KR101103022B1 - Organophosphorus Hydrolase Variants and Method for Preparing the Same - Google Patents

Abstract

The present invention relates to an organophosphorus hydrolase (OPH) variant having increased organophosphate compound resolution and a method for preparing the same, and more particularly, to an organophosphate hydrolase selected through an aromatic molecular evolution technique. It relates to a variant, a method for producing the same and a biosensor for detecting an organic phosphate compound comprising the same.

The organophosphate compound hydrolase variant according to the present invention has an excellent hydrolytic activity of organophosphate compounds compared to the conventional OPH, and thus can be applied to environmental and biosensors such as the reaction to decompose organophosphate compounds and to detect organophosphate compounds. It can be used for various industrial purposes such as detection of residual pesticides.

Organophosphate Hydrolase, Organophosphate Pesticide, Screening, Enzyme Improvement, Biosensor

Description

Organophosphate Hydrolase Variants and Method for Preparing the Same {Organophosphorus Hydrolase Variants and Method for Preparing the Same}

The present invention relates to an organophosphorus hydrolase variant having increased organophosphate compound resolution and a method of preparing the same, and more particularly, to an organophosphate compound hydrolase variant selected through aromatic molecular evolution, It relates to a manufacturing method and a biosensor for detecting an organic phosphate compound comprising the same.

OPH is one of the para oxone (paraoxon) in a ρ -nitrophenol and diethyl phosphate, and the hydrolysis of the reaction solution pH decrease occurs, the reaction of the acid organophosphorus pesticide and electron (e ^-) is generated for. The ρ- nitrophenol produced in this process shows yellow coloration. The color reaction can be analyzed by measuring the absorbance at a wavelength of 412 nm. In addition, since the electrons are generated as shown in Equation 1 below, it may be used as a biosensor quantitatively indicating the content of the organophosphate pesticide through electrochemical analysis.

_{Paraoxon + H 2 O → ρ -nitrophenol} + diethyl phosphate + e - ( formula 1)

Organophosphate compounds are the most abundant pesticides currently in use, and more than 100 are marketed for the purpose of pesticides for pest control. Compared with organochlorine, it has less persistence, less risk of residue, stronger insecticides and a wider range of applied pests. However, many drugs are highly toxic to humans and livestock, and because of their low sustainability, they require a relatively large amount of use. Dichlorovos, parathion, malathion, EPN, diazinon, etc. are used as the kinds of compounds.

On the other hand, mutant library generation techniques continue to be developed, such as error-prone PCR (Cadwell and Joyce, Mutagenic PCR, Cold Spring Harbor Laboratory), chemical mutagenesis, UV irradiation, and the use of mutagenic host cells. There are many methods, such as random mutagenesis and combinatorial mutagenesis that cause mutation through recombination such as DNA shuffling (Kuchner and Arnold, Trends Biotech., 15: 523,1997). ). In addition, kits for easily preparing enzyme libraries are commercially available, which researchers can use for their own purposes.

After obtaining gene diversity through enzymatic variant libraries, a technique is needed to search for variants with desired properties. In general, the search technology of the library is based on the resistance to antibiotics, the search for growth due to the complementarity of nutritional strains, the formation of transparent rings on the agar medium, the expression of fluorescent substances, the solid phase selection by colored expression, and the robotic system. There is a wellplate method that can be automated and sensitively recognizes the difference in activity (Olsen et al., Curr. Opin. Biotechnol., 11 (4): 331, 2000).

As a method for searching and selecting OPH variants, 96-well plate-based methods were used to generate products through enzymatic reactions and to find variants with high color development through organophosphate compounds. In the enzymatic reaction, ρ -nitrophenol is produced by the enzyme reaction of OPH. The ρ- nitrophenol produced here is yellow in color.

Accordingly, the present inventors have made diligent efforts to study new methods that can be repeatedly applied to screening and directional evolution of microorganisms for synthesizing OPH, an industrial enzyme used to degrade organophosphate compounds, and which can be useful for improving enzymes. As a result, by developing a method for improving the activity of OPH and using the same for high-speed screening of a variant having excellent activity, an OPH variant having excellent organophosphate compound hydrolytic activity was obtained, thereby completing the present invention.

In order to achieve the above object, the present invention, in the organophosphorus hydrolase having the amino acid sequence of SEQ ID NO: 4, (1) 95th amino acid H (histidine) is substituted with Q (glutamine) (2) The 131th amino acid K (lysine) is substituted with E (glutamic acid), and (3) The 145th amino acid T (threonine) is substituted with A (alanine). Provide degrading enzyme variants.

In the present invention, the OPH variant may be an organophosphate compound hydrolase variant, which further has any one or more of the following substitution characteristics:

(4) the 24th amino acid T (threonine) is substituted with K (lysine); (5) the 126th amino acid I (isoleucine) is substituted with S (serine); (6) the 141th amino acid K (lysine) is substituted with M (methionine); And (7) the 280th amino acid S (serine) is substituted with C (cysteine).

In the present invention, the OPH variant may be an organophosphate compound hydrolase variant, which further has any one or more of the following substitution characteristics:

(8) the 11th amino acid T (threonine) is substituted with A (alanine); (9) the 33rd amino acid S (serine) is substituted with N (asparagine); (10) the 52nd amino acid A (alanine) is substituted with S (serine); (11) 75th amino acid T (threonine) is substituted with S (serine); (12) the 118th amino acid L (leucine) is substituted with F (phenylalanine); (13) 165th amino acid A (alanine) is substituted with V (valine); (14) the 210th amino acid S (serine) is substituted with R (arginine); And (15) 246 th amino acid I (isoleucine) is substituted with N (asparagine).

In the present invention, the OPH variant may be an organophosphate hydrolase variant further having any one or more of the following substitution characteristics:

(16) the 105th amino acid D (aspartic acid) is substituted with V (valine); (17) the 196th amino acid S (serine) is substituted with P (proline); (18) the 199th amino acid C (cysteine) is substituted with G (glycine); And (19) 313rd amino acid V (valine) is substituted with F (phenylalanine).

In the present invention, the OPH variant may be an organophosphate compound hydrolase variant, which further has any one or more of the following substitution characteristics:

(20) seventh amino acid D (aspartic acid) is substituted with N (asparagine); (21) the 21st amino acid A (alanine) is substituted with T (threonine); (22) the 29th amino acid H (histidine) is substituted with V (valine); (23) the 39th amino acid R (arginine) is substituted with L (leucine); (24) the 44th amino acid F (phenylalanine) is substituted with L (leucine); (25) the 46th amino acid G (glycine) is substituted with C (cysteine); (26) the 57th amino acid R (arginine) is substituted with S (serine); (27) the 64 th amino acid A (alanine) is substituted with S (serine); (28) the 106th amino acid P (proline) is substituted with L (leucine); (29) the 122nd amino acid F (phenylalanine) is substituted with L (leucine); (30) the 163th amino acid S (serine) is substituted with R (arginine); (31) the 174th amino acid T (threonine) is substituted for S (serine); (32) the 202th amino acid H (histidine) is substituted with L (leucine); (33) 216th amino acid A (alanine) is substituted with D (aspartic acid); (34) the 236th amino acid D (aspartic acid) is substituted with G (glycine); (35) 248th amino acid S (serine) is substituted with W (tryptophan); (36) the 254th amino acid L (leucine) is substituted with F (phenylalanine); (37) 259th amino acid L (leucine) is substituted with I (isoleucine); (38) 271st amino acid S (serine) is substituted with W (tryptophan); (39) the 280th amino acid S (serine) is substituted with R (arginine); (40) the 286th amino acid M (methionine) is substituted with T (threonine); (41) the 289th amino acid M (methionine) is substituted with L (leucine); (42) the 291th amino acid R (arginine) is substituted with G (glycine); (43) the 295th amino acid D (aspartic acid) is substituted with A (alanine); And (44) 324 th amino acid T (threonine) is substituted with N (asparagine).

The present invention also provides a gene encoding the organophosphate compound hydrolase variant.

The present invention also provides a recombinant vector containing a gene encoding the organophosphate compound hydrolase variant.

The present invention also provides a recombinant microorganism in which the gene or the recombinant vector is inserted into a host cell selected from the group consisting of bacteria, fungi and yeasts.

The present invention also comprises the steps of culturing the recombinant microorganism to express an organophosphate compound hydrolase variant; And it provides a method for producing an organophosphate compound hydrolase variant comprising recovering an organophosphate compound hydrolase variant from the culture.

The present invention also provides a biosensor for detecting an organophosphate compound in which the organophosphate compound hydrolase variant is immobilized.

The present invention also provides a method for detecting an organophosphate compound using the organophosphate compound hydrolase variant.

As described above, the present invention provides an organophosphate compound hydrolase variant, which has been selected through the high-speed screening method of the OPH, has an excellent hydrolytic activity as compared to the activity of the existing OPH, so that the organic phosphate-based compound is excellent. It has the effect of providing a biocatalyst to be removed, it is applicable to the environment and biosensors such as the reaction to decompose the organic phosphate compound and the organic phosphate compound detection, and can be used for various industrial purposes such as residual pesticide detection.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

In the present invention, "organophosphorus compounds" are known to be very toxic substances for inhibiting acetylcholine esterase in the nervous system, thereby impairing nerve function and eventually leading to death. Several studies have been conducted on how to remove pesticides and pesticides that contain compounds. Examples of such organophosphate compounds include paraoxon, parathion, azametiphos, azinphos, bensulide, malaoxon, malaoxon, malathion, Heptetophos, dichlorvos, dialilifos, diazinon, diclofenthion, dimethoate, dimethylvinphos, dimethylvinphos Dioxabenzofos, disulfoton, demethon S, edifenphos, ethephon, ethion, ethoprophos, clofenbinfoss (chlorfenvinphos), chlorpyrifos, fenitrothion, penthion, fenthion, fonofos, formothion, heptenophos, iprobenfos, Isazofos, isoxathion, mecarbam, metamidophos, metty Methidathion, monocrotophos, naled, omethoate, phoxim, pirimphos, profenofos, propaphos, Prothiofos, pyraclofos, pyrazophos, pyridaphenthion, quinalphos, terbufos, tetrachlorvinphos, thio Thimeton, tolclofos, triazophos, trichlorphon, vamidothion, and coumaphos.

In the present invention, "organophosphorus hydrolase (OPH)" is called organophosphosphate-degrading enzyme or phosphotriestrase, Pseudomonas diminuta MG (Pseudomonas diminuta MG) and Flavobacterium spp. (Flavobacterium sp. strain ATCC 27551). Since the enzyme hydrolyzes various insecticides as well as toxic chemical-warfare agents, including cattle and VX, research on this is active.

The present inventors used the high speed screening method of OPH in order to obtain the organophosphate compound hydrolase variant excellent in the hydrolytic activity. Specifically, in the 96 deep-well plate, microorganisms expressing the OPH gene library by IPTG induction were crushed with a cell lysis solution, and then the supernatant was separated and the substrate was prepared to have an organic phosphate compound in the form of microcrystals. The reaction was carried out by mixing to measure the absorbance at 412nm, through which a microorganism expressing OPH with increased activity was selected to obtain a variant. Here, the microorganisms may be all kinds of microorganisms such as enteric microorganisms and thermophilic microorganisms prepared for screening microorganisms having OPH activity, which are industrially useful enzymes, and soils prepared for separating microorganisms having OPH activity, Samples derived from ponds, rivers, hot springs, seawater, wastewater, food, and beverages can be used.

In the present invention, the process of obtaining the variant is provided by directed evolution, and the gene encoding the protein of interest is randonm using error-prone PCR and DNA shuffling technique. Mutation and recombination to construct a genetic library of genetic variants (Diversification step), to obtain variants with the desired activity from the gene (Selection step), and to find out where the mutations occurred from the variants. Sequencing step (Amplification step). Therefore, even in the present invention, the microorganism can isolate a variant having increased activity by using E. coli transformed with an enzyme variant library obtained by using an error-prone PCR method using a normal OPH template. have.

 In one aspect, the present invention relates to an organophosphorus hydrolase having an amino acid sequence of SEQ ID NO: 4, wherein (1) the 95th amino acid H (histidine) is substituted with Q (glutamine), (2 ) 131th amino acid K (lysine) is substituted with E (glutamic acid), and (3) 145th amino acid T (threonine) is substituted with A (alanine) will be.

Here, the amino acid sequence of SEQ ID NO: 4 is an amino acid sequence of OPH isolated from Flavoacterium sp. Strain ATCC 27551, and the gene sequence encoding the same has the nucleotide sequence of SEQ ID NO: 3.

In the present invention, the OPH variant may be an organophosphate compound hydrolase variant, which further has any one or more of the following substitution characteristics:

(4) the 24th amino acid T (threonine) is substituted with K (lysine); (5) the 126th amino acid I (isoleucine) is substituted with S (serine); (6) the 141th amino acid K (lysine) is substituted with M (methionine); And (7) the 280th amino acid S (serine) is substituted with C (cysteine).

For example, in addition to having the substitution characteristics of (1) to (3), a variant additionally including all of the substitution characteristics of (4) to (7) will also be one example of the present invention, wherein the OPH variant is It may have an amino acid sequence of SEQ ID NO: 9.

In the present invention, the OPH variant may be an organophosphate compound hydrolase variant, which further has any one or more of the following substitution characteristics:

(8) the 11th amino acid T (threonine) is substituted with A (alanine); (9) the 33rd amino acid S (serine) is substituted with N (asparagine); (10) the 52nd amino acid A (alanine) is substituted with S (serine); (11) 75th amino acid T (threonine) is substituted with S (serine); (12) the 118th amino acid L (leucine) is substituted with F (phenylalanine); (13) 165th amino acid A (alanine) is substituted with V (valine); (14) the 210th amino acid S (serine) is substituted with R (arginine); And (15) 246 th amino acid I (isoleucine) is substituted with N (asparagine).

For example, in addition to having the substitution characteristics of (1) to (3), a variant additionally including all of the substitution characteristics of (8) to (15) will also be one example of the present invention, wherein the OPH variant is It may have an amino acid sequence of SEQ ID NO: 10.

In the present invention, the OPH variant may be an organophosphate compound hydrolase variant, which further has any one or more of the following substitution characteristics:

(16) the 105th amino acid D (aspartic acid) is substituted with V (valine); (17) the 196th amino acid S (serine) is substituted with P (proline); (18) the 199th amino acid C (cysteine) is substituted with G (glycine); And (19) 313th amino acid V (valine) is substituted with F (phenylalanine)

For example, in addition to having the substitution characteristics of (1) to (3), a variant additionally including all of the substitution characteristics of (16) to (19) will also be an example of the present invention, wherein the OPH variant is It may have the amino acid sequence of SEQ ID NO: 11.

In the present invention, the OPH variant may be an organophosphate compound hydrolase variant, which further has any one or more of the following substitution characteristics:

(20) seventh amino acid D (aspartic acid) is substituted with N (asparagine); (21) the 21st amino acid A (alanine) is substituted with T (threonine); (22) the 29th amino acid H (histidine) is substituted with V (valine); (23) the 39th amino acid R (arginine) is substituted with L (leucine); (24) the 44th amino acid F (phenylalanine) is substituted with L (leucine); (25) the 46th amino acid G (glycine) is substituted with C (cysteine); (26) the 57th amino acid R (arginine) is substituted with S (serine); (27) the 64 th amino acid A (alanine) is substituted with S (serine); (28) the 106th amino acid P (proline) is substituted with L (leucine); (29) the 122nd amino acid F (phenyl alanine) is substituted with L (leucine); (30) the 163th amino acid S (serine) is substituted with R (arginine); (31) the 174th amino acid T (threonine) is substituted for S (serine); (32) the 202th amino acid H (histidine) is substituted with L (leucine); (33) 216th amino acid A (alanine) is substituted with D (aspartic acid); (34) the 236th amino acid D (aspartic acid) is substituted with G (glycine); (35) 248th amino acid S (serine) is substituted with W (tryptophan); (36) the 254th amino acid L (leucine) is substituted with F (phenylalanine); (37) 259th amino acid L (leucine) is substituted with I (isoleucine); (38) 271st amino acid S (serine) is substituted with W (tryptophan); (39) the 280th amino acid S (serine) is substituted with R (arginine); (40) the 286th amino acid M (methionine) is substituted with T (threonine); (41) the 289th amino acid M (methionine) is substituted with L (leucine); (42) the 291th amino acid R (arginine) is substituted with G (glycine); (43) the 295th amino acid D (aspartic acid) is substituted with A (alanine); And (44) 324 th amino acid T (threonine) is substituted with N (asparagine).

For example, in addition to having the substitution characteristics of (1) to (3), a variant additionally including all of the substitution characteristics of (20) to (44) will also be one example of the present invention, wherein the OPH variant is It may have an amino acid sequence of SEQ ID NO: 12.

In another aspect, the present invention relates to a gene encoding the organophosphate compound hydrolase variant. Here, the gene may have a nucleotide sequence of any one of SEQ ID NO: 5 to SEQ ID NO: 8.

In addition, the amino acid or nucleotide sequence of any one of the above sequences is substituted, deleted, inserted and added, resulting in mutation, thereby at least 70%, 80%, 90%, or An amino acid sequence or nucleotide sequence having 95% or more of sequence identity may also be included in the present invention.

The "sequence identity" refers to sequence similarity of two polynucleotide residues. The "sequence identity" can be determined by comparing two nucleotide sequences aligned optimally over a region of the base sequence to be compared. Here, the polynucleotide to be compared may have additions or deletions (for example, gaps, overhangs, etc.) as compared with reference sequences (for example, consensus sequences, etc.) for optimal alignment of two sequences. The numerical value of sequence identity determines the same nucleic acid base which exists in both sequences, determines the number of suitable sites, and then divides the number of suitable sites by the total number of bases in the sequence region to be compared, By multiplying 100, it can be calculated. Sequence identity between nucleic acids is measured using, for example, sequence analysis software, specifically BLASTN, FASTA, and the like. The BLASTN is http: //www.ncbi.nlm.nih. Generally available at gov / BLAST /.

In another aspect, the present invention relates to a recombinant vector containing a gene encoding the organophosphate compound hydrolase variant.

In the present invention, "vector" means a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing DNA in a suitable host. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into the appropriate host, the vector can replicate and function independently of the host genome, or in some cases can be integrated into the genome itself. Since plasmids are currently the most commonly used form of vectors, "plasmid" and "vector" are sometimes used interchangeably in the context of the present invention. For the purposes of the present invention, it is preferred to use plasmid vectors. Typical plasmid vectors that can be used for this purpose include (a) a replication initiation point that allows for efficient replication to include hundreds of plasmid vectors per host cell, and (b) host cells transformed with the plasmid vector. It has a structure comprising an antibiotic resistance gene and a restriction enzyme cleavage site (c) allows foreign DNA fragments to be inserted. Although no suitable restriction enzyme cleavage site is present, the use of synthetic oligonucleotide adapters or linkers according to conventional methods can facilitate ligation of the vector and foreign DNA.

In addition, the genes are "operably linked" when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to enable gene expression when appropriate molecules (eg, transcriptional activating proteins) are bound to regulatory sequence (s). For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when it affects the transcription of the sequence; Or the ribosomal binding site is operably linked to a coding sequence when positioned to facilitate translation. In general, "operably linked" means that the linked DNA sequences are in contact, and in the case of a secretory leader, are in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.

Of course, not all vectors function equally to express the DNA sequences of the present invention, and likewise not all hosts function equally for the same expression system. However, one of ordinary skill in the art may, without departing from the scope of the present invention without undue experimental burden, select appropriately among other vectors, expression control sequences and hosts. For example, in selecting a vector, a host should be considered, since the vector must be replicated therein, and the number of copies of the vector, the ability to control the number of copies and other proteins encoded by the vector, e.g. Expression of antibiotic markers should also be considered.

In another aspect, the present invention relates to a recombinant microorganism in which the gene is inserted into a host cell selected from the group consisting of bacteria, fungi and yeasts. More specifically, the present invention relates to a recombinant microorganism in which a gene encoding the organophosphate hydrolase variant is inserted on a chromosome or transformed with a recombinant vector including the variant gene.

After ligation described above, the recombinant vector must be transformed into a suitable host cell. Here, preferred host cells are prokaryotic cells and suitable prokaryotic host cells may be E. coli , for example E. coli JM109, E. coli DH5α, E. coli JM101, E. coli K12, E. coli W3110, E. coli X1776, E. coli XL1-Blue (Stratagene, USA), E. coli B, E. coli B21 (DE3), E. coli TOP10, and the like. E. coli strains such as FMB101, NM522, NM538 and NM539 and other prokaryotic species and genera may also be used. In addition to the aforementioned E. coli , strains of the genus Agrobacterium, such as Agrobacterium A4, bacilli, such as Bacillus subtilis , Salmonella typhimurium or Serratia marghesen another variety of enteric bacteria and Pseudomonas (Pseudomonas) in strains such as marcescens) may be used as host cells. It is also possible to use eukaryotic cells such as yeast and fungi as hosts.

The transformed recombinant microorganism may be prepared according to any transformation method. In the present invention, "transformation" refers to a phenomenon in which DNA is introduced into a host so that DNA can be reproduced as a factor of a chromosome or by completion of chromosome integration, thereby causing an artificial genetic change by introducing external DNA into a cell. Means. In general, transformation methods, in particular, transformation of plasma cells can be readily accomplished using a heat shock method using calcium chloride as described in section 1.82 in the book 'Molecular Cloning' of Sambrook et al. Optionally, electroporaton (Neumann et al., EMBO J., 1: 841, 1982), calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, lithium acetate -DMSO method is available. In addition, in the present invention, as a method of inserting the gene on the chromosome of the host cell, a commonly known gene manipulation method may be used. For example, a retroviral vector, an adenovirus vector, an adeno-associated virus vector, and herpes simplex. And viral viral vectors, poxvirus vectors, lentiviral vectors or non-viral vectors.

In another aspect, the present invention comprises the steps of culturing the recombinant microorganism to express an organophosphate compound hydrolase variant; And it relates to a method for producing an organophosphate compound hydrolase variant comprising recovering an organophosphate compound hydrolase variant from the culture.

Cultivation of the transformed recombinant microorganism is carried out according to well-known methods, conditions such as the culture temperature and time, the pH of the medium can be appropriately adjusted.

Recovery of organophosphate compound hydrolase variants from the cultured recombinant microorganisms is accomplished by conventional biochemical separation techniques such as treatment with protein precipitants (salting), centrifugation, ultrasonic disruption, ultrafiltration, dialysis, molecular sieves. Various chromatography such as chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc. can be used, and in general, these can be used in combination to separate proteins of high purity.

In still another aspect, the present invention relates to a biosensor for detecting an organophosphate compound in which the organophosphate compound hydrolase variant is immobilized.

Here, the biosensor includes components of the conventionally disclosed biosensor, but also includes a biochip, a bioarray, or the like, for example, a detection kit. For example, beads, particles, dipsticks, fibers, It may be provided as a sensor for detection fixed to a conventional support such as a filter, a membrane and a glass slide or a silane or silicate solid support.

The solid support may comprise at least one substantially hard surface, on which the variants may be immobilized. At this time, it may be immobilized by any conventional chemical coupling method. As an example, the biotin is bonded to the ends of the variant to form a complex, and streptavidin is immobilized on the surface of the substrate such as the chip, and the variant is formed by the interaction between the biotin and the Streptavidin immobilized on the surface of the substrate. It may also be immobilized on the substrate surface.

The biosensor may be provided in the form of a kit and, if necessary, may include a buffer solution, containers for performing and analyzing detection, such as a tub, a small sachet, an envelope, and a tube. , Ampoules, and the like, which may be formed, in part or in whole, from plastic, glass, paper, foil, wax, and the like. The container may be equipped with a fully or partially separable stopper, which may initially be part of the container or attached to the container by mechanical, adhesive, or other means. The container may also be equipped with a stopper, which may be accessed by its needle. The kit may include an external package, which may include instructions for use of the components.

In still another aspect, the present invention relates to a method for detecting an organophosphate compound, which uses the biosensor for detecting the organophosphate compound.

Here, the organophosphate compound may be paraoxon, parathion, azametiphos, azinphos, bensulide, malaoxon, malaoxon, malathion or hex. Hetenophos, dichlorvos, dialilifos, diazinon, diclofenthion, dimethoate, dimethylvinphos, dioxa Benzoxa, disulfoton, demethon S, edifenphos, ethephon, ethion, ethoprophos, clofenbinfoss chlorfenvinphos, chlorpyrifos, phenitrothion, penthion, fenthion, fonofos, formothion, heptenophos, iprobenfos, director Isazofos, isoxathion, mecarbam, metamidophos, metidati (methidathion), monocrotophos, naled, omethoate, phoxim, pirimphos, profenofos, propaphos, pro Thiophos, pyraclofos, pyrazophos, pyridaphenthion, quinalphos, terbufos, tetrachlorvinphos, thiomethone (thiometon), tolclofos, triazophos, trichlorphon, trimidone, vamidothion and coumaphos.

Here, the detection method comprises the steps of reacting an organophosphate compound hydrolase variant fixed to the biosensor with a sample containing an organophosphate compound to be detected; And measuring the reaction of the organophosphate compound hydrolase combined with the organophosphate compound to be detected using absorbance, amount of current, amount of color development, and the like.

This detection method can also be used to detect residual pesticides such as pesticides containing the organophosphate compounds.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples. In particular, in the following examples, the activity was tested only for some of the variants having all of the above-mentioned substitution characteristics, but it will be apparent to those skilled in the art that the variants optionally containing such substitution characteristics also have increased organophosphate hydrolysis activity. will be. In addition, although an increase in activity for paraoxone has been confirmed as an organophosphate compound, it will be apparent to those skilled in the art that the variant of the present invention has excellent decomposition activity for other organophosphate compounds including the same.

Example 1: Cloning OPH Gene and Confirming OPH Expression

For general cloning, the host strain was E. coli TOP10 (Invitrogen, USA), and the host strain for expression and production of OPH protein was E. coli BL21 (DE3) (Novagen, USA) was used. Flavobacterium from the Korea Institute of Bioscience and Biotechnology, Biological Resource Center (http://www.brc.re.kr; Korea) sp. (KCTC2480, ATCC27551) genomic DNA as a template,

Mature OPH gene fragment (NCBI access No.) amplified by PCR using a forward primer (SEQ ID NO: 5'-GGAATTC CATATG GGATCGATCGGCACAGGC-3 ') and a reverse primer (SEQ ID NO: 2: 5'-AAAA CTCGAG TGACGCCCGCAAGGTCGGTGA-3) AY766084, SEQ ID NO: 3) was obtained. A plasmid pET-6HmOPH-6H was prepared to express mature OPH using T4 DNA ligase at the Nde I and Xho I sites of the pET-22b (+) vector (Novagen, USA) having a T7 promoter (FIG. 1). . At the N-terminus and C-terminus of mature OPH, there is a gene encoding 6 histidine for purification using Ni-column.

The pET-6HmOPH-6H plasmid (about 6.46 kb) was transformed using a heat shock method in E. coli BL21 (DE3) was incubated at 37 ℃, 200 rpm in a 250 ml flask containing 100 ml of LB medium. . When the absorbance was 0.4 at a wavelength of 600 nm, IPTG was treated with a final concentration of 0.1 mM, and then incubated at 30 ° C. and 200 rpm for 6 hours. Each cultured cell was crushed by sonication electroshock method. After crushing, the protein was divided into soluble fraction and insoluble fraction by centrifugation and analyzed by 12% (w / v) SDS-PAGE electrophoresis. SDS-PAGE was stained using Coomassie brilliant blue R-250.

The result was as shown in FIG. Since mature OPH has 6 his-taq, the purification of protein was carried out under the condition that the protein was not denatured using resin having affinity for Ni-NTA. Flavobacterium sp. The size of the derived mature OPH protein was about 36 kDa, confirming that it was mostly expressed as an insoluble protein.

Each protein was isolated and purified using a Ni-NTA column. The isolated protein was quantified by Lowry method and used in this study. Insoluble protein can change the structure of the protein, which can cause problems in the enzyme activity, so that the enzyme maintains its structural activity through the enzyme denaturation and renaturation process.

After denaturation through 8 M urea was passed through the Ni-NTA column and elution using 0.5 M imidazole. Each fraction was analyzed via 12% (w / v) SDS-PAGE. As shown in FIG. 3, about 90% or more of the expressed protein could be harvested through purification, and almost no protein escaped through the washing process, indicating that most proteins have high affinity for the Ni-NTA column. It was found to combine.

Example 2: Preparation of OPH Variant Gene Library

The analysis of OPH activity using LB liquid medium in 96 deep well plate of the present invention was developed by the technique of directional molecular evolutionary element for the improvement of organophosphate compound resolution activity. In the preparation of the enzyme variant library, mutation-induced PCR (error-prone PCR) was used for random mutagenesis. As a template, Flavobacterium sp. Genomic DNA of (KCTC2480, ATCC27551) was used, and a forward primer (SEQ ID NO: 5'-GGAATTC CATATG GGATCGATCGGCACAGGC-3 ') and a reverse primer (SEQ ID NO: 2: 5'-AAAA CTCGAG TGACGCCCGCAAGGTCGGTGA-3') were used. . For mutagenesis PCR, mutagenesis PCR was performed with MgCl ₂ concentrations of 3 mM, 5 mM, and 7 mM, respectively, from the table below.

In the forward primer used for the mutation-induced PCR, a gene encoding 6 histidine was inserted to purify the protein through the Ni-NTA column. Mutation-induced PCR was performed by repeating the procedure of inactivation for 5 minutes at 95 ° C, inactivation at 95 ° C for 30 seconds, primer binding at 55 ° C for 30 seconds, and DNA synthesis for 1 minute at 72 ° C. It was carried out by performing a DNA synthesis reaction for 7 minutes at ℃. The purified PCR products and the constant expression vector pET-22b (+) (Novagen, USA) were sequentially treated with restriction enzymes Nde I and Xho I. The prepared introduction DNA and the vector were mixed at a molar ratio of 4: 1 and treated with T4 DNA ligase to prepare a library vector containing OPH varient.

Preparation of cells for the introduction of genes (competent cells) is a very important process to maintain a stable library of high quality is always a new cell was prepared and used and the following procedure was followed. First, a single colony of Escherichia coli BL21 (DE3) (Novagen, USA) was inoculated in 4 ml of LB medium and incubated overnight at 37 ° C. After about 3 hours, when the absorbance (OD ₆₀₀ ) of the culture solution reached 0.5 to 0.6, the mixture was left for 20 minutes on ice, and the culture solution was placed in a pre-cooled centrifuge container and centrifuged (5,000 × g, 20 minutes, 4 ° C.). Suspended by adding cold water to the cells from which the supernatant was completely removed, the cells were collected again by centrifugation, suspended again by adding cold 10% (w / v) glycerol, and centrifuged. 10% (w / v) glycerol was added to the cells obtained above to give a cell concentration (OD ₆₀₀ ) of 100, and 50 µl aliquots were used.

Electroporation was performed by mixing DNA with the prepared cells, adding an electric shock (18 kV / cm, 25 Hz), and adding 1 ml of LB medium and incubating at 200 rpm at 37 ° C for 1 hour.

The above gene library manufacturing process is optimized based on repeated research, and has been used as a base technology for library production in molecular evolution research. The quality evaluation of the prepared mutagenic PCR library was carried out in a large plate medium (24 cm × 24 cm, Espiel, Korea) containing 50 μg / ml ampicillin and transforming the transformant recovery solution for 24 hours at 37 ° C. About 1,104 colonies were inoculated into LB medium of 96 deep well plates and shaken for 24 hours. The cells were inoculated again in a new 96 deep well plate by 1%, and the absorbance (OD ₆₀₀ ) was 0.3-0.4, which was added to 1 mM IPTG to induce the transformed microorganism to produce OPH while incubating for 12 hours. The transformed microorganisms were centrifuged (3,500 rpm, 4 ° C., 30 min) to discard the culture supernatant, leaving only the cells. 1 ml of PBS was added to a 96 deep well plate in which only the cells remained, and the cells were suspended and centrifuged again (3,500rpm, 4 ° C, 30min) to wash the cells so that there was no culture solution (repeated twice).

Example 3: Activity Determination Test of OPH

(1) Preparation of Organophosphate Compound Reagent and Measurement of OPH Activity

Paraoxon (Supelco, USA), an example of organophosphate compounds, was dissolved in primary distilled water to make 1 ppm. 30 μl phosphate buffer (pH 8.0), 20 μl substrate (1 ppm paraoxon) and 50 μl OPH solution were used. In this reaction, the blank consists of the remaining phosphate buffer and substrate except the enzyme solution. In the graph of absorbance change using SpectraMax M2 multi-reader (Molecular Devices, USA), the slope between points can be measured to find the point with the highest change. As shown in Figure 4, by confirming the yellow product showing a strong absorbance at 412 nm, it was possible to measure the OPH enzyme activity.

(2) Confirmation of proportional relationship between decomposition of organophosphate compound and OPH activity

200 μl of 6M urea and 20 μl of cell lysis solution were added to the cells and shaken (200 rpm) for 3 hours, followed by centrifugation (3,500 rpm, 4 ° C., 1 hour), and the supernatant was transferred to a 96 well plate (clear coster plate). 50 μl each was transferred, and 50 μl of an organic phosphate compound (paraoxon, 1 ppm) was added thereto, and the absorbance was measured at 30 seconds intervals at 410 nm for 20 minutes (FIG. 5).

A total of 1,104 colonies were analyzed in the same manner as above to investigate the ratio and linear distribution of gene mutations by mutation-induced PCR. As a result of analysis, 30 positive candidates were selected to analyze DNA sequences. From the sequencing results, the final four positive clones out of 30 clones were reselected (Table 1).

[Table 1]

"Increase activity" shown in Table 1 is a value of the activity increase rate compared to the enzyme activity of the wild type (wild type) control group as a result of comparing the absorbance based on the reaction time 10 minutes, affecting the enzyme reaction rate during the reaction To exert.

In addition, the sequencing results of the four clones (# 19, 53, 61, 777) (SEQ ID NOS: 5 to 8), clone # 19 had a mutation of 7 amino acids (SEQ ID NO: 9), 10 In the minute, the activity was improved by 5.1 times compared with the control wild type OPH. In addition, clone # 53 had a mutation of 11 amino acids (SEQ ID NO: 10), which also improved about 12-fold compared to control, and clone # 61 had a mutation of 7 amino acids (SEQ ID NO: 11). Activity was 6.9-fold, # 777 had 26 amino acid mutations (SEQ ID NO: 12), and activity was increased by 16.1-fold. When comparing the results of sequencing and kinetic assay, clones # 53 and # 777 showed the highest activity compared to wild type OPH. It has also been shown that this method can be effectively used for high speed screening of OPH variants with high activity for organophosphate compounds.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a genetic map of pET-mOPH6H to produce mature OPH.

Figure 2 is a protein electrophoresis picture to confirm the expression of mature OPH protein (Figure 2, SM: protein size marker, 1: soluble fraction of E. coli BL21 (DE3) as a negative control, 2: E. as a negative control) . Insoluble fraction of coli BL21 (DE3), 3: Whole cell fraction sample of E. coli BL21 (DE3) (clone No.3) expressing mature OPH, 4: E. coli BL21 (DE3) expressing mature OPH ( Sample of soluble fraction of clone No. 3), E. coli BL21 (DE3) expressing 5: mature OPH (sample of insoluble fraction of clone No. 3), 6: E. coli BL21 (DE3) expressing mature OPH ( Sample No. 4, whole cell fraction of clone No. 4) 7: E. coli expressing mature OPH E. coli soluble fraction sample # 4, clone of BL21 (DE3) (clone No. 4) E. coli expressing mature OPH Insoluble fraction sample # 4 of BL21 (DE3) (clone No.4).

3 is protein electrophoresis of mature OPH protein purified through Ni-NTA column (SM: protein size marker, 1: mature OPH purified through Ni-NTA column from insoluble fraction of E. coli BL21 (DE3)). , 2: sample passed through Ni-NTA column).

Figure 4 is a graph of the spectroscopic characterization of OPH through the absorption spectrum analysis.

5 is a graph of activity comparison of selected mutant OPH enzymes.

<110> KAIST <120> Organophosphorus Hydrolase Variants and Method for Preparing the          Same <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 1 ggaattccat atgggatcga tcggcacagg c 31 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 2 aaaactcgag tgacgcccgc aaggtcggtg a 31 <210> 3 <211> 1014 <212> DNA <213> Organophosphorus Hydrolase from Flavobacterium sp. <400> 3 ggatcgatcg gcacaggcga tcggatcaat accgtgcgcg gtcctatcac aatctctgaa 60 gcgggtttca cactgactca cgagcacatc tgcggcagct cggcaggatt cttgcgtgct 120 tggccagagt tcttcggtag ccgcaaagct ctagcggaaa aggctgtgag aggattgcgc 180 cgcgccagag cggctggcgt gcgaacgatt gtcgatgtgt cgactttcga tatcggtcgc 240 gacgtcagtt tattggccga ggtttcgcgg gctgccgacg ttcatatcgt ggcggcgacc 300 ggcttgtggt tcgacccgcc actttcgatg cgattgagga gtgtagagga actcacacag 360 ttcttcctgc gtgagattca atatggcatc aaagacaccg gaattagggc gggcattatc 420 aaggtcgcga ccacaggcaa ggcgaccccc tttcaggagt tagtgttaaa ggcggccgcc 480 cgggccagct tggccaccgg tgttccggta accactcaca cggcagcaag tcagcgcgat 540 ggtgagcagc aggccgccat ttttgagtcc gaaggcttga gcccctcacg ggtttgtatt 600 ggtcacagcg atgatactga cgatttgagc tatctcaccg ccctcgctgc gcgcggatac 660 ctcatcggtc tagaccacat cccgcacagt gcgattggtc tagaagataa tgcgagtgca 720 tcagccctcc tgggcatccg ttcgtggcaa acacgggctc tcttgatcaa ggcgctcatc 780 gaccaaggct acatgaaaca aatcctcgtt tcgaatgact ggctgttcgg gttttcgagc 840 tatgtcacca acatcatgga cgtgatggat cgcgtgaacc ccgacgggat ggccttcatt 900 ccactgagag tgatcccatt cctacgagag aagggcgtcc cacaggaaac gctggcaggc 960 atcactgtga ctaacccggc gcggttcttg tcaccgacct tgcgggcgtc atga 1014 <210> 4 <211> 337 <212> PRT <213> Organophosphorus Hydrolase from Flavobacterium sp. <400> 4 Gly Ser Ile Gly Thr Gly Asp Arg Ile Asn Thr Val Arg Gly Pro Ile   1 5 10 15 Thr Ile Ser Glu Ala Gly Phe Thr Leu Thr His Glu His Ile Cys Gly              20 25 30 Ser Ser Ala Gly Phe Leu Arg Ala Trp Pro Glu Phe Phe Gly Ser Arg          35 40 45 Lys Ala Leu Ala Glu Lys Ala Val Arg Gly Leu Arg Arg Ala Arg Ala      50 55 60 Ala Gly Val Arg Thr Ile Val Asp Val Ser Thr Phe Asp Ile Gly Arg  65 70 75 80 Asp Val Ser Leu Leu Ala Glu Val Ser Arg Ala Ala Asp Val His Ile                  85 90 95 Val Ala Ala Thr Gly Leu Trp Phe Asp Pro Pro Leu Ser Met Arg Leu             100 105 110 Arg Ser Val Glu Glu Leu Thr Gln Phe Phe Leu Arg Glu Ile Gln Tyr         115 120 125 Gly Ile Lys Asp Thr Gly Ile Arg Ala Gly Ile Ile Lys Val Ala Thr     130 135 140 Thr Gly Lys Ala Thr Pro Phe Gln Glu Leu Val Leu Lys Ala Ala Ala 145 150 155 160 Arg Ala Ser Leu Ala Thr Gly Val Pro Val Thr Thr His Thr Ala Ala                 165 170 175 Ser Gln Arg Asp Gly Glu Gln Gln Ala Ala Ile Phe Glu Ser Glu Gly             180 185 190 Leu Ser Pro Ser Arg Val Cys Ile Gly His Ser Asp Asp Thr Asp Asp         195 200 205 Leu Ser Tyr Leu Thr Ala Leu Ala Ala Arg Gly Tyr Leu Ile Gly Leu     210 215 220 Asp His Ile Pro His Ser Ala Ile Gly Leu Glu Asp Asn Ala Ser Ala 225 230 235 240 Ser Ala Leu Leu Gly Ile Arg Ser Trp Gln Thr Arg Ala Leu Leu Ile                 245 250 255 Lys Ala Leu Ile Asp Gln Gly Tyr Met Lys Gln Ile Leu Val Ser Asn             260 265 270 Asp Trp Leu Phe Gly Phe Ser Ser Tyr Val Thr Asn Ile Met Asp Val         275 280 285 Met Asp Arg Val Asn Pro Asp Gly Met Ala Phe Ile Pro Leu Arg Val     290 295 300 Ile Pro Phe Leu Arg Glu Lys Gly Val Pro Gln Glu Thr Leu Ala Gly 305 310 315 320 Ile Thr Val Thr Asn Pro Ala Arg Phe Leu Ser Pro Thr Leu Arg Ala                 325 330 335 Ser     <210> 5 <211> 1014 <212> DNA <213> OPH variant clone No. 19 <400> 5 ggatcgatcg gcacaggcga tcggatcaat accgtgcgcg gtcctatcac aatctctgaa 60 gcgggtttca aactgactca cgagcacatc tgcggcagct cggcaggatt cttgcgtgct 120 tggccagagt tcttcggtag tcgcaaagct ctagcggaaa aggctgtgag aggattgcgc 180 cgcgccagag cggctggcgt gcgaacgatt gtcgatgtgt cgactttcga tatcggtcgc 240 gacgtcagtt tattggccga ggtttcgcgg gctgccgacg ttcaaatcgt ggcggcgacc 300 ggcttgtggt tcgacccgcc actttcgatg cgattgagga gtgtagagga actcacacag 360 ttcttcctgc gtgagagtca atatggtatc gaagacaccg gaattagggc gggcattatc 420 atggtcgcga ccgcaggcaa ggcgaccccc tttcaggagt tagtgttaaa ggcggccgcc 480 cgggccagct tggccaccgg tgttccggta accactcaca cggcagcaag tcagcgcgat 540 ggtgagcagc aggccgccat ttttgagtcc gaaggcttga gcccctcacg ggtttgtatt 600 ggtcacagcg atgatactga cgatttgagc tatctcaccg ccctcgctgc gcgcggatac 660 ctcatcggtc tagaccacat cccgcacagt gcgattggtc tagaagataa tgcgagcgca 720 tcagccctcc tgggcatccg ttcgtggcaa acacgggctc tcttgatcaa ggcgctcatc 780 gaccaaggct acatgaaaca aatcctcgtt tcgaatgact ggctgttcgg gttttcgtgc 840 tatgtcacca acatcatgga cgtgatggat cgcgtgaacc ccgacgggat ggccttcatt 900 ccactgagag tgatcccatt cctacgagag aagggcgtcc cacaggaaac gctggcaggc 960 atcactgtga ctaacccggc gcggttcttg tcaccgacct tgcgggcgtc ataa 1014 <210> 6 <211> 1014 <212> DNA <213> OPH variant clone No. 53 <400> 6 ggatcgatcg gcacaggcga tcggatcaat gccgtgcgcg gtcctatcac agtctctgaa 60 gcgggtttca cactgactca cgagcacatc tgtggcaact cggcaggatt cttgcgtgct 120 tggccacagt tcttcggtag tcgcaaagct ctatcggaaa aggctgtgag aggattgcgc 180 cgcgccagag cggctggcgt gcgaacgatt gtcgatgtgt cgtctttcga tatcggtcgc 240 gacgtcagtt tattggccga ggtttcgcgg gctgccgacg ttcaaatcgt ggcggcgacc 300 ggcttgtggt tcgacccgcc actttcgatg cgattgagga gtgtagagga attcacacag 360 ttcttcctgc gtgagattca atatggcatc gaagacaccg gaattagggc gggcattatc 420 aaggtcgcga ccgcaggcaa ggcgaccccc tttcaggagt tagtgttaaa ggcggccgcc 480 cgggccagct tggtcaccgg tgttccggta accactcaca cggcagcaag tcagcgcgat 540 ggtgagcagc aggccgccat ttttgagtcc gaaggcttga gcccctcacg ggtttgtatt 600 ggtcacagcg atgatactga cgatttgaga tatctcaccg ccctcgctgc gcgcggatac 660 ctcatcggtc tagaccacat cccgcacagt gcgattggtc tagaagataa tgcgagcgca 720 tcagccctcc tgggcaaccg ttcgtggcaa acacgggctc tcttgatcaa ggcgctcatc 780 gaccaaggct acatgaaaca aatcctcgtt tcgaatgact ggctgttcgg gttttcgagc 840 tatgtcacca acatcatgga cgtgatggat cgcgtgaacc ccgacgggat ggccttcatt 900 ccactgagag tgatcccatt cctacgagag aagggcgtcc cacaggaaac gctggcaggc 960 atcactgtga ctaacccggc gcggttcttg tcaccgacct tgcgggcgtc ataa 1014 <210> 7 <211> 1014 <212> DNA <213> OPH variant cloen No. 61 <400> 7 ggatcgatcg gcacaggcga tcggatcaat accgtgcgcg gtcctatcac aatctctgaa 60 gcgggtttca cactgactca cgagcacatc tgcggtagct cggcaggatt cttgcgtgct 120 tggccagagt tcttcggtag tcgcaaagct ctagcggaaa aggctgtgag aggattgcgc 180 cgcgccagag cggctggcgt gcgaacgatt gtcgatgtgt cgactttcga tatcggtcgc 240 gacgtcagtt tattggccga ggtttcgcgg gctgccgacg ttcaaatcgt ggcggcgacc 300 ggcttgtggt tcgtcccgcc actttcgatg cgattgagga gtgtagagga actcacacag 360 ttcttcctgc gtgagattca atatggcatc gaagacaccg gaattagggc gggcattatc 420 aaggtcgcga ccgcaggcaa ggcgaccccc tttcaggagt tagtgttaaa ggcggccgcc 480 cgggccagct tggccaccgg tgttccggta accactcaca cggcagcaag tcagcgcgat 540 ggtgagcagc aggccgccat ttttgagtcc gaaggcttga gccccccacg ggttggtatt 600 ggtcacagcg atgatactga cgatttgagc tatctcaccg ccctcgctgc gcgcggatac 660 ctcatcggtc tagaccacat cccgcacagt gcgattggtc tagaagataa tgcgagcgca 720 tcagccctcc tgggcatccg ttcgtggcaa acacgggctc tcttgatcaa ggcgctcatc 780 gaccagggct acatgaaaca aatcctcgtt tcgaatgact ggctgttcgg gttttcgagc 840 tatgtcacca acatcatgga cgtgatggat cgcgtgaacc ccgacgggat ggccttcatt 900 ccactgagag tgatcccatt cctacgagag aagggcttcc cacaggaaac gctggcaggc 960 atcactgtga ctaacccggc gcggttcttg tcaccgacct tgcgggcgtc ataa 1014 <210> 8 <211> 1014 <212> DNA <213> OPH variant cloen No. 777 <400> 8 ggatcgatcg gcacaggcaa tcggattaat accgtgcgcg gtcctatcac aatctctgaa 60 acgggtttca cactgactca cgaggtcatc tgcggcagct cggcaggatt cttgcttgct 120 tggccacagc tcttctgtag tcgcaaagct ctagcggaaa aggctgtgag tggactgcgc 180 cgcgccagat cggctggcgt gcgaacgata gtcgatgtgt cgactttcga aatcggtcgt 240 gacgtcagtt tattggccga ggtttcgcgg gctgccgacg ttcaaatagt ggcggcgacc 300 ggcttgtggt tcgacctgcc actttcgatg cgtttgagga gtgtagagga actcacacag 360 ttcctcctgc gtgagattca atatggcatc gaagacaccg gaattagggc gggcattatc 420 aaggtcgcga ctgcaggcaa ggcgaccccc tttcaggagt tagtgttaaa ggcggccgcc 480 cgggccagat tggccaccgg tgttccggta accactcact cggcagcaag tcagcgcgat 540 ggtgagcagc aggccgccat ttttgagtcc gaaggcttga gcccctcacg ggtttgtatt 600 ggtctcagcg atgatactga tgatttgagc tatctcaccg ccctcgatgc gcgcggatac 660 ctcatcggtc tagaccacat cccgcacagt gcgattggtc tagaaggtaa tgcgagcgca 720 tcagccctcc tgggcatccg ttggtggcaa acacgggctt ttttgatcaa ggcgatcatc 780 gaccaaggat acatgaaaca aatcctcgtt tggaatgact ggctgttcgg gttttcgaga 840 tatgtcacca acatcacgga cgtgttggat ggcgtgaacc ccgccgggat ggccttcatt 900 ccactgagag tgatcccatt cctacgagag aagggcgtcc cacaggaaac gctggcaggc 960 atcactgtga ataacccggc gcggttcttg tcaccgacct tgcgggcgtc ataa 1014 <210> 9 <211> 337 <212> PRT <213> OPH variant clone No. 19 <400> 9 Gly Ser Ile Gly Thr Gly Asp Arg Ile Asn Thr Val Arg Gly Pro Ile   1 5 10 15 Thr Ile Ser Glu Ala Gly Phe Lys Leu Thr His Glu His Ile Cys Gly              20 25 30 Ser Ser Ala Gly Phe Leu Arg Ala Trp Pro Glu Phe Phe Gly Ser Arg          35 40 45 Lys Ala Leu Ala Glu Lys Ala Val Arg Gly Leu Arg Arg Ala Arg Ala      50 55 60 Ala Gly Val Arg Thr Ile Val Asp Val Ser Thr Phe Asp Ile Gly Arg  65 70 75 80 Asp Val Ser Leu Leu Ala Glu Val Ser Arg Ala Ala Asp Val Gln Ile                  85 90 95 Val Ala Ala Thr Gly Leu Trp Phe Asp Pro Pro Leu Ser Met Arg Leu             100 105 110 Arg Ser Val Glu Glu Leu Thr Gln Phe Phe Leu Arg Glu Ser Gln Tyr         115 120 125 Gly Ile Glu Asp Thr Gly Ile Arg Ala Gly Ile Ile Met Val Ala Thr     130 135 140 Ala Gly Lys Ala Thr Pro Phe Gln Glu Leu Val Leu Lys Ala Ala Ala 145 150 155 160 Arg Ala Ser Leu Ala Thr Gly Val Pro Val Thr Thr His Thr Ala Ala                 165 170 175 Ser Gln Arg Asp Gly Glu Gln Gln Ala Ala Ile Phe Glu Ser Glu Gly             180 185 190 Leu Ser Pro Ser Arg Val Cys Ile Gly His Ser Asp Asp Thr Asp Asp         195 200 205 Leu Ser Tyr Leu Thr Ala Leu Ala Ala Arg Gly Tyr Leu Ile Gly Leu     210 215 220 Asp His Ile Pro His Ser Ala Ile Gly Leu Glu Asp Asn Ala Ser Ala 225 230 235 240 Ser Ala Leu Leu Gly Ile Arg Ser Trp Gln Thr Arg Ala Leu Leu Ile                 245 250 255 Lys Ala Leu Ile Asp Gln Gly Tyr Met Lys Gln Ile Leu Val Ser Asn             260 265 270 Asp Trp Leu Phe Gly Phe Ser Cys Tyr Val Thr Asn Ile Met Asp Val         275 280 285 Met Asp Arg Val Asn Pro Asp Gly Met Ala Phe Ile Pro Leu Arg Val     290 295 300 Ile Pro Phe Leu Arg Glu Lys Gly Val Pro Gln Glu Thr Leu Ala Gly 305 310 315 320 Ile Thr Val Thr Asn Pro Ala Arg Phe Leu Ser Pro Thr Leu Arg Ala                 325 330 335 Ser     <210> 10 <211> 337 <212> PRT <213> OPH variant clone No. 53 <400> 10 Gly Ser Ile Gly Thr Gly Asp Arg Ile Asn Ala Val Arg Gly Pro Ile   1 5 10 15 Thr Val Ser Glu Ala Gly Phe Thr Leu Thr His Glu His Ile Cys Gly              20 25 30 Asn Ser Ala Gly Phe Leu Arg Ala Trp Pro Gln Phe Phe Gly Ser Arg          35 40 45 Lys Ala Leu Ser Glu Lys Ala Val Arg Gly Leu Arg Arg Ala Arg Ala      50 55 60 Ala Gly Val Arg Thr Ile Val Asp Val Ser Ser Phe Asp Ile Gly Arg  65 70 75 80 Asp Val Ser Leu Leu Ala Glu Val Ser Arg Ala Ala Asp Val Gln Ile                  85 90 95 Val Ala Ala Thr Gly Leu Trp Phe Asp Pro Pro Leu Ser Met Arg Leu             100 105 110 Arg Ser Val Glu Glu Phe Thr Gln Phe Phe Leu Arg Glu Ile Gln Tyr         115 120 125 Gly Ile Glu Asp Thr Gly Ile Arg Ala Gly Ile Ile Lys Val Ala Thr     130 135 140 Ala Gly Lys Ala Thr Pro Phe Gln Glu Leu Val Leu Lys Ala Ala Ala 145 150 155 160 Arg Ala Ser Leu Val Thr Gly Val Pro Val Thr Thr His Thr Ala Ala                 165 170 175 Ser Gln Arg Asp Gly Glu Gln Gln Ala Ala Ile Phe Glu Ser Glu Gly             180 185 190 Leu Ser Pro Ser Arg Val Cys Ile Gly His Ser Asp Asp Thr Asp Asp         195 200 205 Leu Arg Tyr Leu Thr Ala Leu Ala Ala Arg Gly Tyr Leu Ile Gly Leu     210 215 220 Asp His Ile Pro His Ser Ala Ile Gly Leu Glu Asp Asn Ala Ser Ala 225 230 235 240 Ser Ala Leu Leu Gly Asn Arg Ser Trp Gln Thr Arg Ala Leu Leu Ile                 245 250 255 Lys Ala Leu Ile Asp Gln Gly Tyr Met Lys Gln Ile Leu Val Ser Asn             260 265 270 Asp Trp Leu Phe Gly Phe Ser Ser Tyr Val Thr Asn Ile Met Asp Val         275 280 285 Met Asp Arg Val Asn Pro Asp Gly Met Ala Phe Ile Pro Leu Arg Val     290 295 300 Ile Pro Phe Leu Arg Glu Lys Gly Val Pro Gln Glu Thr Leu Ala Gly 305 310 315 320 Ile Thr Val Thr Asn Pro Ala Arg Phe Leu Ser Pro Thr Leu Arg Ala                 325 330 335 Ser     <210> 11 <211> 337 <212> PRT <213> OPH variant clone No. 61 <400> 11 Gly Ser Ile Gly Thr Gly Asp Arg Ile Asn Thr Val Arg Gly Pro Ile   1 5 10 15 Thr Ile Ser Glu Ala Gly Phe Thr Leu Thr His Glu His Ile Cys Gly              20 25 30 Ser Ser Ala Gly Phe Leu Arg Ala Trp Pro Glu Phe Phe Gly Ser Arg          35 40 45 Lys Ala Leu Ala Glu Lys Ala Val Arg Gly Leu Arg Arg Ala Arg Ala      50 55 60 Ala Gly Val Arg Thr Ile Val Asp Val Ser Thr Phe Asp Ile Gly Arg  65 70 75 80 Asp Val Ser Leu Leu Ala Glu Val Ser Arg Ala Ala Asp Val Gln Ile                  85 90 95 Val Ala Ala Thr Gly Leu Trp Phe Val Pro Pro Leu Ser Met Arg Leu             100 105 110 Arg Ser Val Glu Glu Leu Thr Gln Phe Phe Leu Arg Glu Ile Gln Tyr         115 120 125 Gly Ile Glu Asp Thr Gly Ile Arg Ala Gly Ile Ile Lys Val Ala Thr     130 135 140 Ala Gly Lys Ala Thr Pro Phe Gln Glu Leu Val Leu Lys Ala Ala Ala 145 150 155 160 Arg Ala Ser Leu Ala Thr Gly Val Pro Val Thr Thr His Thr Ala Ala                 165 170 175 Ser Gln Arg Asp Gly Glu Gln Gln Ala Ala Ile Phe Glu Ser Glu Gly             180 185 190 Leu Ser Pro Pro Arg Val Gly Ile Gly His Ser Asp Asp Thr Asp Asp         195 200 205 Leu Ser Tyr Leu Thr Ala Leu Ala Ala Arg Gly Tyr Leu Ile Gly Leu     210 215 220 Asp His Ile Pro His Ser Ala Ile Gly Leu Glu Asp Asn Ala Ser Ala 225 230 235 240 Ser Ala Leu Leu Gly Ile Arg Ser Trp Gln Thr Arg Ala Leu Leu Ile                 245 250 255 Lys Ala Leu Ile Asp Gln Gly Tyr Met Lys Gln Ile Leu Val Ser Asn             260 265 270 Asp Trp Leu Phe Gly Phe Ser Ser Tyr Val Thr Asn Ile Met Asp Val         275 280 285 Met Asp Arg Val Asn Pro Asp Gly Met Ala Phe Ile Pro Leu Arg Val     290 295 300 Ile Pro Phe Leu Arg Glu Lys Gly Phe Pro Gln Glu Thr Leu Ala Gly 305 310 315 320 Ile Thr Val Thr Asn Pro Ala Arg Phe Leu Ser Pro Thr Leu Arg Ala                 325 330 335 Ser     <210> 12 <211> 337 <212> PRT <213> OPH variant clone No. 777 <400> 12 Gly Ser Ile Gly Thr Gly Asn Arg Ile Asn Thr Val Arg Gly Pro Ile   1 5 10 15 Thr Ile Ser Glu Thr Gly Phe Thr Leu Thr His Glu Val Ile Cys Gly              20 25 30 Ser Ser Ala Gly Phe Leu Leu Ala Trp Pro Gln Leu Phe Cys Ser Arg          35 40 45 Lys Ala Leu Ala Glu Lys Ala Val Ser Gly Leu Arg Arg Ala Arg Ser      50 55 60 Ala Gly Val Arg Thr Ile Val Asp Val Ser Thr Phe Glu Ile Gly Arg  65 70 75 80 Asp Val Ser Leu Leu Ala Glu Val Ser Arg Ala Ala Asp Val Gln Ile                  85 90 95 Val Ala Ala Thr Gly Leu Trp Phe Asp Leu Pro Leu Ser Met Arg Leu             100 105 110 Arg Ser Val Glu Glu Leu Thr Gln Phe Leu Leu Arg Glu Ile Gln Tyr         115 120 125 Gly Ile Glu Asp Thr Gly Ile Arg Ala Gly Ile Ile Lys Val Ala Thr     130 135 140 Ala Gly Lys Ala Thr Pro Phe Gln Glu Leu Val Leu Lys Ala Ala Ala 145 150 155 160 Arg Ala Arg Leu Ala Thr Gly Val Pro Val Thr Thr His Ser Ala Ala                 165 170 175 Ser Gln Arg Asp Gly Glu Gln Gln Ala Ala Ile Phe Glu Ser Glu Gly             180 185 190 Leu Ser Pro Ser Arg Val Cys Ile Gly Leu Ser Asp Asp Thr Asp Asp         195 200 205 Leu Ser Tyr Leu Thr Ala Leu Asp Ala Arg Gly Tyr Leu Ile Gly Leu     210 215 220 Asp His Ile Pro His Ser Ala Ile Gly Leu Glu Gly Asn Ala Ser Ala 225 230 235 240 Ser Ala Leu Leu Gly Ile Arg Trp Trp Gln Thr Arg Ala Phe Leu Ile                 245 250 255 Lys Ala Ile Ile Asp Gln Gly Tyr Met Lys Gln Ile Leu Val Trp Asn             260 265 270 Asp Trp Leu Phe Gly Phe Ser Arg Tyr Val Thr Asn Ile Thr Asp Val         275 280 285 Leu Asp Gly Val Asn Pro Ala Gly Met Ala Phe Ile Pro Leu Arg Val     290 295 300 Ile Pro Phe Leu Arg Glu Lys Gly Val Pro Gln Glu Thr Leu Ala Gly 305 310 315 320 Ile Thr Val Asn Asn Pro Ala Arg Phe Leu Ser Pro Thr Leu Arg Ala                 325 330 335 Ser      

Claims (18)

delete In organophosphorus hydrolase having the amino acid sequence of SEQ ID NO: 4, the organophosphate hydrolase variant having the following substitution characteristics: (1) the 95th amino acid H (histidine) is substituted with Q (glutamine); (2) the 131th amino acid K (lysine) is substituted with E (glutamic acid); (3) the 145th amino acid T (threonine) is replaced with A (alanine); (4) the 24th amino acid T (threonine) is substituted with K (lysine); (5) the 126th amino acid I (isoleucine) is substituted with S (serine); (6) the 141th amino acid K (lysine) is substituted with M (methionine); And (7) 280th amino acid S (serine) is substituted with C (cysteine). In organophosphorus hydrolase having the amino acid sequence of SEQ ID NO: 4, the organophosphate hydrolase variant having the following substitution characteristics: (1) the 95th amino acid H (histidine) is substituted with Q (glutamine); (2) the 131th amino acid K (lysine) is substituted with E (glutamic acid); (3) the 145th amino acid T (threonine) is replaced with A (alanine); (8) the 11th amino acid T (threonine) is substituted with A (alanine); (9) the 33rd amino acid S (serine) is substituted with N (asparagine); (10) the 52nd amino acid A (alanine) is substituted with S (serine); (11) 75th amino acid T (threonine) is substituted with S (serine); (12) the 118th amino acid L (leucine) is substituted with F (phenylalanine); (13) 165th amino acid A (alanine) is substituted with V (valine); (14) the 210th amino acid S (serine) is substituted with R (arginine); And (15) 246th amino acid I (isoleucine) is substituted for N (asparagine). In organophosphorus hydrolase having the amino acid sequence of SEQ ID NO: 4, the organophosphate hydrolase variant having the following substitution characteristics: (1) the 95th amino acid H (histidine) is substituted with Q (glutamine); (2) the 131th amino acid K (lysine) is substituted with E (glutamic acid); (3) the 145th amino acid T (threonine) is replaced with A (alanine); (16) the 105th amino acid D (aspartic acid) is substituted with V (valine); (17) the 196th amino acid S (serine) is substituted with P (proline); (18) the 199th amino acid C (cysteine) is substituted with G (glycine); And (19) 313th amino acid V (valine) is substituted for F (phenylalanine). In organophosphorus hydrolase having the amino acid sequence of SEQ ID NO: 4, the organophosphate hydrolase variant having the following substitution characteristics: (1) the 95th amino acid H (histidine) is substituted with Q (glutamine); (2) the 131th amino acid K (lysine) is substituted with E (glutamic acid); (3) the 145th amino acid T (threonine) is replaced with A (alanine); (20) seventh amino acid D (aspartic acid) is substituted with N (asparagine); (21) the 21st amino acid A (alanine) is substituted with T (threonine); (22) the 29th amino acid H (histidine) is substituted with V (valine); (23) the 39th amino acid R (arginine) is substituted with L (leucine); (24) the 44th amino acid F (phenylalanine) is substituted with L (leucine); (25) the 46th amino acid G (glycine) is substituted with C (cysteine); (26) the 57th amino acid R (arginine) is substituted with S (serine); (27) the 64 th amino acid A (alanine) is substituted with S (serine); (28) the 106th amino acid P (proline) is substituted with L (leucine); (29) the 122nd amino acid F (phenylalanine) is substituted with L (leucine); (30) 163rd amino acid S (serine) is substituted with R (arginine); (31) the 174th amino acid T (threonine) is substituted for S (serine); (32) the 202th amino acid H (histidine) is substituted with L (leucine); (33) 216th amino acid A (alanine) is substituted with D (aspartic acid); (34) 236 th amino acid D (aspartic acid) is substituted with G (glycine); (35) 248th amino acid S (serine) is substituted with W (tryptophan); (36) the 254th amino acid L (leucine) is substituted with F (phenylalanine); (37) 259th amino acid L (leucine) is substituted with I (isoleucine); (38) 271st amino acid S (serine) is substituted with W (tryptophan); (39) the 280th amino acid S (serine) is substituted with R (arginine); (40) the 286th amino acid M (methionine) is substituted with T (threonine); (41) the 289th amino acid M (methionine) is substituted with L (leucine); (42) the 291th amino acid R (arginine) is substituted with G (glycine); (43) the 295th amino acid D (aspartic acid) is substituted with A (alanine); And (44) 324 th amino acid T (threonine) is substituted for N (asparagine). The organophosphate compound hydrolase variant according to claim 2, which has an amino acid sequence represented by SEQ ID NO: 9. The organophosphate compound hydrolase variant according to claim 3, which has an amino acid sequence represented by SEQ ID NO: 10. 5. An organophosphate compound hydrolase variant according to claim 4, having an amino acid sequence of SEQ ID NO. The organophosphate compound hydrolase variant according to claim 5, which has an amino acid sequence represented by SEQ ID NO: 12. A gene encoding the organophosphate compound hydrolase variant of any one of claims 2 to 5. The gene of claim 10, wherein the gene has any one of SEQ ID NOs: 5 to 8. A recombinant vector containing a gene encoding the organophosphate compound hydrolase variant of claim 10. The recombinant microorganism in which the gene of claim 10 is inserted into a host cell selected from the group consisting of bacteria, fungi and yeasts. The recombinant microorganism of claim 13, wherein the host cell is Escherichia coli. Culturing the recombinant microorganism of claim 13 to express the organophosphate compound hydrolase variant; And recovering the organophosphate compound hydrolase variant from the culture. In the biosensor for detecting an organophosphate compound, A biosensor for detecting an organophosphate compound according to any one of claims 2 to 5, wherein the organophosphate compound hydrolase variant is immobilized. The method of claim 16, wherein the organophosphate compound is paraoxon, parathion, azametiphos, azinphos, bensulide, malaoxon, malitone (18). malathion, heptenophos, dichlorvos, dialifos, diazinon, dichlofenthion, dimethoate, dimethylvinphos ), Dioxabenzofos, disulfoton, demethon S, edifenphos, ethephon, ethion, ethoprophos, Chlorfenvinphos, chlorpyrifos, fenitrothion, fenthion, phonofos, formothion, heptenophos, and iprobenfos iprobenfos, isazofos, isoxathion, mecarbam, metamidophos, Metidathion, monocrotophos, naled, omethoate, phoxim, pirimphos, profenofos, propaphos ), Prothiofos, pyraclofos, pyrazophos, pyridaphenthion, quinalphos, terbufos, tetrachlorvinphos , Thiometon, tolclofos, triazophos, trichlorphon, vamidothion, and coumaphos Biosensor for organophosphate compound detection. A method for detecting an organophosphate compound, comprising using the biosensor for detecting the organophosphate compound of claim 16.

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