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
group
polypeptides
enzyme
recombinant
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PCT/IB2014/064616
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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
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • 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
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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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    • 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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    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2305/00—Use of specific compounds during water treatment
    • C02F2305/08—Nanoparticles or nanotubes

Abstract

The present invention provides novel polynucleotide sequences for high level expression in Escherichia coli and encoding rSsoPox(wt) enzyme and its variant having enhanced organophosphate (OP)-hydrolyzing activity. The invention also provides a novel method to produce the recombinant enzymes in highly pure and active form in an unprecedented high yield. The invention also provides novel compositions for long-term storage of purified rSsoPox enzymes. The invention also provides PEGylated-rSsoPox conjugate having decreased protease sensitivity. The invention also provides a method to generate reusable nanobiocatalyst by covalently immobilizing pure r Sso Pox enzyme onto magnetic nanoparticles. The reusable nanobiocatalyst can be recovered from the reaction mixture after its use and can be stored and reused multiple times to degrade the OP-compounds. The rSsoPox enzymes and rSsoPox-immobilized nanobiocatalyst can be used as safe, effective and environmental-friendly OP-compound degrading agent.

Description

"RECOMBINANT AND STABLE SSOPOX ENZYMES, METHOD OF GENERATION THEREOF AND REUSABLE NANOBIOCATALYST OF
THE SAME"
FIELD OF INVENTION:
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.
BACKGROUND OF THE INVENTION:
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. Because of their widespread use, their inadequate storage and disposal and their accidental and intentional spillage, 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). However, these treatments are considered as unsatisfactory and inadequate. Thus, there is an urgent need for the development of more effective treatment for OP-poisoning. Similarly, various technologies currently available for the decontamination (clean-up) of OP-contaminated objects and areas suffer from serious limitations. For e.g., various physico-chemical methods available for decontamination (clean-up) of OP-contaminated objects and areas are not only corrosive and inflammable in nature but also require sophisticated instruments and trained professionals. Moreover, these methods generate various products during OP- decontamination that are equally or more toxic. Thus, there is an urgent need of safe, effective, and environmental-friendly means for decontamination (clean-up) of OP- contaminated objects and areas.
Further, bacterial infections are major public health issue and the development of resistance to existing antibiotics (antimicrobial agents) by biofilm-forming bacteria is a key problem. Also, 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. 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). Thus, 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.
Therapeutic use of 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:
There are numerous limitations regarding large scale production of SsoPox enzyme for commercial use, as discussed below:
i. 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.
ii. Low OP-hydrolyzing activity of native SsoPox enzyme. Native SsoPox does not have sufficiently high hydrolyzing activity against a variety of OP-compounds and there is a need to engineer improved variant of SsoPox enzyme that exhibits enhanced OP- hydrolyzing activity.
Hi Poor storage stability (shelf-life) of purified enzyme. Long-term storage stability of purified rSsoPox enzyme is poor.
iv. Protease sensitivity of purified enzyme: Sensitivity of SsoPox enzyme towards proteolytic degradation and potential immunogenicity is a major challenge for its therapeutic use.
v. Lack of efficient recovery means of SsoPox enzyme from the application environment for their reuse. Prior Arts do not describe a simple and economical method for the efficient recovery of the SsoPox enzyme from its application environment for reuse.
Production of rSsoPox enzymes has been attempted by various workers but the procedures used in the prior art have one limitation or other, as discussed below. Also, none of the prior arts disclosed composition(s) for long-term storage stability of pure rSsoPox enzymes, PEGylated-r SsoPox conjugate having decreased protease sensitivity and reduced immunogenicity, and rSsoPox-immobilized biocatalyst. In the present invention, these limitations have been overcome. A comparison of the prior arts and the present invention is given in Table 1.
Table 1: Comparison of the Prior Arts with the present invention.
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
Extremophiles, the production of rSsoPox enzymes and have the production of rSsoPox
9, 297 following limitations: enzymes. This system has ) Afriat, L., et • Low yield: following advantages over the al , 2006, The yield of active and purified rSsoPox expression systems disclosed in
Biochemistry, obtained using these systems is very low. For prior arts 1 and 2.
45, 13,677 e.g., prior arts 1, 2, and 9 discloses the final • Simple system.
) 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
Biol, 379, 1071 cell mass of Pseudomonas putida, production of rSsoPox enzymes ) Merone, L., respectively. which do not uses additional et al. 2008, • Complex systems: 'helper' plasmid(s) and which is
Curr. Chem. Prior art 2, 7, 8 and prior art 9 discloses easy to handle, easy to
Biol, 2, 237 expression and purification of rSsoPox by manipulate and scale-up, and ) Vecchio, using specialized and complex E. coli cells permit rapid generation and
P.D., et al, (that contains additional 'helper' plasmids screening of improved variant of
2009, along with the one encoding for rSsoPox) rSsoPox.
Extremophiles, and Pseudomonas putida, respectively. These • High yield:
13, 461 are complex expression systems that are The present invention discloses ) Merone, L., difficult to scale up and results in low yield a method to produce rSsoPox et al, 2010, of recombinant proteins. enzymes in highly pure and
Bior. Tech., active form in an unprecedented
101, 9204 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
Rep., 2, 779 suffer from following limitations. • Novel method of production ) Hiblot, J., et of rSsoPox enzymes:
al, 2013, PLoS These prior arts 1-9 do not disclose improved The present invention provides
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
Appl. Environ. These prior arts 1-9 do not disclose E. coli and isolating Microbiol, 77, production of rSsoPox by refolding the enzymatically active enzymes.
1181, inclusion bodies. This method of production of
SsoPox enzyme is not disclosed
These prior arts 1-9 do not disclose in the arts.
composition(s) for long-term storage • Novel improved variant: stabilization of purified SsoPox enzyme. The invention discloses novel variant of r SsoPox enzyme
These prior arts 1-9 do not disclose PEG- having enhanced phosphor- conjugated SsoPox enzyme. triesterase (OP- hydrolyzing) activity. This variant is not
These prior arts 1-9 do not discloses disclosed in the arts.
generation of reusable nanobiocatalyst • Stable compositions obtained from the immobilization of rSsoPox The present invention discloses onto magnetic nanoparticles. compositions for long-term storage stability of purified
10) Chabriere Prior art 10 discloses mutated rSsoPox enzymes.
and Elias, (U.S. hyperthermophilic enzymes having • Low-protease sensitivity:
Pat. Appl. No. phosphotriesterase and lactonase activity The present invention discloses
12/597,847) using an E. coli expression system and N-terminal mono-PEGylated- suffers from following limitations: rSsoPox conjugate which has
• Low yield: decreased protease sensitivity
The yield of active and purified r SsoPox and reduced immunogenicity. produced is very low (5-9 mg/1 of E. coli Reusable nanobiocatalyst: The culture). present invention discloses
The prior art do not discloses improved generation of reusable variant of r SsoPox enzyme having enhanced nanobiocatalyst by covalently
OP-hydrolyzing activity disclosed in the immobilizing the pure rSsoPox current invention. enzyme onto magnetic
The prior art do not discloses production of nanoparticles. These nano- rSsoPox by refolding the inclusion bodies. biocatalysts can be isolated form
The prior art do not disclose composition(s) the reaction mixture after their for long-term storage stabilization of purified use and stored and can be reused
SsoPox enzyme. multiple times to degrade OP- compounds. The prior art do not disclose PEG-conjugated
SsoPox enzyme.
The prior art do not discloses generation of
reusable nanobiocatalyst obtained from the
immobilization of rSsoPox onto magnetic
nanoparticles.
Thus, none of the prior arts disclose the present invention, which provides:
i. Novel polynucleotide sequence encoding rSsoPox enzyme having enhanced hydrolytic activities towards at least one SsoPox- substrate,
ii. A novel method for the production of rSsoPox enzyme in highly pure and active form in an unprecedented high yield, by refolding the recombinant enzyme expressed as inclusion bodies in E. coli,
iii. Compositions for long-term storage stabilization of pure rSsoPox enzyme, iv. N-terminal mono-PEGylated-r SsoPox conjugate having decreased protease sensitivity, and
v. 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.
OBJECTS OF THE INVENTION:
One object of the present invention is to provide a novel bacterial construct for the generation and production of rSsoPox enzymes.
Another object of the present invention is to provide novel polynucleotide sequence encoding rSsoPox enzyme having increased hydrolytic activities towards at least one SsoPox- substrate. Another object of the present invention is to provide a novel method for the production of rSsoPox enzymes in highly pure and active form and in a high yield.
Another object of the present invention is to provide compositions for long-term storage stability (shelf-life) of pure rSsoPox enzyme. Another object of the present invention is to provide N-terminal mono-PEGylated-rSsoPox conjugate having decreased protease sensitivity.
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.
SUMMARY OF THE INVENTION:
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. coli, compositions to increase the long-term storage stability of purified r SsoPox enzyme, N-terminal mono- PEGylated-r SsoPox conjugate having decreased protease sensitivity, and a novel reusable nanobiocatalyst generated by covalently immobilizing pure and active rSsoPox enzymes 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.
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.
Still another aspect of this invention provide an isolated and optimized nucleic acid sequence encoding rSsoPox(wt) enzyme (i) having at least one hydrolytic activity identical to naturally occurring SsoPox, and (ii) expressible in high quantity in bacterial cells and comprises of SEQ ID NO. 1. Still another aspect of this invention provide an amino acid sequence encoding rSsoPox(wt) enzyme (i) having at least one hydrolytic activity identical to naturally occurring SsoPox, and (ii) expressible in high quantity in bacterial cells and comprises of SEQ ID NO. 3. Another aspect of the present invention is to provide the polypeptides wherein at least one polypeptide chain has increased hydrolytic activity towards at least one SsoPox- substrate.
In still another aspect of present invention, the SsoPox- substrate is selected from a group consisting of organophosphates, lactones and acylhomoserine lactones.
Another aspect of the present invention provides a method for production of recombinant polypeptide comprising amino acid sequence of rSsoPox and variant thereof comprising SEQ ID NOs. 3-4 by refolding the polypeptide expressed as inclusion bodies m E. coli. Still another aspect of the present invention provides a method for production of rSsoPox polypeptides, comprising at least one amino acid sequence selected from the group comprising SEQ ID NOs: 3-4, wherein the method result into high level expression of rSsoPox polypeptides as enzymatically non-functional aggregated inclusion bodies, wherein the said method comprises the steps of inducing the host cell culture, the host cell preferably being E. coli.
Another aspect of the present invention provides nucleic acid constructs comprising the isolated polynucleotides of SEQ ID NOs. 1-2. Another aspect of the present invention provides 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.
Another aspect of the present invention provide stabilized 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. Still another aspect of the present invention provide stabilized pharmaceutical composition comprises of recombinant polypeptide, wherein said recombinant polypeptide exhibits OP- hydrolyzing activity and can be rSsoPox(wt) or its variant. Still another aspect 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. In yet another aspect of the present invention the PEG molecule is a linear molecule selected among the group of linear and branched PEGs.
In yet another aspect of the present invention the PEG molecule of is methoxy PEG aldehyde selected from a group of PEG-succinimidyl carbonate, PEG-pN02 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.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS OR FIGURES:
The accompanying drawings illustrate some of the embodiments of the invention and, together with the description, serve to explain the invention. These drawings are offered by way of illustration and not by way of limitation.
Figure 1. 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. Figure 3. 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. Symbols: lane M, protein molecular weight markers; lane 1, cell lysate; lane 2 and 3, supernatants of the inclusion bodies washings- 1 and -2; and lane 4, purified inclusion bodies. 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) was mixed with varying concentrations of paraoxon (0-2 mM) and the paraoxon hydrolysis was determined as described in Example 4. The hydrolysis data were fitted by Lineweaver-Burk equation and the R2 values were 0.94- 0.99. Legends: (- -), rSsoPox(Wt) and (~A~), rSsoPox(variant-A)- 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. Symbols: lane M: protein molecular weight markers; lane 1 : unmodified rSsoPox(variant-A) and lane 2: N-terminal mono- PEGylated-rSsoPox(variant-A)- Panel C shows protease sensitivity of unmodified rSsoPox(variant- A) (bars i and ii) and N-terminal mono-PEGylated-rSsoPox(variant-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. Symbols; bar i: unmodified rSsoPox(variant-A); bar ii: unmodified rSsoPox(variant-A) + trypsin; bar iii : N-terminal mono- PEGylated-rSsoPox(variant-A); and bar iv: N-terminal mono-PEGylated-rSsoPox(variant-A) + trypsin.
Figure 6. Panel A show a schematic representation of the chemistry employed for covalent immobilization of pure rSsoPox(variant-A) enzyme onto magnetic nanoparticles. Panels B and C shows paraoxon-hydrolyzing activity of (i) equal amount of free rSsoPox(variant-A), (ϋ) free rSsoPox(variant-A) present in the supernatant of immobilization reaction after centrifugation, and (iii) resuspended rSsoPox(variant-A)-MNPs (nanobiocatalyst). Panels D and E are the image depicting dispersion of rSsoPox(variant-A)-immobilized nanobiocatalyst in TBS buffer (20 mM Tris-HCl, pH 8.0 containing 150 mM NaCl and 0.2 mM CoCl2) 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(variant-A)- immobilized nanobiocatalyst in aqueous TBS buffer. The rSsoPox(variant-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(variant-A)-irnmobilized nanobiocatalyst was recovered from the reaction mixture by applying external magnetic field (i.e., a magnet) and the OD405 of the reaction mixture was measured in a spectrophotometer to determine the amount of paraoxon hydrolyzed by the rSsoPox(variant-A)-immobilized nanobiocatalyst. The values obtained were corrected for the non-enzymatic hydrolysis of paraoxon in control. The recovered rSsoPox-immobilized nanobiocatalyst was washed with phosphate buffered saline (PBS), pH 7.5, kept at 4°C for 6-8 h in same buffer and used again to hydrolyze paraoxon by suspending the washed rSsoPox(variant-A)-immobilized nanobiocatalyst into fresh TBS buffer containing 1 mM paraoxon. The experiments were conducted in duplicate and the mean values are presented. DETAILED DESCRIPTION OF THE INVENTION
In the detailed description of the present invention, numerous specific details are described to provide a thorough understanding of the various embodiments of the present invention. However, a person skilled in the relevant art will recognize that an embodiment of the present invention can be practiced without one or more of the specific details, or with other apparatus, systems, assemblies, methods, components, materials, parts, and/or the like. In other instances, well-known structures, materials, or operations are not specifically shown or described in detail to avoid obscuring aspects of embodiments of the present invention.
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of person skill in the art. Some of the terms are defined briefly here below. The definitions should not be construed in a limiting sense. 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. The term '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 (IBs), 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 (MNPs): 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.
Potential 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, parathion-methyl, pirimiphos-ethyl, pirimiphos-methyl, pyrazophos, quinalphos, ronnel, sulfopros, sulfotepp, trichloronate, tabun, soman, sarin, cyclosarin, GX, VX, R-VX, diisopropylflourophospate, etc, or a combination thereof; lactones: such as γ-butyrolactone, γ-caprolactone, γ-octanoiclactone, γ- nonanoiclactone, γ-decaanoiclactone, γ-undecaanoic lactone, nonanoic-5-lactone, undecanoic-5-lactone, dodecanoic-5-lactone, ε-caprolactone, etc, or a combination thereof; acyl homoserine lactones: such as 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- oxododecanoyal-DL-homoserine lactone, N-oxotetradecanoyal-DL-homoserine lactone, N-3- ketocaproyal homoserine lactone, N-3 -hydroxy decanoyal homoserine lactone, etc, or a combination thereof
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 H2 groups in the protein molecule.
Recombinant SsoPox (rSsoPox): 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(wt). 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). One variant of the enzyme is disclosed in the present invention and coding of the same has been done as below:
rSsoPoX(variant-A)- refers to a rSsoPox enzyme containing two amino acid substitutions (Y40N and N297D) in rSsoPox(wt). (MTCC accession # MTCC5841; GenBank accession # KF924250).
Site-specific/N-terminal PEGylation: refers to attachment of PEG molecule at N-terminal end of the protein molecule.
Stability of protein, the term 'stability of protein' in this invention 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. coli, novel compositions to increase the long-term storage stability of purified rSsoPox enzyme in aqueous solution and freeze dried form, N-terminal mo no-PEGylated-r SsoPox conjugate having decreased protease sensitivity, and a novel reusable nanobiocatalyst generated by covalently immobilizing pure and active rSsoPox enzymes onto magnetic nanoparticles. Accordingly, in one embodiment of the present invention is provided a recombinant polynucleotide (rSsoPox) and variant thereof comprising at least one nucleotide sequence selected from the group comprising SEQ ID NOs: 1-2.
In another embodiment of the present invention is provided a recombinant polynucleotide, wherein the said polynucleotide encodes at least one polypeptide chain, wherein the polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 3-4.
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.
In another embodiment of the present invention is provided a recombinant polynucleotide (rSsoPox) 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.
In another embodiment of the present invention is provided 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. In another embodiment of the present invention is provided 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, fenthion, fenthrothion, isofenphos, jodfenphos, leptophos-oxon, malathion, methyl-parathion, mevinphos, parathion, parathion-methyl, pirimiphos-ethyl, pirimiphos-methyl, pyrazophos, quinalphos, ronnel, sulfopros, sulfotepp, trichloronate, tabun, soman, sarin, cyclosarin, GX, VX, R-VX, diisopropylflourophospate, etc.
In another embodiment of the present invention is provided 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 γ- butyrolactone, γ-caprolactone, γ-octanoiclactone, γ-nonanoiclactone, γ-decaanoiclactone, γ- nonanoic-5-lactone, undecanoic-5-lactone, dodecanoic-5-lactone, ε-caprolactone, etc.
In another embodiment of the present invention is provided 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-oxododecanoyal-DL-homoserine lactone, N- oxotetradecanoyal-DL-homoserine lactone, N-3-ketocaproyalhomoserine lactone, N-3- hydroxydecanoyal homoserine lactone, etc.
In another embodiment of the present invention is provided a recombinant polynucleotide (rSsoPox) 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 (kcat/Km ratio) by at least 40-folds for paraoxon substrate with respect to the rSsoPox^).
In another embodiment of the present invention is provided 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,
• culturing the host cells expressing rSsoPox polypeptides,
• inducing high level of expression of rSsoPox polypeptides,
• collecting the cell mass expressing rSsoPox polypeptides,
• lysing the cells,
· purifying the inclusion bodies containing rSsoPox polypeptides,
• solubilizing the inclusion bodies with the help of chaotropic agents,
• refolding the rSsoPox enzymes using refolding buffer,
• isolating the enzymatically active rSsoPox using chromatography,
• obtaining enzymatically active rSsoPox polypeptides.
In one embodiment of the present invention is provided 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. Preferably the host cell is E. coli. According to yet another embodiment of the present invention 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. According to yet another embodiment of the present invention 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. According to yet another embodiment of the present invention 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. In yet another embodiment of the present invention the purity of the isolated inclusion bodies varies from at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100%.
In another embodiment of the present invention is provided 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.
According to still another embodiment of the present invention 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.
In another embodiment of the present invention is provided 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.
In another embodiment of the present invention is provided 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.
In another embodiment of the present invention is provided 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 CaCl2, CoCl2, MgCl2, and ZnCl2. In another embodiment of the present invention is provided 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.
In another embodiment of the present invention is provided 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.
In another embodiment of the present invention is provided 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:
• purifying the inclusion bodies containing rSsoPox polypeptides,
• solubilizing the inclusion bodies containing rSsoPox polypeptides with the help of urea,
• refolding the denatured rSsoPox polypeptides by diluting in refolding buffer,
• isolating the enzymatically active rSsoPox polypeptides using chromatography,
• obtaining the enzymatically active rSsoPox polypeptides.
In another embodiment of the present invention is provided 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 CaCl2, CoCl2, MgCl2, and ZnCl2 and the refolding additives are selected from the group of salts, sugars, polymers, polyols, detergents and surfactants. In another embodiment of the present invention is provided 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%.
According to still another embodiment of the present invention the final concentrations of rSsoPox enzymes used in the refolding reaction varies from less than 1 mg/ml including less than 300 μg/ml, such as less than 100 μg/ml, including less than 40 μg/ml. According to yet another feature of the present invention the optimum time of refolding varies from 2 to 24 h depending upon the refolding condition and rSsoPox enzymes.
According to yet another embodiment of this invention 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.
In yet another embodiment of this invention there is provided a method to separate/isolate enzymatically active rSsoPox enzymes from inactive enzymes present in the refolding mixture.
According to yet another feature of the present invention 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.
According to yet another embodiment of the present invention, the optimum refolding conditions can be same or different for rSsoPox enzymes.
According to yet another embodiment of the present invention 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.
In another embodiment of the present invention is provided the 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(variant-A).
According to yet another embodiment of the present invention, the storage-stable rSsoPox compositions of the present invention are fully solubilized in aqueous solution, i.e., in a water-based solution. However, 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.
According to yet another embodiment of the present invention, the storage-stable rSsoPox compositions are stably stored in ready-to-use fluid form and in a freeze-dried form.
According to still another embodiment of the present invention, 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).
According to yet another embodiment of the present invention, 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.
According to yet another embodiment of the present invention, the composition of this invention contains at least one excipient or component. The 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. The 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. According to yet another embodiment of the present invention, the pharmaceutical excipients in the composition can be replaced by any of the similar excipients such as:
Buffer: For e.g. Tris-HCl can be replaced with HEPES, EPPS, TAPS and like.
Amino acids/proteins: For e.g. 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.
According to still another embodiment of the present invention, the 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.
According to yet another embodiment of this 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.
In yet another embodiment of the present invention, no antimicrobial agent is added to the storage stable composition of rSsoPox.
In yet another embodiment of the present invention, antimicrobial agent(s) can be added to avoid microbial contamination of the storage stable composition of rSsoPox enzyme over long-term storage.
In yet another embodiment of the present invention, in various compositions, the concentration of enzyme may vary from 1 nM to 1M.
In another embodiment the present invention provides a mono-PEGylated-rSsoPox, wherein the rSsoPox can be rSsoPox(wt) or any of its variant and preferably is rSsoPox(variant-A).
In yet another embodiment, the rSsoPox is modified with site specific PEGylation selected from a group of site specific and random PEGylation. In yet another embodiment 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.
In yet another embodiment, 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.
In yet another embodiment, the PEG attached is a linear molecule selected among the group of linear and branched PEGs. In yet another embodiment, the PEG attached is methoxy PEG aldehyde selected from a group of PEG-succinimidyl carbonate, PEG-pN02 phenyl carbonate, PEG-AA-NHS and PEG-carbonylimidazole.
In yet another aspect, the N-terminal mono-PEGylated-rSsoPox has decreased protease sensitivity as compared to unmodified enzyme.
In yet another aspect, N-terminal mono-PEGylated-rSsoPox is particularly suitable for treatment of OP-poisoning in human and other live stocks. In another embodiment of the present invention are provided nucleic acid constructs comprising the isolated polynucleotides of the present invention.
In another embodiment of the present invention are provided nucleic acid constructs comprising the isolated polynucleotides encoding polypeptides of the present invention.
In another embodiment of the present invention is provided an isolated host cell comprising the nucleic acid constructs of the present invention.
According to yet another embodiment of present invention, the rSsoPox enzyme having enhanced OP-hydrolyzing activity can be used for decontaminating OP-contaminated surfaces. Thus, 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.
According to yet another embodiment of the present invention, the rSsoPox variant having enhanced OP-hydrolyzing activity can be used to treat or prevent OP-poisoning in OP- poisoned subjects. In another embodiment of the present invention is provided 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.
In yet another embodiment of the present invention is provided 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(variant-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.
According to yet another embodiment of the present invention is provided a method for degrading OP-compounds in water system using the rSsoPox-immobilized magnetic nanobiocatalyst described above. According to yet another embodiment of the present invention is provided a use of rSsoPox- immobilized nanobiocatalyst described above for
• decontaminating OP-contaminated water
• coating on the electrode surfaces of biosensors for toxicity analysis, environmental monitoring, food quality control, and military investigations. According to yet another embodiment of the present invention, 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. According to another aspect of present invention, 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.
According to still another aspect of the present invention any method for immobilization of protein (e.g. covalent linkage, entrapment and non-covalent interactions) onto the surface can be selected by person skilled in the art. The method of immobilization selected here for magnetic nanoparticles, is covalent linkage.
According to yet another aspect of the present invention, the magnetic particles chosen for enzyme coating essentially contain CMX as functional group among a group of amines, carboxylic acids, chitosan and the like.
According to yet another aspect of present invention the percentage binding of enzyme to MNPs vary from at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100%.
According to yet another aspect of present invention 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.
According to still another aspect of this invention, the enzyme coated MNPs are reusable and the reusability varies to at least 1 cycle to 5, 10, 100 and 1000 cycles.
According to yet another embodiment of present invention 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. Although the present invention has been described in detail for specific embodiments thereof, it is apparent to those skilled in the art that various alterations and modifications are conductible without departing from the spirit and scope of the present invention. The present disclosure with reference to the accompanying examples describe the present invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. It is understood that the examples are provided for the purpose of illustrating the invention only, and are not intended to limit the scope of the invention in any way.
Example 1: Generation of nucleic acid construct (plasmid) containing novel polynucleotide sequence encoding rSsoPox(wt).
Results: Amino acid sequence of naturally occurring (native) SsoPox enzyme (GenBank: AAW47234.1) was used to design a gene encoding rSsoPox(wt) enzyme. In the designed gene, the 5'end of the open reading frame (ORF) is flanked by Ndel restriction site. The 3'end of the ORF is flanked by a Xhol restriction site (FIG. 1A). The designed gene was custom- synthesized, cloned into pUC57 plasmid and was purchased commercially (from GenScript, NJ, USA). The gene was sub-cloned into expression plasmid, pET23a(+) between Ndel and Xhol restriction sites (FIG. IB), by following standard molecular biology techniques known in the art. The plasmid containing gene encoding rSsoPox(wt) (pET23a(+)-rSsoPox(wt)) was transformed into E. coli BL21(DE3) cells and the recombinant protein was expressed.
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. At nucleic acid level, the designed gene for rSsoPox^) exhibits only 34.6% similarity with the gene of naturally occurring SsoPox. However, comparison of the deduced amino acid sequences of rSsoPox^) and naturally occurring SsoPox enzyme indicated that both share 99.9% identity at the amino acid level. 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(variaiit-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. Thus, 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 ν·. w .b i nionnai i org pri merx/) and were T7 f-(5 -TAATACGACTC ACT AT AGGG-3 λ ) 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. In a typical experiment, a library of 300-500 colonies (containing randomly mutated rSsoPox(Wt) gene) were observed. To each plate was then added 3-4 ml of fresh LB-media containing 50 g/ml carbenicillin and the colonies were scraped with the help of scraper and the suspension of the E. coli DH5a cells were added into fresh LB-media containing 50 g/ml carbenicillin. The cultures were then allowed to grow at 37°C, 200 rpm for 8-24 h. The cells were collected by centrifugation and used to purify the plasmid.
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 OD6oo of the cultures reached to 0.6-0.8. At this point the cultures were induced with 0.5 mM isopropyl β-D-l-thiogalactopyranoside (IPTG) and 0.2 mM CoCl2 was added to each culture tubes. 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:
Out of all screened colonies, one variant named rSsoPox(variant-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(variant-A) was found to contain following two amino acid substitutions: Y40N and N297D.
Example 3: Production of rSsoPox enzymes.
A. Culturing of the recombinant E. coli cells containing gene for rSsoPox enzyme and purification of inclusion bodies: Glycerol stock of recombinant E. coli BL21(DE3) cells containing gene for rSsoPox enzyme was streaked on LB-agar plate containing 50 g/ml carbenicillin and 0.2 mM CoCl2 and the plate was incubated overnight at 37°C. A single colony from the plates was used to initiate the seed culture in LB-broth supplemented with 50 μg/ml carbenicillin and 0.2 mM CoCl2 and the seed culture was grown at 37°C overnight at 200 rpm. One % of this seed culture was then inoculated into fresh LB-broth supplemented with 50 μg/ml carbenicillin and 0.2 mM CoCl2 and the main culture was grown at 37°C till OD6oo reached 0.6 - 0.8. The culture was then induced with 1 mM IPTG and was allowed to grow further at 37°C for 8 h. The bacterial cells were then harvested by centrifugation (10,000 g, 10 min, 4°C) and the cell pellet was used to purify the inclusion bodies by following the procedure described in (Middelberg, A. P., 2001, Trends in Biotechnology, 20, 437), with small modification. Briefly, the cells were re-suspended in ice-cold lysis buffer. 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 MgCl2 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.
Results: Inclusion bodies enriched in the rSsoPox enzyme were isolated using a simple procedure and the purification of the inclusion bodies was assessed by SDS-PAGE and western blot analysis. Mouse anti-His antibody was used as a primary antibody in the western blot analysis. Representative results are presented in FIG. 3. Small amount of rSsoPox enzyme was always observed in the cell lysate (soluble) fraction and washings while majority of the rSsoPox enzyme was present in pure form in inclusion bodies. Same procedure was followed for the purification of inclusion bodies of rSsoPox(variant-A)-
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 CaCl2, CoCl2, MgCl2, and ZnCl2, a salt selected from the group of NaCl, KC1 and H4CI, 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, histidine, glutamic acid, aspartic acid, glycine, alanine, proline, serine, threonine, tryptophan, phenylalanine, cysteine, methonine, valine, leucine, isoleucine, tyrosine, asparagine and glutamine, a sugar selected from a group of maltose, glucose, mannose, trehalose, sucrose, dextrose, lactose, glycerol, sorbitol, mannitol, myo-inositol, xylitol and ethylene glycol, a polymer selected from a group of cyclodextrins and polyethylene glycols, a surfactant selected from a group of NDSB201 and NDSB256, and a reducing agent selected from GSH, TCEP and DTT.
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 CoCl2. 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.
Results: Multiple refolding buffer were checked to identify the refolding buffer enabling maximum refolding of rSsoPox enzymes, one of which is illustrated here. In one set of conditions, the refolding buffer comprised of 50 mM Tris-HCl, 9.6 mM NaCl, 0.4 mM KC1, 2 mM MgCl2, 2 mM CaCl2, 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.
C. Separation of enzymatically active rSsoPox enzyme from inactive enzyme present in refolding mixture using ion-exchange chromatography: The refolding mixture was applied onto a anion-exchange gel chromatographic column pre-equilibrated with buffer containing 20 mM HEPES, pH 8.0, 0.2 mM CoCl2. After washing the column with same buffer, the bound proteins were eluted using increasing concentrations of NaCl (0.1-1 M) in the same buffer. Eluted fractions were analyzed for both protein contents (OD280) and enzyme activity (using paraoxon as substrate) and fractions containing proteins were pooled, concentrated using Amicon concentrator (MWCO 3 kDa) and stored at 4°C. Fractions were analyzed by SDS-PAGE.
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)
Figure imgf000032_0001
a Protein amount and activity were measured after dissolving the inclusion bodies in 8M urea. b Protein amount and activity were measured in the pooled fraction after concentration. c Specific activity is for rSsoPoxivanant-A) .
Example 4: Characterization of OP- hydro lyzing activity of rSsoPox enzymes.
Enzymatic properties of purified rSsoPox(wt) and rSsoPox(variant-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. The amount of substrate hydrolyzed {i.e., product formed) was calculated from the initial linear rates of hydrolysis using extinction coefficient (9100 M-1 cm-1) (Khersonsky, O., and Tawfik, D. S., 2005, Biochemistry, 44, 6371). Values of initial velocity (v0) of the enzyme catalysed reaction and substrate concentrations were used to obtain the kinetic parameters by fitting the data with Lineweaver-Burk (1 /v0 = Km I Vmax 1/S
+ 1 /Kmax) plots.
Results:
The Lineweaver-Burk plots and the kinetic parameters are given in FIG. 4. The Km and kcat / Km values of rSsoPox(Wt) for paraoxon obtained in this study are in close agreement with the values reported for the native SsoPox enzyme (Hiblot, J., et ol., 2012, Sci. Rep., 2, 779 and Ng, F.S.W., et cil, 2010, Appl. Environ. Microbiol, 77, 1181). Compared to rSsoPox(wt), rSsoPox(variant-A) exhibited >40-folds higher kcat/Km 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 CoCl2) 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. For lyophilization, 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. For samples stored at 25°C in aqueous buffer (as liquid formulation), 10 1 of samples were directly added to the activity buffer (20 mM Tris-HCl, pH 8.0, 0.2 mM CoCl2) containing paraoxon and the paraoxonase activity was measured at 405 nm. For lyophilized samples (freeze-dried formulation), the samples were reconstituted to the original volume by adding appropriate amount of distilled water and 10 1 of the reconstituted samples were used to determine the paraoxonase activity. Activity of the enzyme in the storage buffer at 0 day was taken as control and was assigned 100% and the relative activity of the enzyme in various compositions was then calculated.
Results:
Stable compositions identified enable long-term storage stability of rSsoPox(variaiit-A) enzyme and comprise of purified rSsoPox(variaiit-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 CaCl2, CoCl2,MgCl2, and ZnCl2, wherein the salt is selected from the group of NaCl, KC1 and NH4C1, 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 amino acid is selected from the group of arginine, lysine, histidine, glutamic acid, aspartic acid, glycine, alanine, proline, serine, threonine, tryptophan, phenylalanine, cysteine, methonine, valine, leucine, isoleucine, tyrosine, asparagine and glutamine, wherein a sugar is selected from a group of maltose, glucose, mannose, trehalose, sucrose, dextrose, lactose, glycerol, sorbitol, mannitol, myoinositol, xylitol and ethylene glycol. a) Storage stability of rSsoPoX(variant-A) when stored in aqueous solution at 25° C: Purified rSsoPox(variaiit-A) enzyme stored in buffer containing no added excipients showed considerable loss of activity (-60% decrease in the activity in 120 days). However, the stable compositions as prepared in the present invention, retained 75-100% of the original activity of enzyme after 120 days of storage at 25°C. b) Storage stability of rSsoPoX(variant-A) when stored in freeze-dried form at 25°C: Purified rSsoPox(variaiit-A) enzyme in buffer containing no added excipients when freeze-dried and stored at 25°C showed considerable loss of activity (~ 70% decrease in the activity in 120 days). However, stable compositions as prepared in the present invention retained -100% of the original activity of enzyme after 120 days of storage of freeze-dried form at 25°C.
Example 6: Stabilization of rSsoPox(Variant-A) by PEGylation
A) N-terminal mono-PEGylation of rSsoPox(variaiit-A) and purification of N-terminal mono-PEGylated-rSsoPox(variaiit-A) by ion-exchange chromatography: The pure rSsoPox(variant-A) was mixed with 15-folds molar excess of mPEG-propionaldehyde (Mw = 5 kDa) in 100 mM sodium phosphate buffer, pH, 6.0 containing 20 mM sodium cyanoborohydride (as a reducing agent) and the reaction was incubated at 8°C for 7 days with gentle mixing. 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 CoCl2. 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 (OD28o) 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.
B) Protease sensitivity assay: 0.1 mg of unmodified rSsoPox(variant-A) and N-terminal mono- PEGylated-rSsoPox(variant-A) were separately mixed with 70 μΐ of 1 mM HCl. Then, 10 μΐ of trypsin solution (0.1 mg/ml in 1 mM HCl) and 20 μΐ 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. Aliquots were taken out from the reaction mixtures and used to determine the protease sensitivity of the enzymes by measuring the residual activity of the enzyme. Enzyme samples (in which trypsin was not added) was taken as control and the activity of control enzymes was assigned 100%. Results:
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. The protein in the eluted fractions were pooled, concentrated, dialyzed, and subjected to 12.5%) SDS-PAGE analysis (Fig. 5B). Unmodified rSsoPox(variant-A) migrated as a single band corresponding to an apparent molecular weight of -35 kDa (lane 1). In contrast, N-terminal mono-PEGylated-rSsoPox(variant-A) showed a single electrophoretic band with an apparent molecular weight of -40 kDa (lane 2).
To see the effect of PEGylation on the protease sensitivity of the enzyme, equal amounts of unmodified rSsoPox(variant-A) and mono-PEGylated-rSsoPox(variant-A) were exposed separately to trypsin (proteolytic enzyme) and the stability of proteins was determined by measuring the paraoxonase activity of the enzyme (Fig. 5C). The enzyme, not exposed to trypsin, was taken as the control and was assigned 100% activity. Incubation of unmodified rSsoPox with trypsin resulted in a significant decrease in the activity of the enzyme (compare bar i and ii). In contrast, mono-PEGylated-rSsoPox(variant-A) retained - 88%> of its activity under similar conditions (compare bar iii and iv). This suggests that mono- PEGylation of rSsoPox(variant-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(variant-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). Briefly, 5 mg of the homogenously dispersed MNPs were taken in a tube, washed five-six times with double-distilled water (dd-H20), and were re-suspended in dd-H20. Separately, 10 mg of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was freshly dissolved in dd-H20 (150 1). The MNPs were then activated by adding a freshly prepared solution of EDC to the suspension of MNPs in dd-H20 and by gently mixing the samples for 10 min at 25°C. The activated MNPs were then washed several times with dd-H20 to remove any unreacted EDC. To this activated MNPs suspension was then added 50 μg 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. During the immobilization reaction 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. To measure the efficiency of immobilization, 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.
Results:
The purified rSsoPox(variant-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 NH2 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 (reaction in which MNPs were not activated by EDC) 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). The decrease in the paraoxon-hydrolyzing activity (and the protein content) in the supernatant of the coupling reaction in which activated-MNPs were used indicated that the enzyme was immobilized onto the MNPs. The efficiency of the immobilization was found to be 60-100% using this procedure.
To see whether immobilization of rSsoPox(variant-A) has affected the catalytic properties of the immobilized enzyme, specific activity of rSsoPox-MNPs (nanobiocatalyst) were compared with the specific activity of the free enzyme under identical assay conditions. Immobilized enzyme was found to be 35 ±10% active as compared to the free enzyme (FIG. 6C). To see the effect of external magnetic field on the separation of rSsoPox-MNPs (nanobiocatalyst) in aqueous buffer, vial containing rSsoPox-MNPs suspension in TBS was exposed to a magnet. In the absence of magnet (magnetic field) the rSsoPox-MNP nanobiocatalyst were well dispersed in the aqueous buffer (FIG. 6D). The rSsoPox-MNP nanobiocatalyst quickly (within 3-5 min) aggregated on the wall of the vial when the magnetic field was applied (FIG. 6E) and re-dispersed in the aqueous buffer when the magnetic field was removed.
Example 8: Reusability of rSsoPox-MNP nanobiocatalyst to degrade OP-compound in aqueous media.
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. Separately, MNPs (which do not contain immobilized rSsoPox enzyme) was used as a control to see the non-enzymatic hydrolysis of the paraoxon. After completion of the incubation time, the reaction was stopped by separating and recovering the rSsoPox-MNPs nanobiocatalyst from the reaction mixture by using magnet. Hydrolysis of the paraoxon was determined by measuring the yellow color formation at 405 nm in the supernatant of the reaction mixture. Extent of paraoxon-hydrolysis was calculated from the molar extinction coefficient of ?-nitro phenol (ε4ο5 = 9100 M_1cm_1) and corrected for the non-enzymatic hydrolysis. The recovered rSsoPox-MNP nanobiocatalyst were washed with PBS and stored (as suspension) in the same buffer at 4°C for 6-8h. To check the stability and reusability rSsoPox-MNP nanobiocatalyst, 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. Again after completion of the reaction the rSsoPox-MNP nanobiocatalyst were recovered from the reaction mixture by using magnet and stored as described above. Results: 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 above examples and the technical descriptions are illustrative of the invention and any variations to the same should be regarded as within the scope of the invention. ADVANTAGES OF PRESENT SYSTEM:
• Easy method of rSsoPox enzymes production: 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.
• High level expression: 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.
• Economical: Owing to easy method of production, high yield and enhanced activity the method disclosed is economical.
•Long-term storage stability: 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.
· Decreased protease sensitivity: 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. Some of the novel features of the present invention are:
• A novel polynucleotide sequences (gene) encoding rSsoPox(wt) enzyme has been designed for high level expression of enzyme in the E. coli expression system.
• Novel enzyme variant of rSsoPox(wt) have been generated that possesses enhanced OP-hydrolyzing activity.
· 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.
For coating of surfaces: 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.

Claims

The Claims:
1. A recombinant polynucleotide (rSsoPox) and variant thereof comprising at least one nucleotide sequence selected from the group comprising SEQ ID NOs: 1-2.
2. A recombinant polynucleotide as claimed in claim 1, wherein said polynucleotide encodes at least one polypeptide chain, wherein the polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 3-4.
3. A recombinant polypeptide as claimed in claim 2 wherein 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, wherein said
SsoPox-substrate may be selected from the group of but not limited to phosphotriesters and lactones.
4. A recombinant polynucleotide (rSsoPox) and variants 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 the SsoPox-substrate may be selected from the group of but not limited to phosphotriesters and lactones.
5. A recombinant polynucleotide (rSsoPox) and variant thereof as claimed in claim 4, wherein the SsoPox-substrate is selected from synthetic or natural chemical selected from a group of but not limited to phosphotriesters and lactones or a combination thereof.
6. A recombinant polynucleotide (rSsoPox) and variant thereof as claimed in claim 5, wherein the phosphotriesters are selected from the group comprising paraoxon, diazoxon, chlorpyrifosoxon, soman, sarin, diisopropylflourophospate, 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, parathion-methyl, pirimiphos-ethyl, pirimiphos-methyl, pyrazophos, quinalphos, ronnel, sulfopros, sulfotepp, trichloronate, tabun, soman, sarin, cyclosarin, GX, VX, R-VX, diisopropylflourophospate.
7. A recombinant polynucleotide (rSsoPox) and variant thereof as claimed in claim 5, wherein the lactones are selected from the group comprising γ-butyrolactone, γ- caprolactone, γ-octanoiclactone, γ-nonanoiclactone, γ-decaanoiclactone, γ-undecaanoic lactone, nonanoic-5-lactone, undecanoic-5-lactone, dodecanoic-5-lactone, ε-caprolactone, etc; acyl homoserine lactones, such as 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- oxododecanoyal-DL-homoserine lactone, N-oxotetradecanoyal-DL-homoserine lactone, N-3- ketocaproyal homoserine lactone, N-3-hydroxydecanoyal homoserine lactone.
8. The recombinant polypeptides of claim 4, wherein at least one of the said polypeptide chain possesses increased hydrolytic activity (kcat/Km ratio) by at least 40-folds for paraoxon substrate compared to rSsoPox(wt).
9. A method for production of recombinant polypeptide comprising amino acid sequence of rSsoPox(wt) and variant thereof by refolding inclusion bodies, wherein the recombinant polypeptides are enzymatically active rSsoPox polypeptides, comprising the steps of- a. designing rSsoPox gene and subjecting it to random mutagenesis,
b. culturing the host cells expressing rSsoPox polypeptides,
c. inducing high level of expression of rSsoPox polypeptides,
d. collecting the cell mass expressing rSsoPox polypeptides,
e. lysing the cells,
f. purifying the inclusion bodies containing rSsoPox polypeptides,
g. solubilizing the inclusion bodies with the help of chaotropic agents,
h. refolding the rSsoPox enzymes or polypeptides using refolding buffer,
i. isolating the enzymatically active rSsoPox using chromatography,
j. obtaining enzymatically active rSsoPox polypeptides,
wherein the composition of refolding buffer used for refolding the misfolded/inactive enzymes comprise the ingredients and their concentrations not limiting to a buffering agent, a cofactor, a salt, a detergent, an amino acid, a sugar, a polymer, a surfactant, and a reducing agent and possible variations thereof.
10. A method for production of recombinant polypeptides as claimed in claim 9, wherein the chaotropic agent is selected from the group comprising organic compounds, solvents, salts, detergents and is preferably urea.
11. A method for production of recombinant polypeptides as claimed in claim 9, wherein the refolding buffer comprises at least one buffering agent selected from the group of Tris-
HC1, CHES, EPPS, HEPES, Glycine-NaOH, Phosphate, TAPS, MOPS, and MES.
12. A method for production of recombinant polypeptides as claimed in claim 9, 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.
13. A method for production of recombinant polypeptides as claimed in claim 9, wherein the cofactor is selected from the group comprising CaCl2, CoCl2, MgCl2, and ZnCl2.
14. A method for production of recombinant polypeptides as claimed in claim 9, wherein the refolding buffer further comprises at least one refolding additives selected from the group of salts, sugars, polymers, polyols, detergents and surfactants.
15. A method for production of recombinant polypeptides as claimed in claim 9, wherein the salt is selected from the group comprising NaCl, KC1 and H4CI.
16. A method for production of recombinant polypeptides as claimed in claim 9, wherein the detergent is selected from the group comprising tween-20, tween-20, NP-10, P-40, triton X- 100, CHAPS and Brij 35.
17. A method for production of recombinant polypeptides as claimed in claim 9, wherein the amino acid is selected from the group comprising arginine, lysine, histidine, glutamic acid, aspartic acid, glycine, alanine, proline, serine, threonine, tryptophan, phenylalanine, cysteine, methonine, valine, leucine, isoleucine, tyrosine, asparagine and glutamine.
18. A method for production of recombinant polypeptides as claimed in claim 9, wherein the sugar is selected from a group comprising maltose, glucose, mannose, trehalose, sucrose, dextrose, lactose, glycerol, sorbitol, mannitol, myo-inositol, xylitol and ethylene glycol.
19. A method for production of recombinant polypeptides as claimed in claim 9, wherein the polymer is selected from a group comprising cyclodextrins and polyethylene glycols.
20. A method for production of recombinant polypeptides as claimed in claim 9, wherein the surfactant is selected from a group comprising NDSB201 and DSB256, and a reducing agent selected from TCEP and DTT and possible variation thereof.
21. A method for production of rSsoPox polypeptides, wherein said polypeptides 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.
22. A method of production of active rSsoPox enzymes from inclusion bodies, wherein said polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 3-4, said method comprising the steps of:
a. purifying the inclusion bodies containing rSsoPox polypeptides,
b. solubilizing the inclusion bodies containing rSsoPox polypeptides with the help of urea,
c. refolding the denatured rSsoPox polypeptides by diluting in refolding buffer, d. isolating the enzymatically active rSsoPox polypeptides using chromatography, e. obtaining the enzymatically active rSsoPox polypeptides.
23. A method of production of enzymatically active rSsoPox polypeptides as claimed in claim 22, 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 CaCl2, CoCl2, MgCl2, and ZnCl2 and the refolding additives are selected from the group of salts, sugars, polymers, polyols, detergents and surfactants.
24. The method of claim 22 wherein the purity of enzymatically active rSsoPox polypeptides is at least 80%.
25. Stabilized 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, wherein said polypeptide is derived from at least one nucleotide sequence selected from the group comprising SEQ ID NOs: 1-2 and said rSsoPox polypeptide chain comprises at least one amino acid sequence selected from the group comprising SEQ ID NOs: 3-4.
26. PEGylated-rSsoPox conjugate having decreased protease sensitivity, wherein the PEGylated-rSsoPox enzyme is mono-PEGylated-rSsoPox, wherein PEG is covalently linked to rSsoPox enzyme at its N-terminal end.
27. The PEGylated-rSsoPox conjugate of claim 26 wherein the molecular weight of conjugated PEG selected among the molecular weights of 2 kDa, 5 kDa, 10 kDa, 20 kDa, preferably 5 kDa.
28. The PEG molecule of claim 26 is a linear molecule selected among the group of linear and branched PEGs.
29. The PEG molecule of claim 26 is methoxy PEG aldehyde selected from a group of PEG- succinimidyl carbonate, PEG-pN02 phenyl carbonate, PEG-AA-NHS and PEG- carbonylimidazole.
30. Nucleic acid constructs comprising the isolated polynucleotides of claim 1.
31. Nucleic acid constructs comprising the isolated polynucleotides encoding polypeptide of claim 2.
32. An isolated host cell comprising the nucleic acid constructs of claim 31.
33. Use of polynucleotides of claim 1 and polypeptides of claims 2 and 26, in isolation or in combination as an effective antidote, anti-toxicant and anti-chemical warfare agents for prevention or treatment of organophosphate poisoning in humans as well as in other animals.
34. Use of polynucleotides and polypeptides of claim 1 and 2, respectively, in isolation or in combination with other agents or process or methods, for decontaminating organophosphate-contaminated surfaces and objects including but not limited to land and water bodies, equipments, instruments, and machineries used in various industries, farm produce, etc.
35. Use of polynucleotides and polypeptides of claim 1 and 2, respectively, in isolation or in combination with other agents, for coating various objects like membranes and filters in water purification and air purification systems, fabrics, wearables, biosensors.
36. Use of polynucleotides and polypeptides of claim 1 and 2, respectively, in isolation or in combination with other agents, for making various enzyme-based decontamination formulations for OP-decontamination including soaps, foam, aerosols, tissue papers, hydrogels, paints, silicate etc.
37. A SsoPox-immobilized magnetic nanobiocatalyst comprising a magnetic nanoparticles coated with rSsoPox enzyme of SEQ No. 4 encoded by polynucleotide SEQ No. 2, wherein
a. the immobilization surfaces can be carrier particles, nanomaterials, fabrics, membranes, discs, metals and more but preferably are magnetic nanoparticles (MNPs),
b. the immobilization can be done by either covalent binding, non-covalent binding and adsorption method preferably by covalent binding, wherein SsoPox enzymes immobilized on the surface of nanoparticles are used for degradation of organophosphate-compound.
38. A method for degrading OP-compounds in water system using the rSsoPox-immobilized magnetic nanobiocatalyst described in claim 37.
39. Use of r SsoPox-immobilized nanobiocatalyst of claim 37 for a. decontaminating OP-contaminated water bodies b. coating on the electrode surfaces of biosensors for toxicity analysis, environmental monitoring, food quality control, and military investigations.
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US10260061B2 (en) 2011-03-10 2019-04-16 Cornell University Mesoporous catalysts of magnetic nanoparticles and free-radical-producing enzymes, and methods of use
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