variant-A) were separately mixed with 70 ⁇ of 1 mM HCl. Then, 10 ⁇ of trypsin solution (0.1 mg/ml in 1 mM HCl) and 20 ⁇ Tris HCl buffer (2.5 M Tris-HCl, pH 8.5 containing 250 mM NaCl) was added to the reaction mixtures and the mixtures were incubated at 37°C for 4 h. After incubation, 1 mM PMSF was added to stop the reaction.
  • trypsin solution 0.1 mg/ml in 1 mM HCl
  • Tris HCl buffer 2.5 M Tris-HCl, pH 8.5 containing 250 mM NaCl
  • the rSsoPox(variant-A) was reacted with mPEG-ALD (enzyme : PEG molar ratio of 1 : 15) in buffer containing 20 mM sodium cyanoborohydride and the reaction was incubated at 8°C for 7 days. After 7 days of reaction, a homogenous conjugate of N-terminal mono- PEGylated-rSsoPox(variant-A) was obtained. PEGylated enzyme was purified using ion- exchange chromatography. Typical chromatogram showing resolution of protein is shown in Fig. 5A. One peak containing active enzyme was eluted at 0.4-0.5 M NaCl concentration.
  • rSsoPox( va riant-A) migrated as a single band corresponding to an apparent molecular weight of -35 kDa (lane 1).
  • N-terminal mono-PEGylated-rSsoPox(variant-A) showed a single electrophoretic band with an apparent molecular weight of -40 kDa (lane 2).
  • mono-PEGylated-rSsoPox( va riant-A) retained - 88%> of its activity under similar conditions (compare bar iii and iv). This suggests that mono- PEGylation of rSsoPox( va riant-A) enhanced the resistance of the enzyme towards proteolytic degradation (i.e., decreased protease sensitivity), by shielding the protease sensitive area(s) of the enzyme.
  • Example 7 Generation of reusable nanobiocatalyst by covalently immobilizing pure rSsoPox enzyme onto magnetic nanoparticles (MNPs).
  • Application of immobilized enzymes onto magnetic nanoparticles offers a distinct advantage over soluble enzymes, because immobilized enzyme can be easily recovered from the reaction mixture after its use with the help of external magnetic field and can be reused.
  • the purified rSsoPox( va riant-A) was covalently immobilized on the surface of fluidMAG-CMX MNPs (hydrodynamic diameter of 200 nm) by following the procedure recommended by the manufacturer (Chemicell, Berlin, Germany).
  • dd-H 2 0 double-distilled water
  • EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
  • the activated MNPs were then washed several times with dd-H 2 0 to remove any unreacted EDC.
  • To this activated MNPs suspension was then added 50 ⁇ g of pure rSsoPox enzyme (diluted in HEPES buffer) and the suspension was mixed gently for 2h at 25°C.
  • a control reaction was also done in which the enzyme was added to an equal amount of (inactive) MNPs which were not activated with EDC.
  • the MNPs were then washed several times with PBS to remove any unbound proteins. Finally the MNPs were washed with TBS to block any unreacted groups on the MNPs surface and were stored in PBS at 4°C for further use.
  • the separation of MNPs from the supernatants was carried out by the application of a magnetic field and the supernatants (washes) were collected in separate tubes.
  • different fractions were checked for their paraoxon-hydrolyzing activity and the total protein content.
  • the amount of the immobilized protein was calculated from the difference between the paraoxon-hydrolyzing activity of control (supernatant of sample containing inactive MNPs and enzyme) and supernatant of the sample containing activated MNPs.
  • the purified rSsoPox( va riant-A) was immobilized covalently onto fluidMAG-CMX MNPs using a two-step reaction (procedure) i.e. formation of activated ester and coupling with rSsoPox(variant-A) enzyme (FIG. 6A).
  • the MNPs were activated with EDC to convert the carboxyl group of the CMX present on the surface of MNPs into an activated ester.
  • the ester groups on the surface of the activated MNPs were then reacted with the NH 2 groups of rSsoPox(variant-A) enzyme to form -CO- H- (covalent) linkage between them.
  • the paraoxon- hydrolyzing activity in the supernatant of the control reaction was determined and was assigned 100%. Comparison of the activity in the supernatant of the control and the coupling reaction (i.e., reaction in which activated MNPs were used) indicated that the activity in the supernatant of coupling reaction was considerably less than the activity in the supernatant of the control reaction (FIG. 6B). Similar pattern was observed when the total protein content in the supernatants of control and coupling reactions were compared by a Bradford assay (data not shown).
  • Example 8 Reusability of rSsoPox-MNP nanobiocatalyst to degrade OP-compound in aqueous media.
  • rSsoPox-MNP nanobiocatalyst The stability and reusability of rSsoPox-MNP nanobiocatalyst to degrade OP- compound was studied in aqueous buffer and paraoxon was used as a representative OP- compound in this assay.
  • Stored rSsoPox-MNP nanobiocatalyst suspension in TBS was washed with the same buffer and the cake of rSsoPox-MNP nanobiocatalyst was resuspended in TBS containing 1 mM paraoxon. The reaction was incubated at 40°C for 5-7 h with gentle mixing.
  • MNPs which do not contain immobilized rSsoPox enzyme
  • MNPs which do not contain immobilized rSsoPox enzyme
  • the reaction was stopped by separating and recovering the rSsoPox-MNPs nanobiocatalyst from the reaction mixture by using magnet.
  • the recovered rSsoPox-MNP nanobiocatalyst were washed with PBS and stored (as suspension) in the same buffer at 4°C for 6-8h.
  • the rSsoPox-MNP nanobiocatalyst were collected from the stored suspension and the wet cake of rSsoPox-MNP nanobiocatalyst was again suspended in TBS containing 1 mM paraoxon and the hydrolysis of the paraoxon was determined.
  • FIG. 7 shows the operational stability of rSsoPox-MNP nanobiocatalyst during repeated use for paraoxon-hydrolysis in aqueous buffer.
  • the rSsoPox-MNP nanobiocatalyst retained significant paraoxon-hydrolyzing activity even after 8 cycles of usage (retained -60% of its initial activity after 8 cycles). This suggests that the immobilized rSsoPox enzyme is stable on the MNPs surface and the external magnet could efficiently recover the rSsoPox-MNP nanobiocatalyst from the reaction mixture.
  • the invention provides E. coli expression system (E. coli BL21DE3 cells) for the production of rSsoPox enzymes which has following qualities: easy to handle, inexpensive, less time consuming, easy to manipulate and scale-up, high growth rate and rapid biomass accumulation, and rapid generation and screening of improved variant of rSsoPox enzymes.
  • the invention provides optimized polynucleotide sequences for high level expression of rSsoPox enzymes iri E. coli cells. • Increased OP-hydrolyzing activity: The invention provides variant of rSsoPox(wt) enzyme having enhanced (>40-folds) OP-hydrolyzing activity.
  • the invention provides novel compositions to increase the long- term storage stability (shelf-life) of purified rSsoPox enzymes.
  • the composition contains pharmaceutically acceptable excipients that do not interfere with the functional activity of the enzyme. These compositions can be used for the economical industrial scale production, storage and transportation of rSsoPox enzymes.
  • the invention provides an economical means to decrease the protease sensitivity and reduce potential immunogenicity of purified rSsoPox enzyme by PEG-conjugation.
  • PEGs are pharmaceutically acceptable excipients that are well characterized in the art and used in various compositions and that do not interfere with the biological activity of the enzyme.
  • Nanobiocatalyst The invention provides a method to generate reusable nanobiocatalyst by covalently immobilizing pure rSsoPox enzyme onto magnetic nanoparticle.
  • the reusable nanobiocatalyst efficiently hydrolyzes OP-compounds in aqueous solution and can be recovered from the reaction mixture after its use and can be stored and reused multiple times to degrade OP-compounds in different samples.
  • the method of the present invention has not been disclosed in the prior art.
  • the present invention successfully overcomes the technical hurdles of the prior art and discloses a novel method for the production of (a) rSsoPox enzymes, (b) compositions to increase the storage stability of purified enzymes (c) N-terminally mono-PEGyalted-rSsoPox conjugate, and (d) rSsoPox-immobilized nanobiocatalyst, in a very easy and economical manner.
  • a novel polynucleotide sequences (gene) encoding rSsoPox(wt) enzyme has been designed for high level expression of enzyme in the E. coli expression system.
  • a novel, simple, easy, low cost, less time consuming and high yield giving method is developed for the production of rSsoPox enzymes. •
  • the novel compositions have been developed for long-term storage stability of purified rSsoPox enzyme under various storage conditions like in aqueous solution at 25°C and in freeze-dried form at 25°C.
  • N-terminal mono-PEGylated-rSsoPox conjugate has decreased protease sensitivity and expected to have low potential immunogenicity as compared to the unmodified enzyme.
  • a novel rSsoPox-immobilized nanobiocatalyst is disclosed that can hydrolyze OP- compounds in aqueous solution and that can be recovered from the reaction mixture after its use and can be stored and reused multiple times to degrade OP-compounds in different samples.
  • the process described in the present invention has tremendous industrial applications and can be used for economical industrial scale production of rSsoPox enzymes having enhanced OP- hydrolytic activity, in high yield and purity.
  • the products described in the present invention has tremendous application in various biotechnological applications e.g., decontamination of OP-contaminated objects and areas.
  • the product described in the present invention may also have application in biopharmaceutical industries as therapeutic agent for e.g., treatment of OP-poisoning in humans and other animals.
  • Industrial applications of pure rSsoPox enzymes and rSsoPox-immobilized nanobiocatalyst
  • Biological control measures Pure rSsoPox enzymes (unmodified and PEG-conjugated) having enhanced OP-hydrolyzing activity can be used as an effective biological control measure against OP-poisoning (both prophylactic and post exposure) in human and other animals.
  • Treatment for OP-poisoning Pure rSsoPox enzymes (unmodified and PEG-conjugated) having enhanced OP-hydrolyzing activity can be used alone in pharmaceutical composition or along with classical treatment for OP-poisoning, like acetylcholinesterase reactivators, antimuscarines and anticonvulsant, to improve the treatment.
  • Decontaminating agent Pure rSsoPox enzyme having enhanced OP-hydrolyzing activity can be used as agent to generate various formulations for decontaminating surfaces, objects and areas contaminated with OP-compounds.
  • Pure rSsoPox enzyme having enhanced OP-hydrolyzing activity can be used as agent to prepare coated wearables (likes cloths, gloves, etc), laboratory hardware, devices as well as other items like sponges, wipes etc. to be used at the site contaminated with OP-compounds.
  • Biosensors Pure rSsoPox enzyme having enhanced OP-hydrolyzing activity can be used as agent to develop biosensors for detection of OP-compounds (CWNAs, pesticides, etc.) in various samples.
  • rSsoPox-immobilized nanobiocatalyst as bio-decontaminating agent The rSsoPox- immobilized nanobiocatalyst can be used as reusable bio-decontaminating agent in various applications of OP-cleanup e.g., in decontaminating water contaminated with OP- compounds, decontaminating farm produce contaminated with OP-compounds, etc.

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US9597672B2 (en) 2011-03-10 2017-03-21 Cornell University Mesoporous catalysts of magnetic nanoparticles and free-radical-producing enzymes, and methods of use
WO2018151649A1 (en) 2017-02-15 2018-08-23 Pharem Biotech Ab Identification of enzymes degrading recalcitrant environmentally hazardous organic compounds
US10792649B2 (en) 2015-07-15 2020-10-06 Zymtronix, Llc Automated bionanocatalyst production
US10881102B2 (en) 2015-05-18 2021-01-05 Zymtronix, Llc Magnetically immobilized microbiocidal enzymes
US10993436B2 (en) 2016-08-13 2021-05-04 Zymtronix Catalytic Systems, Inc. Magnetically immobilized biocidal enzymes and biocidal chemicals

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CA2895901A1 (en) * 2012-12-20 2014-06-26 Olivier-Georges BOUMENDIL Enzyme having a nmda receptor antagonist activity and/or an anticholinergic activity

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US9597672B2 (en) 2011-03-10 2017-03-21 Cornell University Mesoporous catalysts of magnetic nanoparticles and free-radical-producing enzymes, and methods of use
US10260061B2 (en) 2011-03-10 2019-04-16 Cornell University Mesoporous catalysts of magnetic nanoparticles and free-radical-producing enzymes, and methods of use
US10316313B2 (en) 2011-03-10 2019-06-11 Cornell University Mesoporous catalysts of magnetic nanoparticles and free-radical-producing enzymes, and methods of use
US10881102B2 (en) 2015-05-18 2021-01-05 Zymtronix, Llc Magnetically immobilized microbiocidal enzymes
US11517014B2 (en) 2015-05-18 2022-12-06 Zymtronix, Inc. Magnetically immobilized microbiocidal enzymes
US10792649B2 (en) 2015-07-15 2020-10-06 Zymtronix, Llc Automated bionanocatalyst production
US10993436B2 (en) 2016-08-13 2021-05-04 Zymtronix Catalytic Systems, Inc. Magnetically immobilized biocidal enzymes and biocidal chemicals
US12127557B2 (en) 2016-08-13 2024-10-29 Zymtronix Catalytic Systems, Inc. Magnetically immobilized biocidal enzymes and biocidal chemicals
WO2018151649A1 (en) 2017-02-15 2018-08-23 Pharem Biotech Ab Identification of enzymes degrading recalcitrant environmentally hazardous organic compounds

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