KR101892358B1 - Prolidase mutant, and preparation method and use thereof - Google Patents

Abstract

The present invention relates to a pyrrolidase mutant through the amino acid substitution of an organic phosphoric acid hydrolase, Prolidase, a method for preparing the same, and the use of the pyrrolidase mutant prepared as described above, More particularly, the present invention relates to a method for the detection of a follidase mutation in which the amino acid of the 252 th residue or the amino acid of the 365 th residue is substituted with arginine from the N-terminus of the follidylase through a database-based in silico analysis And a recombinant expression vector containing the corresponding prolylderase mutant gene, a prokaryotic cell transformed from the recombinant expression vector, and a method for producing a prolylase mutant using the same, and a method for producing a wild- Lt; RTI ID = 0.0 > of frolidase < / RTI > As a solvent for the active inhibitory composition to inhibit the poisoning symptoms of nerve agents there is an effect that can be applied to prevention and treatment of nerve agents.

Description

PROLIDASE MUTANT AND AND PREPARATION METHOD AND USE THEREOF FIELD OF THE INVENTION The present invention relates to novel fulleridase mutants,

The present invention relates to a pyrrolidase mutant through the amino acid substitution of a human-derived pyrrolidase (Prolidase), an organophosphate hydrolase, and a method for preparing the same, and more particularly to a database-based in silico- The follolidase through the analysis has been found to be useful as a mutant of frolidase having an improved enzyme activity over wild-type frolidase by attempting various amino acid mutations, a method for producing the same and a method for producing the frolidase mutant .

Nerve agents are organophosphate compounds containing organic phosphorus ester groups and are mainly sarin, soman, cyclosarin, VX (O-Ethyl S- (2-methylpropoxy) phosphoryl) sulfanylethanamine (VR) are well known. Depending on the type of R group bonded to the phosphate group, G-type and V -type. These nerve agents bind to acetylcholinesterase in the nervous tissue to inhibit the degradation of acetylcholine, a neurotransmitter, resulting in lethal killing by inducing muscle spasm, rigidity, muscle contraction and cardiopulmonary dysfunction.

When exposed to neuroleptics, current therapies are combined with atropine and oxime-based antidotes, but they have the disadvantage that they should be administered in a short time after exposure to various side effects and exposure. Therefore, recent studies have focused not only on the detoxification of nerve agents, but also on the improvement of enzymatic activity through the discovery of biocatalysts using preventable protein enzymes.

Prolidase is an enzyme that hydrolyzes peptide bonds by recognizing two isotypes such as kidney-derived type and skin-derived type, and recognizing the C-terminal of proline in protein. Roldridae has been known to have high hydrolytic activity.

Frolidase has 28% similarity to Organophosphorus acid anhydrolase (OPAA), one of the bacterial enzymes that have excellent neuroleptic resolution.

Transgenic mice overexpressing frolidase have been reported to show increased resistance to diisopropylfluorophosphate (DFP) when exposed to a neurokinetic agent, diisopropylfluorophosphate (DFP) Enzyme activity of the protein was found to be lower than that of OPAA. Therefore, in order to use frolidase for detoxifying neuroleptics, it is necessary to prepare an improved form of frolidase for the activity of a nerve agent, so that it can be utilized as a biocatalyst.

Billecke et al., 1999

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a novel type of follicle-producing follidase which is capable of increasing the enzyme activity through amino acid mutation corresponding to the enzyme active site through in silico analysis, And a method for producing the same.

In addition, the prepared follidase mutant was purified from Escherichia coli as a fulleridase A252R and a fulleridase P365R mutant enzyme, and the enzyme activity against diisopropylfluorophosphate (DFP), a neural agent similar substance, was measured It is found that the fulleridase mutant can be provided as a neuroleptic antidote or as a novel biocatalyst agent through the use of fulleridase mutants.

In order to achieve the above object, the present invention provides a follidase mutant comprising a protein wherein the 252nd amino acid or the 365th amino acid is substituted with arginine from the N-terminus of the wild type follidase do.

Specifically, in the human-derived pyrrolidase amino acid sequence represented by the amino acid sequence of SEQ ID NO: 6, the amino acid sequence of SEQ ID NO: 7 in which alanine, which is the amino acid of the 252nd residue from the N-terminus, is substituted with arginine Or an amino acid sequence of SEQ ID NO: 8 substituted with arginine, which is an amino acid of the 365th residue from the N-terminus in the amino acid sequence of human-derived pyrrolidase represented by the amino acid sequence of SEQ ID NO: 6 .

And the gene encoding the pyrrolidase mutant consisting of the amino acid sequence of SEQ ID NO: 7 comprises the nucleotide sequence of SEQ ID NO: 4, or the gene encoding the pyrrolidase mutant of the amino acid sequence of SEQ ID NO: 8 The gene consists of the nucleotide sequence of SEQ ID NO: 5.

The gene coding for the pyrrolidase mutant for producing such a follidase mutant has the nucleotide sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5, and a gene comprising the follidase mutant gene And recombined in an expression vector.

It is preferable that the recombinant expression vector of the present invention has a structure necessary for expression in a base sequence including a promoter, a pyrrolidase gene, an electron-terminating sequence and the like, and the expression vector itself can be autonomously replicated in a host cell.

Although pET21b was used as an expression vector used in the present invention, it is not limited thereto and other expression vectors which satisfy the above-described requirements can be used.

The method for producing a follidase mutant of the present invention comprises the steps of transforming prokaryotic cells with a recombinant expression vector comprising a follidase off-variant gene having the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5, Culturing the transformed prokaryotic cell, and isolating and purifying the target protein expressed from the transformant, the pluridase mutant. At this time, the prokaryotic cell used for producing the mutant is preferably, for example, E. coli.

The production of the follidase mutant of the present invention can be carried out by culturing a transformant obtained by transforming a suitable host cell with a recombinant expression vector containing a gene encoding a follidase mutant, By producing a gene product, a follolidase mutant, in a culture supernatant, a follidase mutant is obtained from the culture.

The step of separating and purifying is a step of finally obtaining a target protein, a pyrrolidase mutant. The method of recovering the target protein expressed as a follolidase mutant in the transformant is separated and purified through column chromatography . Here, the column chromatography can be carried out by separation or purification using ion exchange chromatography, gel-filtration chromatography, HPLC, reverse phase-HPLC, adsorption chromatography and affinity column chromatography, alone or in combination.

The method for recovering the protein of the present invention is not limited thereto, but can be carried out through various separation and purification methods known in the art. In order to remove cell debris and culture impurities, the cell lysate is centrifuged After the separation, precipitation, for example, salting out (ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, isopropyl alcohol, etc.) Various column chromatography and the like can be carried out.

In addition, to achieve the above object, the present invention provides a composition for inhibiting neuronal agonist activity comprising a pyrrolidase mutant produced through the above-described method as an active ingredient.

That is, the use of a follidase mutant can be used as a composition for preventing and treating diseases caused by a nerve agent including an active ingredient thereof.

As used herein, the term " treatment " refers to any action that reduces neuroprotective agents, particularly the diseases caused by the poisoning of diisopropylfluorophosphate (DFP) and the pathological conditions of the diseases caused thereby do.

The composition for the prevention and treatment of diseases caused by the nerve agent of the present invention may comprise at least one immunopotentiator selected from cholerotoxin, aluminum hydroxide, carbopol, mineral oil and biodegradable oil May be further included.

The composition of the present invention may be prepared in the form of a unit dose by being formulated using a pharmaceutically acceptable carrier and / or an excipient according to a method which can be easily carried out by those skilled in the art, It may be manufactured by inserting it into a container.

The pharmaceutically acceptable carrier to which the composition is incorporated is a carrier usually used at the time of formulation and may be selected from lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, But are not limited to, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil .

Herein, the term "pharmaceutically acceptable carrier" means a carrier or diluent which does not irritate the organism and does not inhibit the biological activity and properties of the administered compound.

The present invention relates to a method for producing a fibrillary enzyme by substituting arginine at the 252nd amino acid or the 365th amino acid from the N-terminus among the amino acids present in the enzyme active site of the follidase through in- silico analysis based on a database, The present invention relates to novel follidase mutants which improve the enzymatic activity of the enzyme and increase the protein stability of the protein.

The present invention can obtain a follidase mutant from Escherichia coli in a high yield. The follidase mutant thus prepared is used for preventing and treating neuroprotective agents to suppress the addiction to nerve agents, A biocatalyst, and the like.

Figure 1 shows the amino acid corresponding to the enzyme active site based on the wild-type (WT) pyrrolidase protein structure.
Figures 2 and 3 show the predicted site of follidase mutation formation through an arginine substitution mutation in the linus and amino acid sequence on the enzyme active site based on the protein structure of the follidase.
FIG. 4 through FIG. 6 show expression vectors for the production of a follidase mutant according to an embodiment of the present invention.
FIGS. 7 and 8 are the result of confirming the substitution of the base sequence by the site-directed mutagenesis method.
FIG. 9 shows the result of confirming expression of wild-type (WT) follolidase protein.
FIGS. 10 and 11 are the results of confirming the expression of the follidase protein of the follidase mutations, respectively.
FIG. 12 shows the results of 10% SDS-PAGE electrophoresis of the separated and purified proteins according to one embodiment of the present invention and staining with Coomassie blue.
FIG. 13 shows the results of Western blot analysis of proteins separated and purified according to an embodiment of the present invention using an anti-his antibody.
FIGS. 14 and 15 show the enzyme activity results of the plastidase mutant on diisopropylfluorophosphate (DFP).

The technical terms used in the present invention are to be interpreted in a sense generally understood by a person having ordinary skill in the art to which the present invention belongs, And shall not be construed as an oversimplified meaning. In addition, the general terms used in the present invention should be construed in accordance with the predefined or prior context.

Furthermore, the singular expressions used in the present invention include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "consisting of", "consisting of" or "including" shall not be construed as necessarily including the various components or steps described in the specification, Or some steps may not be included, or may be interpreted to include additional components or steps.

The present invention relates to a method for improving the enzyme activity of a wild-type follidase by preparing a mutant in which the 252nd amino acid or the 365th amino acid is substituted with arginine from the N-terminus of the wild type follidase .

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are merely examples of the present invention, and the scope of the present invention is not limited to these Examples.

Example 1 analyzes the sequence and amino acid sequence of a human-derived prolidase to design a pyrrolidase mutant and uses it to perform enzyme activity through database-based in silico analysis Lt; RTI ID = 0.0 > of follidase < / RTI > mutants.

In Example 1, pET21b-prolidase in which a wild-type pluridase gene was cloned into an E. coli protein expression vector pET21b was first prepared to produce a prodrillase mutant, and specifically, a prolidase obtained from a human skin cell line ) Is amplified by polymerase chain reaction (PCR) using the cDNA as a template. In this amplification process, an omnidirectional primer 5'-AAGCTTACCATGGCGGCGGCCACCGGACC-3 '(SEQ ID NO: 13) and a reverse primer 5'-CTCGAGCTTGGGGCCAGAGAAGGGGG-3' based on the nucleotide sequence of known prolidase SEQ ID NO: (SEQ ID NO: 14), and inserted into restriction enzyme recognition sites HindIII and XhoI of E. coli expression vector pET21b (Novagen, Madison, WI, USA) Vector pET21b-prolidase is prepared.

The follidase mutant of the present invention is a database-based in silico analysis method which sets the nucleotide sequence and amino acid of the gene of follidase provided from a known database and determines the enzyme activity in the follidase protein The base sequences and amino acids corresponding to the enzyme active sites which are expected to increase are defined.

The pyrrolidase mutant of the present invention comprises an amino acid sequence of SEQ ID NO: 7 substituted with arginine (arginine, Arg) alanine (alanine, Ala) which is the 252nd amino acid from the N-terminal of the pyrrolidase represented by SEQ ID NO: (Amino acid sequence of SEQ ID NO: 8) substituted with arginine (Arg) at position 365 from Prolinease A252R mutant (hereinafter also referred to as 'prolidase-A252R') or N-terminal proline (Hereinafter also referred to as " prolidase-P365R ").

In the case of the follidase A252R mutant, the pET21b-prolidase prepared as described above was used as a template and was prepared using a site-directed mutagenesis method (Stratagene Corp.) A primer set consisting of an omnidirectional primer 5'-CAGTGGTGAGAACTCACGCGTGCTACACTACGGCGTGCTACACTACGGAC-3 '(SEQ ID NO: 15) and a reverse primer 5'-GTCCGTAGTGTAGCACGCGTGAGTTCTCACCACTG-3' (SEQ ID NO: 16) was used to prepare the daazae A252R mutant. The A252R mutant was amplified by polymerase chain reaction (PCR), and the PCR amplified mutant was treated with Dpn I as a restriction enzyme for about 1 hour and transformed into Escherichia coli XL1-Blue strain to obtain the nucleotide sequence of SEQ ID NO: 1 Was prepared and named as pET21b-prolidase-A252R.

(SEQ ID NO: 17) and reverse primer 5'-AGTGGCCAAGCCCGTGACGCATAAACACGGCCCCC-3 '(SEQ ID NO: 18) in order to prepare a follidase P365R mutant as another follidase mutant Pyridase P365R mutants were amplified by polymerase chain reaction (PCR) using primer pairs constructed. The plasmid DNA, which is a recombinant expression vector comprising the nucleotide sequence of SEQ ID NO: 2, was prepared by the same procedure as that for producing the above-mentioned follidase A252R mutant, and this product was named pET21b-prolidase-P365R.

In addition, the recombinant expression vector of the present invention may further include other sequences as needed in order to facilitate the production and purification of the target protein finally produced in the present invention, including secretory signal sequences and tag sequences for protein purification .

Therefore, as shown in FIG. 4 to FIG. 6, the gene structure of the recombinant expression vector of the follidase mutant of the present invention is a T7 promoter, a T7 tag coding sequence, a target protein gene, a His tag sequence (His tag coding sequence in order. That is, in the present invention, the recombinant expression vector has a pET21b vector fused with a T7-tag at the N-terminus of the target protein and a His-tag at the C-terminus. Herein, the sequence which can be additionally contained is not limited to the kind of the sequence necessary for purifying the target protein, and the kind of the sequence necessary for purifying the target protein is not limited.

The gene of the recombinant expression vector of the pyrrolidase mutant includes the nucleotide sequence shown in SEQ ID NOS: 9 to 12, and specifically the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, The base includes an ampicillin resistance gene represented by SEQ ID NO: 9 as the antibiotic resistance gene, a T11 promoter of SEQ ID NO: 10 as the 5117th to 5135th bases, a nucleotide sequence of the 5208th to 5237th bases Comprising a T7 tag sequence of SEQ ID NO: 11 and a His tag sequence of SEQ ID NO: 12 with nucleotides 6759 to 6776, respectively.

Example 2 relates to expression of follidase in a transformant obtained by transforming pET21b-prolidase, pET21b-prolidase-A252R and pET21b-prolidase-P365R into a host cell with the recombinant expression vector prepared according to Example 1 above .

Specifically, in Example 2, Escherichia coli BL21 (DE3) transformed with pET21b-prolidase, pET21b-prolidase-A252R and pET21b-prolidase-P365R to induce prolidase expression was added to Luria-Bertani (LB) mg / ml ampicillin and incubate at 37 ° C for 12 hours. In order to establish optimal temperature for expression of Soluble prolidase from cultured Escherichia coli, four temperature conditions of 37 ℃, 30 ℃, 25 ℃ and 20 ℃ were selected and the difference of growth of Escherichia coli The inoculum amount of E. coli was set to OD6000.4 (37 DEG C), OD6000.6 (30 DEG C), OD6000.8 (25 DEG C), and OD6001.0 (20 DEG C).

After incubation for 20 min at each incubation temperature, E. coli was incubated at 37 ° C for 1 h, 30 ° C for 2 h, Lt; 0 > C for 3 hours and 20 [deg.] C for 6 hours. Escherichia coli cells were obtained by centrifugation at 12,000 rpm, 4 ° C for 5 minutes in order to harvest the expression-induced E. coli. To compare the amount of soluble follidase protein produced in Escherichia coli, lysis buffer (20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, 1 mg / ml lysozyme) was added to the harvested E. coli cell body The supernatant was separated from the supernatant by centrifugation at 12000 rpm at 4 ° C for 15 minutes to separate the soluble protein and insoluble protein from the disrupted E. coli. .

Whole cell lysate, soluble protein, and insoluble protein were electrophoresed on 10% SDS-PAGE electrophoresis (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) And then identified by Coomassie brilliant blue (CBB) staining method.

Example 3 relates to the isolation and purification of a follidase expressed from Escherichia coli expressing the transformed follolidase. In order to separate and purify the protein, wild-type wild type follidase, follidase A252R mutant, Escherichia coli expressing each of the P385R mutants was cultured at 20 DEG C culture conditions to induce IPTG expression. The cultured Escherichia coli cells were disrupted using an ultrasonic disrupter, and the expression of the follidase protein expressed after centrifugation And the supernatant is passed through a Ni-NTA column to purify the follidase protein.

Specifically, in Example 3, Escherichia coli expressing follolidase was inoculated with OD6001.0 for isolation and purification of the E. coli protein expressing wild-type pradiolase, and then cultured in IPTG (Isopropyl β-D- 1-thiogalactopyranoside) to induce the expression. Expression - induced cell bodies were harvested by centrifugation and soluble proteins were isolated by ultrasonic disruption. A Ni-NTA column (Quiagen corp.) Purification method using a His-tag added at the C-terminus of the follidase was used to purify the follolidase in the soluble proteins. The soluble proteins were passed through 1 ml of Ni-NTA column and then 5 ml of wash buffer (20 mM TRIS-Cl pH 8.0, 300 mM NaCl, 50 mM Imidazole) to remove proteins that did not bind His-tag. The wild-type pyrrolidase protein bound to the Ni-NTA column was separated into fractions by adding 10 ml of elution buffer (20 mM TRIS-Cl (pH 8.0), 300 mM NaCl, 250 mM Imidazole).

The thus isolated wild-type pyrrolidase protein was dialyzed in dialysis buffer solution (20 mM Tris-HCl (pH 8.0), 300 mM NaCl) for 12 hours. Purified wild-type pravolidase was analyzed by staining with Coomassie brilliant blue (CBB) after performing 10% SDS-PAGE electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis).

On the other hand, the His-tag added to the C-terminus of Prolidase was confirmed by Western blot to verify the proteins isolated and purified according to the examples, and the separated and purified follidase protein was labeled with 10 SDS-PAGE gel electrophoresis. The separated proteins were electrophoresed on a PVDF membrane and transferred to an anti-His antibody (Santa Cruz, CA, USA) as a primary antibody. And Western blotting was performed in order to observe the band to be photographed on the X-ray film by sequentially treating anti-mouse IgG-HRP (Santa Cruz, CA, USA) as a secondary antibody. Proteins were isolated and purified in the same manner for the follidase wild type and the follidase mutant, the follidase A252R mutant and the follidase P365R mutant.

Example 4 relates to a method for measuring the enzyme activity of a follidase mutant through the ability to decompose diisopropylfluorophosphate (DFP), which is one of the organic phosphoric acid compounds, as a nerve agent-like substance.

To determine the enzymatic activity of fibrillase digesting DFP (Sigma, St. Louis, MO, USA), one of the organic phosphate compounds, 1 mM DFP, 0.004% Phenol red, 2.0 mM HEPES (pH 8.0) and 2 mM CaCl ₂ buffer solution were added and the absorbance at 422 nm was measured and the enzyme activity was measured at 25 ° C for 5 minutes (Billecke et al., 1999 ).

The enzyme activity of diisopropylfluorophosphate (DFP) was measured by measuring the absorbance at 422 nm, and the acid production according to the absorbance was calculated as 1.9 × 10 ³ × (Δ absorbance / Δ time) / reaction volume (ul) The enzymatic activity was calculated as μmol / min / ml. To calculate the Kcat and Km values of the enzyme, enzyme activity was measured at 0.1 mM to 1.6 mM, and the Michaelis-Menten equation was used. To calculate the Kcat / Km value.

FIGS. 1 to 3 show in silico analysis results of transforming amino acids corresponding to enzyme active sites on the basis of the structure of the protein of follidase . The wild-type pyrrolidase protein structure of FIG. To identify mutations expected to increase enzyme activity.

As shown in FIG. 2, when alanine alanine (alanine, Ala), the 252th amino acid of frolidase, was changed to arginine (Arg), glutamate and a salt bridge were formed at 249th glutamate The enzyme activity is expected to be increased by increasing the enzyme activity. The ΔG stability is -12.80 kcal / mol, and the ΔG affinity is -0.02 kcal / mol, as compared with the wild-type pyrrolidase in the follidase A252R mutation. .

As shown in FIG. 3, when the 365th proline (Pro) of frolidase is replaced with arginine (Arg), glutamate and salt bridge are formed with 424th glutamate, was predicted to be active is increased, double Rico (in silico) the results of these programs are Raleigh azepin P365R mutation is wild-type loop Raleigh is compared with the stability azepin ΔG is -1.44 kcal / mol, ΔG affinity is -2.98 kcal / mol value Is expected to increase its binding capacity to substrates such as similar neuroactive agents such as diisopropylfluorophosphate (DFP).

FIG. 7 and FIG. 8 show the result of confirming the amino acid sequence of the mutant site by analyzing the nucleotide sequence of the follidase A252R mutant and the pyrrolidase P365R mutant finally prepared according to an embodiment of the present invention will be.

As a result, as shown in FIG. 7, the fibrillase A252R mutant was analyzed by sequencing to find that the nucleotide sequence GCC corresponding to alanine (alanine, Ala) which is the 252th amino acid corresponds to the nucleotide sequence of arginine CGC was replaced with CGC.

Also, as shown in FIG. 8, the follidase P365R mutant has a base sequence CGT corresponding to proline (Pro), which is the 365th amino acid when nucleotide sequence analysis is carried out, with a base sequence of arginine (Arg) Was substituted with CCT corresponding to < RTI ID = 0.0 >

9-11 are graphs depicting the effect of the follidase mutant expressed according to one embodiment of the present invention on the whole cell lysate, soluble protein and insoluble protein in 10% SDS-PAGE electrophoresis, followed by staining of the gel through coomassie brilliant blue (CBB) staining. At this time, the expected protein size of the follidase mutant of the present invention is about 58 kDa.

Expression of follidase in E. coli wild-type pradolase and follidase A252R mutants and follidase P365R mutants resulted in a protein expression band between 45 kD and 60 kDa of the protein marker (M) Was observed, confirming that the protein size was the same as the expected protein size of about 58 kDa.

In order to select the optimal temperature for soluble protein expression in purified protein separation, soluble protein expression patterns were observed while the culture temperature of E. coli was changed from 37 ° C to 20 ° C.

As shown in FIG. 9 and FIG. 10, when the culture temperature gradually decreased from 37 ° C. to 20 ° C. in the wild-type follidase and follidase A252R mutant-expressing E. coli, the follicle expressed by induction of IPTG expression The total amount of the aze protein did not change much, but the supernatant showed that the amount of soluble protein was increased and the amount of insoluble protein was decreased by looking at the pellet.

As shown in FIG. 11, when the culture temperature was gradually decreased from 37 ° C to 20 ° C during the expression of the follidase P365R mutant protein, the amount of soluble protein was increased but the amount of insoluble protein was similar.

These results indicate that the solubility of the wild-type follidase, the follidase A252R mutant, and the follidase P365R mutant at 20 ° C induction temperature is the highest.

FIG. 12 is a graph showing the results of protein quantification after completing purification of a target protein according to an embodiment of the present invention. The result was separated by performing 10% SDS-PAGE electrophoresis, followed by staining with Coomassie brilliant blue (CBB) Is a gel image.

As a result, the yield of protein production of E. coli against wild type pravolidase and mutants was confirmed to be 14 mg / L. The size of the purified protein was in agreement with the expected 58 kDa of the expression vector, and the purity was 99% or more based on the Coomassie Brilliant Blue (CBB) staining.

13 shows the result of analysis using an anti-His antibody in order to confirm the purified protein by Western blot according to an embodiment of the present invention. As a result, The western blot band was observed at the same 58 kDa position observed in Brilliant Blue (CBB) staining, confirming that the expected protein purification was performed correctly.

The following Table 1, FIG. 14 and FIG. 15 show the enzymatic activity of the neurokinetic agent-like substance DFP using the fulleridase A252R mutant protein and the fulleridase P365R mutant protein purified according to one embodiment of the present invention .

V _max
(U / mg) Km (mM) Kcat / Km
^{(× 10 6 M - 1 min} -1) Fold increase WT 60.1 0.90 3.93 A252R 34.8 0.40 4.93 1.25 P365R 92.2 1.05 5.28 1.34

As a result, when the enzymatic activity against DFP was measured using the fulleridase A252R mutation, the Vmax value was 34.8 U / mg and the Km value was 0.40 mM, and the enzyme for DFP was detected using the Pyridase P365R mutation When the activity was measured, the Vmax value was 92.2 U / mg and the Km value was 1.05 mM.

Using the values of Vmax and Km values, the Kcat / Km value indicating the enzyme activity of the A252R mutant protein of follidase was found to be 4.93 × 10 ⁶ M ^{- 1} min ^{- 1} , The Kcat / Km value of the mutant protein of the dAze P365R was 5.28 × 10 ⁶ M ^{- 1} min ^-1 .

 In the present invention, the activity of the wild-type pyrrolidase enzyme isolated and purified is 60.1 U / mg and the Km value is 0.90 mM, indicating that when the His-tagged follidase was isolated from E. coli, 51.2 U / mg, and the Km value was 2.7 mM. The reason why the enzymatic activity of the wild type pradolase is different from that of the wild type pradolase according to this embodiment is that the pradolase used in the conventional studies is isotype- And the frolidase used in the present invention is a skin isolated from the skin because there are 10 amino acid residues difference between two isotypes.

These results indicate that Kcat / Km, which indicates the enzyme activity of the fibrillase A252R mutation, is an increased value of 1.25-fold enzyme activity relative to the wild-type follidase, and Kcat / Km of the follidase P365R mutation is the wild- This is an increase of 1.34 times compared to daika.

As described above, the follidase mutant according to the present embodiment is expected to have increased enzyme activity through amino acid substitution of the enzyme active site in structural analysis through in silico analysis of follidase The enzyme activity was measured by selecting mutants.

As a result, it was predicted that the substitution of arginine (arginine, Arg) for alanine (alanine, Ala), which is the 252th amino acid of the enzyme, increased the stability of the enzyme and the predicted follidase A252R mutant was prepared When the enzyme activity was measured, the Kcat / Km value was 1.25 times higher than that of the wild type flaredase.

The substitution of arginine (Arg) for 365th proline (Pro) with another mutant was predicted to increase the affinity for the substrate. In fact, the Pyridase P365R mutant The Kcat / Km value was 1.34 times higher than that of the wild type follidase when the enzyme activity was measured. We also observed a decrease in the solubility of proteins in the P365R mutation, which suggests that proline (Pro), the 365th amino acid of follidase, is important for protein structure formation do.

In conclusion, in the present invention, a follidase A252R mutant and a follidase P365R mutant were prepared by in silico analysis of a human-derived pravolidase database in which the enzyme activity was increased. It was confirmed that the enzyme activity of the prepared follidase A252R mutant and the follidase P365R mutant was increased compared to the wild type follidase. As a result, the pesticide A252R mutant and the follidase P365R mutant can be used as an antidote to the neural agonist, and can be applied as a novel biocatalyst preparation capable of mass production using E. coli.

It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. The scope of the present invention is not limited thereto. It will be apparent to those skilled in the art that various substitutions, modifications and variations are possible within the scope of the present invention, and it is obvious that those parts easily changeable by those skilled in the art are included in the scope of the present invention .

It is to be noted, however, that the technical terms used in the present invention are used only for explaining the present invention, and are not intended to limit the present invention. Also, the technical terms used in the present invention should be construed in a sense generally understood by a person having ordinary skill in the art to which the present invention belongs, unless otherwise defined in the present invention, and an overly comprehensive It should not be construed as a meaning or an overly reduced meaning. In addition, the general terms used in the present invention should be construed in accordance with the predefined or prior context.

<110> AGENCY FOR DEFENSE DEVELOPMENT <120> PROLIDASE MUTANT, AND PREPARATION METHOD AND USE THEREOF <130> AD160069 <160> 18 <170> Kopatentin 2.0 <210> 1 <211> 6915 <212> DNA <213> Artificial Sequence <220> <223> pET21b-prolidase-A252R <400> 1 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta 3960 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500 gcggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc 5160 cctctagaaa taattttgtt taactttaag aaggagatat acatatggct agcatgactg 5220 gtggacagca aatgggtcgg gatccgaatt cgagctccgt cgacaagctt accatggcgg 5280 cggccaccgg accctcgttt tggctgggga atgaaaccct gaaggtgccg ctggcgctct 5340 ttgccttgaa ccggcagcgc ctgtgtgagc ggctgcggaa gaaccctgct gtgcaggccg 5400 gctccatcgt ggtcctgcag ggcggggagg agactcagcg ctactgcacc gacaccgggg 5460 tcctcttccg ccaggagtcc ttctttcact gggcgttcgg tgtcactgag ccaggctgct 5520 atggtgtcat cgatgttgac actgggaagt cgaccctgtt tgtgcccagg cttcctgcca 5580 gccatgccac ctggatggga aagatccatt ccaaggagca cttcaaggag aagtatgccg 5640 tggacgacgt ccagtacgta gatgagattg ccagcgtcct gacgtcacag aagccctctg 5700 tcctcctcac tttgcgtggc gtcaacacgg acagcggcag tgtctgcagg gaggcctcct 5760 ttgacggcat cagcaagttc gaagtcaaca ataccattct tcacccagag atcgttgagt 5820 gccgagtgtt taagacggat atggagctgg aggttctgcg ctataccaat aaaatctcca 5880 gcgaggccca ccgtgaggta atgaaggctg taaaagtggg aatgaaagaa tatgagttgg 5940 aaagcctctt cgagcactac tgctactccc ggggcggcat gcgccacagc tcctacacct 6000 gcatctgcgg cagtggtgag aactcacgcg tgctacacta cggacacgcc ggagctccca 6060 acgaccgaac gatccagaat ggggatatgt gcctgttcga catgggcggt gagtattact 6120 gcttcgcttc cgacatcacc tgctcctttc ccgccaacgg caagttcact gcagaccaga 6180 aggccgtcta tgaggcagtg ctgcggagct cccgtgccgt catgggtgcc atgaagccag 6240 gtgtctggtg gcctgacatg caccgcctgg ctgaccgcat ccacctggag gagctggccc 6300 acatgggcat cctgagcggc agcgtggacg ccatggtcca ggctcacctg ggggccgtgt 6360 ttatgcctca cgggcttggc cacttcctgg gcattgacgt gcacgacgtg ggaggctacc 6420 cagagggcgt ggagcgcatc gacgagcccg gcctgcggag cctgcgcact gcacggcacc 6480 tgcagccagg catggtgctc accgtggagc cgggcatcta cttcatcgac cacctcctgg 6540 atgaggccct ggcggacccg gcccgcgcct ccttccttaa ccgcgaggtc ctgcagcgct 6600 ttcgcggttt tggcggggtc cgcatcgagg aggacgtcgt ggtgactgac agcggcatag 6660 agctgctgac ctgcgtgccc cgcactgtgg aagagattga agcatgcatg gcaggctgtg 6720 acaaggcctt tacccccttc tctggcccca agctcgagca ccaccaccac caccactgag 6780 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat 6840 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag 6900 gaactatatc cggat 6915 <210> 2 <211> 6915 <212> DNA <213> Artificial Sequence <220> <223> pET21b-prolidase-P365R <400> 2 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140 tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520 cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700 catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760 tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820 ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880 tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940 ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000 aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060 gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120 tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180 acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240 cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300 cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360 gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420 cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480 gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540 tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600 atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720 gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780 tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840 cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900 tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta 3960 atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020 atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080 tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140 cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200 aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260 ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320 tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380 tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440 gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500 gcggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560 gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620 ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680 taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740 ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800 atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860 tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920 gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980 gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040 aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100 ctcgatcccg cgaaattaat acgactcact ataggggaat tgtgagcgga taacaattcc 5160 cctctagaaa taattttgtt taactttaag aaggagatat acatatggct agcatgactg 5220 gtggacagca aatgggtcgg gatccgaatt cgagctccgt cgacaagctt accatggcgg 5280 cggccaccgg accctcgttt tggctgggga atgaaaccct gaaggtgccg ctggcgctct 5340 ttgccttgaa ccggcagcgc ctgtgtgagc ggctgcggaa gaaccctgct gtgcaggccg 5400 gctccatcgt ggtcctgcag ggcggggagg agactcagcg ctactgcacc gacaccgggg 5460 tcctcttccg ccaggagtcc ttctttcact gggcgttcgg tgtcactgag ccaggctgct 5520 atggtgtcat cgatgttgac actgggaagt cgaccctgtt tgtgcccagg cttcctgcca 5580 gccatgccac ctggatggga aagatccatt ccaaggagca cttcaaggag aagtatgccg 5640 tggacgacgt ccagtacgta gatgagattg ccagcgtcct gacgtcacag aagccctctg 5700 tcctcctcac tttgcgtggc gtcaacacgg acagcggcag tgtctgcagg gaggcctcct 5760 ttgacggcat cagcaagttc gaagtcaaca ataccattct tcacccagag atcgttgagt 5820 gccgagtgtt taagacggat atggagctgg aggttctgcg ctataccaat aaaatctcca 5880 gcgaggccca ccgtgaggta atgaaggctg taaaagtggg aatgaaagaa tatgagttgg 5940 aaagcctctt cgagcactac tgctactccc ggggcggcat gcgccacagc tcctacacct 6000 gcatctgcgg cagtggtgag aactcacgcg tgctacacta cggacacgcc ggagctccca 6060 acgaccgaac gatccagaat ggggatatgt gcctgttcga catgggcggt gagtattact 6120 gcttcgcttc cgacatcacc tgctcctttc ccgccaacgg caagttcact gcagaccaga 6180 aggccgtcta tgaggcagtg ctgcggagct cccgtgccgt catgggtgcc atgaagccag 6240 gtgtctggtg gcctgacatg caccgcctgg ctgaccgcat ccacctggag gagctggccc 6300 acatgggcat cctgagcggc agcgtggacg ccatggtcca ggctcacctg ggggccgtgt 6360 ttatgcgtca cgggcttggc cacttcctgg gcattgacgt gcacgacgtg ggaggctacc 6420 cagagggcgt ggagcgcatc gacgagcccg gcctgcggag cctgcgcact gcacggcacc 6480 tgcagccagg catggtgctc accgtggagc cgggcatcta cttcatcgac cacctcctgg 6540 atgaggccct ggcggacccg gcccgcgcct ccttccttaa ccgcgaggtc ctgcagcgct 6600 ttcgcggttt tggcggggtc cgcatcgagg aggacgtcgt ggtgactgac agcggcatag 6660 agctgctgac ctgcgtgccc cgcactgtgg aagagattga agcatgcatg gcaggctgtg 6720 acaaggcctt tacccccttc tctggcccca agctcgagca ccaccaccac caccactgag 6780 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat 6840 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag 6900 gaactatatc cggat 6915 <210> 3 <211> 1479 <212> DNA <213> Artificial Sequence <220> <223> prolidase <400> 3 atggcggcgg ccaccggacc ctcgttttgg ctggggaatg aaaccctgaa ggtgccgctg 60 gcgctctttg ccttgaaccg gcagcgcctg tgtgagcggc tgcggaagaa ccctgctgtg 120 caggccggct ccatcgtggt cctgcagggc ggggaggaga ctcagcgcta ctgcaccgac 180 accggggtcc tcttccgcca ggagtccttc tttcactggg cgttcggtgt cactgagcca 240 ggctgctatg gtgtcatcga tgttgacact gggaagtcga ccctgtttgt gcccaggctt 300 cctgccagcc atgccacctg gatgggaaag atccattcca aggagcactt caaggagaag 360 tatgccgtgg acgacgtcca gtacgtagat gagattgcca gcgtcctgac gtcacagaag 420 ccctctgtcc tcctcacttt gcgtggcgtc aacacggaca gcggcagtgt ctgcagggag 480 gcctcctttg acggcatcag caagttcgaa gtcaacaata ccattcttca cccagagatc 540 gttgagtgcc gagtgtttaa gacggatatg gagctggagg ttctgcgcta taccaataaa 600 atctccagcg aggcccaccg tgaggtaatg aaggctgtaa aagtgggaat gaaagaatat 660 gagttggaaa gcctcttcga gcactactgc tactcccggg gcggcatgcg ccacagctcc 720 tacacctgca tctgcggcag tggtgagaac tcacgcgtgc tacactacgg acacgccgga 780 gctcccaacg accgaacgat ccagaatggg gatatgtgcc tgttcgacat gggcggtgag 840 tattactgct tcgcttccga catcacctgc tcctttcccg ccaacggcaa gttcactgca 900 gccgaagg ccgtctatga ggcagtgctg cggagctccc gtgccgtcat gggtgccatg 960 aagccaggtg tctggtggcc tgacatgcac cgcctggctg accgcatcca cctggaggag 1020 ctggcccaca tgggcatcct gagcggcagc gtggacgcca tggtccaggc tcacctgggg 1080 gccgtgttta tgcctcacgg gcttggccac ttcctgggca ttgacgtgca cgacgtggga 1140 ggctacccag agggcgtgga gcgcatcgac gagcccggcc tgcggagcct gcgcactgca 1200 cggcacctgc agccaggcat ggtgctcacc gtggagccgg gcatctactt catcgaccac 1260 ctcctggatg aggccctggc ggacccggcc cgcgcctcct tccttaaccg cgaggtcctg 1320 cagcgctttc gcggttttgg cggggtccgc atcgaggagg acgtcgtggt gactgacagc 1380 ggcatagagc tgctgacctg cgtgccccgc actgtggaag agattgaagc atgcatggca 1440 ggctgtgaca aggcctttac ccccttctct ggccccaag 1479 <210> 4 <211> 1479 <212> DNA <213> Artificial Sequence <220> <223> Prolidase-A252R <400> 4 atggcggcgg ccaccggacc ctcgttttgg ctggggaatg aaaccctgaa ggtgccgctg 60 gcgctctttg ccttgaaccg gcagcgcctg tgtgagcggc tgcggaagaa ccctgctgtg 120 caggccggct ccatcgtggt cctgcagggc ggggaggaga ctcagcgcta ctgcaccgac 180 accggggtcc tcttccgcca ggagtccttc tttcactggg cgttcggtgt cactgagcca 240 ggctgctatg gtgtcatcga tgttgacact gggaagtcga ccctgtttgt gcccaggctt 300 cctgccagcc atgccacctg gatgggaaag atccattcca aggagcactt caaggagaag 360 tatgccgtgg acgacgtcca gtacgtagat gagattgcca gcgtcctgac gtcacagaag 420 ccctctgtcc tcctcacttt gcgtggcgtc aacacggaca gcggcagtgt ctgcagggag 480 gcctcctttg acggcatcag caagttcgaa gtcaacaata ccattcttca cccagagatc 540 gttgagtgcc gagtgtttaa gacggatatg gagctggagg ttctgcgcta taccaataaa 600 atctccagcg aggcccaccg tgaggtaatg aaggctgtaa aagtgggaat gaaagaatat 660 gagttggaaa gcctcttcga gcactactgc tactcccggg gcggcatgcg ccacagctcc 720 tacacctgca tctgcggcag tggtgagaac tcacgcgtgc tacactacgg acacgccgga 780 gctcccaacg accgaacgat ccagaatggg gatatgtgcc tgttcgacat gggcggtgag 840 tattactgct tcgcttccga catcacctgc tcctttcccg ccaacggcaa gttcactgca 900 gccgaagg ccgtctatga ggcagtgctg cggagctccc gtgccgtcat gggtgccatg 960 aagccaggtg tctggtggcc tgacatgcac cgcctggctg accgcatcca cctggaggag 1020 ctggcccaca tgggcatcct gagcggcagc gtggacgcca tggtccaggc tcacctgggg 1080 gccgtgttta tgcctcacgg gcttggccac ttcctgggca ttgacgtgca cgacgtggga 1140 ggctacccag agggcgtgga gcgcatcgac gagcccggcc tgcggagcct gcgcactgca 1200 cggcacctgc agccaggcat ggtgctcacc gtggagccgg gcatctactt catcgaccac 1260 ctcctggatg aggccctggc ggacccggcc cgcgcctcct tccttaaccg cgaggtcctg 1320 cagcgctttc gcggttttgg cggggtccgc atcgaggagg acgtcgtggt gactgacagc 1380 ggcatagagc tgctgacctg cgtgccccgc actgtggaag agattgaagc atgcatggca 1440 ggctgtgaca aggcctttac ccccttctct ggccccaag 1479 <210> 5 <211> 1479 <212> DNA <213> Artificial Sequence <220> <223> Prolidase-P365R <400> 5 atggcggcgg ccaccggacc ctcgttttgg ctggggaatg aaaccctgaa ggtgccgctg 60 gcgctctttg ccttgaaccg gcagcgcctg tgtgagcggc tgcggaagaa ccctgctgtg 120 caggccggct ccatcgtggt cctgcagggc ggggaggaga ctcagcgcta ctgcaccgac 180 accggggtcc tcttccgcca ggagtccttc tttcactggg cgttcggtgt cactgagcca 240 ggctgctatg gtgtcatcga tgttgacact gggaagtcga ccctgtttgt gcccaggctt 300 cctgccagcc atgccacctg gatgggaaag atccattcca aggagcactt caaggagaag 360 tatgccgtgg acgacgtcca gtacgtagat gagattgcca gcgtcctgac gtcacagaag 420 ccctctgtcc tcctcacttt gcgtggcgtc aacacggaca gcggcagtgt ctgcagggag 480 gcctcctttg acggcatcag caagttcgaa gtcaacaata ccattcttca cccagagatc 540 gttgagtgcc gagtgtttaa gacggatatg gagctggagg ttctgcgcta taccaataaa 600 atctccagcg aggcccaccg tgaggtaatg aaggctgtaa aagtgggaat gaaagaatat 660 gagttggaaa gcctcttcga gcactactgc tactcccggg gcggcatgcg ccacagctcc 720 tacacctgca tctgcggcag tggtgagaac tcacgcgtgc tacactacgg acacgccgga 780 gctcccaacg accgaacgat ccagaatggg gatatgtgcc tgttcgacat gggcggtgag 840 tattactgct tcgcttccga catcacctgc tcctttcccg ccaacggcaa gttcactgca 900 gccgaagg ccgtctatga ggcagtgctg cggagctccc gtgccgtcat gggtgccatg 960 aagccaggtg tctggtggcc tgacatgcac cgcctggctg accgcatcca cctggaggag 1020 ctggcccaca tgggcatcct gagcggcagc gtggacgcca tggtccaggc tcacctgggg 1080 gccgtgttta tgcgtcacgg gcttggccac ttcctgggca ttgacgtgca cgacgtggga 1140 ggctacccag agggcgtgga gcgcatcgac gagcccggcc tgcggagcct gcgcactgca 1200 cggcacctgc agccaggcat ggtgctcacc gtggagccgg gcatctactt catcgaccac 1260 ctcctggatg aggccctggc ggacccggcc cgcgcctcct tccttaaccg cgaggtcctg 1320 cagcgctttc gcggttttgg cggggtccgc atcgaggagg acgtcgtggt gactgacagc 1380 ggcatagagc tgctgacctg cgtgccccgc actgtggaag agattgaagc atgcatggca 1440 ggctgtgaca aggcctttac ccccttctct ggccccaag 1479 <210> 6 <211> 493 <212> PRT <213> Artificial Sequence <220> <223> Prolidase <400> 6 Met Ala Ala Ala Thr Gly Pro Ser Phe Trp Leu Gly Asn Glu Thr Leu   1 5 10 15 Lys Val Pro Leu Ala Leu Phe Ala Leu Asn Arg Gln Arg Leu Cys Glu              20 25 30 Arg Leu Arg Lys Asn Pro Ala Val Gln Ala Gly Ser Ile Val Val Leu          35 40 45 Gln Gly Gly Glu Glu Thr Gln Arg Tyr Cys Thr Asp Thr Gly Val Leu      50 55 60 Phe Leu Gln Glu Ser Phe Phe His Trp Ala Phe Gly Val Thr Glu Pro  65 70 75 80 Gly Cys Tyr Gly Val Ile Asp Val Asp Thr Gly Lys Ser Thr Leu Phe                  85 90 95 Val Pro Arg Leu Pro Ala Ser His Ala Thr Trp Met Gly Lys Ile His             100 105 110 Ser Lys Glu His Phe Lys Glu Lys Tyr Ala Val Asp Asp Val Gln Tyr         115 120 125 Val Asp Glu Ile Ala Ser Val Leu Thr Ser Gln Lys Pro Ser Val Leu     130 135 140 Leu Thr Leu Arg Gly Val Asn Thr Asp Ser Gly Ser Val Cys Arg Glu 145 150 155 160 Ala Ser Phe Asp Gly Ile Ser Lys Phe Glu Val Asn Asn Thr Ile Leu                 165 170 175 His Pro Glu Ile Val Glu Ser Arg Val Phe Lys Thr Asp Met Glu Leu             180 185 190 Glu Val Leu Arg Tyr Thr Asn Lys Ile Ser Ser Glu Ala His Arg Glu         195 200 205 Val Met Lys Ala Val Lys Val Gly Met Lys Glu Tyr Gly Leu Glu Ser     210 215 220 Leu Phe Glu His Tyr Cys Tyr Ser Arg Gly Gly Met Arg His Ser Ser 225 230 235 240 Tyr Thr Cys Ile Cys Gly Ser Gly Glu Asn Ser Ala Val Leu His Tyr                 245 250 255 Gly His Ala Gly Ala Pro Asn Asp Arg Thr Ile Gln Asn Gly Asp Met             260 265 270 Cys Leu Phe Asp Met Gly Gly Glu Tyr Tyr Ser Val Ala Ser Asp Ile         275 280 285 Thr Cys Ser Phe Pro Arg Asn Gly Lys Phe Thr Ala Asp Gln Lys Ala     290 295 300 Val Tyr Glu Ala Val Leu Leu Ser Ser Arg Ala Val Met Gly Ala Met 305 310 315 320 Lys Pro Gly Asp Trp Trp Pro Asp Ile Asp Arg Leu Ala Asp Arg Ile                 325 330 335 His Leu Glu Glu Leu Ala His Met Gly Ile Leu Ser Gly Ser Val Asp             340 345 350 Ala Met Val Gln Ala His Leu Gly Ala Val Phe Met Pro His Gly Leu         355 360 365 Gly His Phe Leu Gly Ile Asp Val His Asp Val Gly Gly Tyr Pro Glu     370 375 380 Gly Val Glu Arg Ile Asp Glu Pro Gly Leu Arg Ser Leu Arg Thr Ala 385 390 395 400 Arg His Leu Gln Pro Gly Met Val Leu Thr Val Glu Pro Gly Ile Tyr                 405 410 415 Phe Ile Asp His Leu Leu Asp Glu Ala Leu Ala Asp Pro Ala Arg Ala             420 425 430 Ser Phe Leu Asn Arg Glu Val Leu Gln Arg Phe Arg Gly Phe Gly Gly         435 440 445 Val Arg Ile Glu Glu Asp Val Val Valle Asp Ser Gly Ile Glu Leu     450 455 460 Leu Thr Cys Val Pro Arg Thr Val Glu Glu Ile Glu Ala Cys Met Ala 465 470 475 480 Gly Cys Asp Lys Ala Phe Thr Pro Phe Ser Gly Pro Lys                 485 490 <210> 7 <211> 493 <212> PRT <213> Artificial Sequence <220> <223> Prolidase-A252R <400> 7 Met Ala Ala Ala Thr Gly Pro Ser Phe Trp Leu Gly Asn Glu Thr Leu   1 5 10 15 Lys Val Pro Leu Ala Leu Phe Ala Leu Asn Arg Gln Arg Leu Cys Glu              20 25 30 Arg Leu Arg Lys Asn Pro Ala Val Gln Ala Gly Ser Ile Val Val Leu          35 40 45 Gln Gly Gly Glu Glu Thr Gln Arg Tyr Cys Thr Asp Thr Gly Val Leu      50 55 60 Phe Leu Gln Glu Ser Phe Phe His Trp Ala Phe Gly Val Thr Glu Pro  65 70 75 80 Gly Cys Tyr Gly Val Ile Asp Val Asp Thr Gly Lys Ser Thr Leu Phe                  85 90 95 Val Pro Arg Leu Pro Ala Ser His Ala Thr Trp Met Gly Lys Ile His             100 105 110 Ser Lys Glu His Phe Lys Glu Lys Tyr Ala Val Asp Asp Val Gln Tyr         115 120 125 Val Asp Glu Ile Ala Ser Val Leu Thr Ser Gln Lys Pro Ser Val Leu     130 135 140 Leu Thr Leu Arg Gly Val Asn Thr Asp Ser Gly Ser Val Cys Arg Glu 145 150 155 160 Ala Ser Phe Asp Gly Ile Ser Lys Phe Glu Val Asn Asn Thr Ile Leu                 165 170 175 His Pro Glu Ile Val Glu Ser Arg Val Phe Lys Thr Asp Met Glu Leu             180 185 190 Glu Val Leu Arg Tyr Thr Asn Lys Ile Ser Ser Glu Ala His Arg Glu         195 200 205 Val Met Lys Ala Val Lys Val Gly Met Lys Glu Tyr Gly Leu Glu Ser     210 215 220 Leu Phe Glu His Tyr Cys Tyr Ser Arg Gly Gly Met Arg His Ser Ser 225 230 235 240 Tyr Thr Cys Ile Cys Gly Ser Gly Glu Asn Ser Arg Val Leu His Tyr                 245 250 255 Gly His Ala Gly Ala Pro Asn Asp Arg Thr Ile Gln Asn Gly Asp Met             260 265 270 Cys Leu Phe Asp Met Gly Gly Glu Tyr Tyr Ser Val Ala Ser Asp Ile         275 280 285 Thr Cys Ser Phe Pro Arg Asn Gly Lys Phe Thr Ala Asp Gln Lys Ala     290 295 300 Val Tyr Glu Ala Val Leu Leu Ser Ser Arg Ala Val Met Gly Ala Met 305 310 315 320 Lys Pro Gly Asp Trp Trp Pro Asp Ile Asp Arg Leu Ala Asp Arg Ile                 325 330 335 His Leu Glu Glu Leu Ala His Met Gly Ile Leu Ser Gly Ser Val Asp             340 345 350 Ala Met Val Gln Ala His Leu Gly Ala Val Phe Met Pro His Gly Leu         355 360 365 Gly His Phe Leu Gly Ile Asp Val His Asp Val Gly Gly Tyr Pro Glu     370 375 380 Gly Val Glu Arg Ile Asp Glu Pro Gly Leu Arg Ser Leu Arg Thr Ala 385 390 395 400 Arg His Leu Gln Pro Gly Met Val Leu Thr Val Glu Pro Gly Ile Tyr                 405 410 415 Phe Ile Asp His Leu Leu Asp Glu Ala Leu Ala Asp Pro Ala Arg Ala             420 425 430 Ser Phe Leu Asn Arg Glu Val Leu Gln Arg Phe Arg Gly Phe Gly Gly         435 440 445 Val Arg Ile Glu Glu Asp Val Val Valle Asp Ser Gly Ile Glu Leu     450 455 460 Leu Thr Cys Val Pro Arg Thr Val Glu Glu Ile Glu Ala Cys Met Ala 465 470 475 480 Gly Cys Asp Lys Ala Phe Thr Pro Phe Ser Gly Pro Lys                 485 490 <210> 8 <211> 493 <212> PRT <213> Artificial Sequence <220> <223> Prolidase-P365R <400> 8 Met Ala Ala Ala Thr Gly Pro Ser Phe Trp Leu Gly Asn Glu Thr Leu   1 5 10 15 Lys Val Pro Leu Ala Leu Phe Ala Leu Asn Arg Gln Arg Leu Cys Glu              20 25 30 Arg Leu Arg Lys Asn Pro Ala Val Gln Ala Gly Ser Ile Val Val Leu          35 40 45 Gln Gly Gly Glu Glu Thr Gln Arg Tyr Cys Thr Asp Thr Gly Val Leu      50 55 60 Phe Leu Gln Glu Ser Phe Phe His Trp Ala Phe Gly Val Thr Glu Pro  65 70 75 80 Gly Cys Tyr Gly Val Ile Asp Val Asp Thr Gly Lys Ser Thr Leu Phe                  85 90 95 Val Pro Arg Leu Pro Ala Ser His Ala Thr Trp Met Gly Lys Ile His             100 105 110 Ser Lys Glu His Phe Lys Glu Lys Tyr Ala Val Asp Asp Val Gln Tyr         115 120 125 Val Asp Glu Ile Ala Ser Val Leu Thr Ser Gln Lys Pro Ser Val Leu     130 135 140 Leu Thr Leu Arg Gly Val Asn Thr Asp Ser Gly Ser Val Cys Arg Glu 145 150 155 160 Ala Ser Phe Asp Gly Ile Ser Lys Phe Glu Val Asn Asn Thr Ile Leu                 165 170 175 His Pro Glu Ile Val Glu Ser Arg Val Phe Lys Thr Asp Met Glu Leu             180 185 190 Glu Val Leu Arg Tyr Thr Asn Lys Ile Ser Ser Glu Ala His Arg Glu         195 200 205 Val Met Lys Ala Val Lys Val Gly Met Lys Glu Tyr Gly Leu Glu Ser     210 215 220 Leu Phe Glu His Tyr Cys Tyr Ser Arg Gly Gly Met Arg His Ser Ser 225 230 235 240 Tyr Thr Cys Ile Cys Gly Ser Gly Glu Asn Ser Ala Val Leu His Tyr                 245 250 255 Gly His Ala Gly Ala Pro Asn Asp Arg Thr Ile Gln Asn Gly Asp Met             260 265 270 Cys Leu Phe Asp Met Gly Gly Glu Tyr Tyr Ser Val Ala Ser Asp Ile         275 280 285 Thr Cys Ser Phe Pro Arg Asn Gly Lys Phe Thr Ala Asp Gln Lys Ala     290 295 300 Val Tyr Glu Ala Val Leu Leu Ser Ser Arg Ala Val Met Gly Ala Met 305 310 315 320 Lys Pro Gly Asp Trp Trp Pro Asp Ile Asp Arg Leu Ala Asp Arg Ile                 325 330 335 His Leu Glu Glu Leu Ala His Met Gly Ile Leu Ser Gly Ser Val Asp             340 345 350 Ala Met Val Gln Ala His Leu Gly Ala Val Phe Met Arg His Gly Leu         355 360 365 Gly His Phe Leu Gly Ile Asp Val His Asp Val Gly Gly Tyr Pro Glu     370 375 380 Gly Val Glu Arg Ile Asp Glu Pro Gly Leu Arg Ser Leu Arg Thr Ala 385 390 395 400 Arg His Leu Gln Pro Gly Met Val Leu Thr Val Glu Pro Gly Ile Tyr                 405 410 415 Phe Ile Asp His Leu Leu Asp Glu Ala Leu Ala Asp Pro Ala Arg Ala             420 425 430 Ser Phe Leu Asn Arg Glu Val Leu Gln Arg Phe Arg Gly Phe Gly Gly         435 440 445 Val Arg Ile Glu Glu Asp Val Val Valle Asp Ser Gly Ile Glu Leu     450 455 460 Leu Thr Cys Val Pro Arg Thr Val Glu Glu Ile Glu Ala Cys Met Ala 465 470 475 480 Gly Cys Asp Lys Ala Phe Thr Pro Phe Ser Gly Pro Lys                 485 490 <210> 9 <211> 861 <212> DNA <213> Artificial Sequence <220> <223> ampicillin resistance gene <400> 9 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540 cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840 tcactgatta agcattggta a 861 <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> T7 promoter <400> 10 taatacgact cactatagg 19 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> T7 tag coding sequence <400> 11 gctagcatga ctggtggaca gcaaatgggt 30 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> His tag coding sequence <400> 12 caccaccacc accaccac 18 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> forward primer 1 <400> 13 aagcttacca tggcggcggc caccggacc 29 <210> 14 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 1 <400> 14 ctcgagcttg gggccagaga aggggg 26 <210> 15 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> forward primer 2 <400> 15 cagtggtgag aactcacgcg tgctacacta cggac 35 <210> 16 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 2 <400> 16 gtccgtagtg tagcacgcgt gagttctcac cactg 35 <210> 17 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> forward primer 3 <400> 17 gggggccgtg tttatgcgtc acgggcttgg ccact 35 <210> 18 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> reverse primer 3 <400> 18 agtggccaag cccgtgacgc ataaacacgg ccccc 35

Claims (9)

A follidase having an amino acid sequence in which the 252nd amino acid or the 365th amino acid from the N-terminal is substituted with arginine in the amino acid sequence of human-derived pyrrolidase represented by the amino acid sequence of SEQ ID NO: 6 Mutant. The method according to claim 1,
Wherein said follidase mutant comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. A follidase mutant gene encoding the follidase mutant of claim 1 or 2. The method of claim 3,
Wherein the follidase mutant gene comprises the nucleotide sequence of SEQ ID NO: 4 or SEQ ID NO: 5. A recombinant expression vector, characterized by comprising the polyhydrolase mutant gene of claim 3. Transforming the recombinant expression vector of claim 5 into prokaryotic cells;
Culturing the transformed prokaryotic cell transformant; And
And isolating and purifying a target protein expressed from the transformant, which is a follidase, and purifying the protein. The method according to claim 6,
Wherein the prokaryotic cell is E. coli. A composition for inhibiting nerve agent activity inhibition comprising the pyrrolidase mutant of claim 1 or 2 as an active ingredient. A pharmaceutical composition for preventing or treating a disease caused by exposure to a nerve agent comprising the pyrrolidase mutant of claim 1 or 2 as an active ingredient.

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