KR102012253B1 - Modified peroxidase gene and method of preparing the same - Google Patents

Abstract

The present invention relates to a codon-optimized peroxidase mutant gene and a method for preparing the same, and more particularly to codon-optimized plant-derived peroxidase nucleic acid molecule, a recombinant vector comprising the same and a prokaryotic cell transformed with the recombinant vector. It is about. The present invention has the advantage of supplying enzymes cheaply and continuously by improving the expression rate of peroxidase in E. coli by replacing genetic agricultural production with conventional agricultural peroxidase production, which requires cultivation conditions and land.

Description

Peroxidase mutant gene and method for preparing the same {MODIFIED PEROXIDASE GENE AND METHOD OF PREPARING THE SAME}

The present invention relates to a peroxidase mutant gene and a method for preparing the same, and more particularly, a nucleic acid molecule modified to be highly expressed in a prokaryotic cell, a recombinant vector comprising the same, and a transformation with the recombinant vector. It relates to a prokaryotic cell, and a method of producing plant-derived peroxidase in prokaryotic cells using the peroxidase mutant gene.

Genetic information of living organisms is stored in DNA, except for some viruses, and recorded by four base sequences: adenine, guanine, cytosine, and thymine. Three consecutive bases are transcribed and translated into one amino acid, leaving about 60 codons at 4X4X4 = 64 possible codons, except for the codons that indicate the start and end of transcription. As a result, the number of codons that determine one amino acid may exist from one to six, depending on the amino acid. By the way, the use frequency of the codon which determines a specific amino acid is not the same, and there exists a some difference. For example, there are six codons that determine the amino acid leucine, CTT, CTC, CTA, CTG, TTA, and TTG.However, when leucine is produced in E. coli, the stomach codons are 10% and 10%, respectively. , 3%, 55%, 11%, 11%. In other words, more than half of the genotypes of phenotypes expressed in leucine use CTG codons. The codon usage of other amino acids is varied, with some amino acids in which TTT and TTC codons are used at about the same frequency as phenylalanine, and AGG codons have only 3% of the codons for the amino acid arginine. .

Trends in codon usage of amino acids have been studied in various ways and show different codon biases depending on mammals, plants, E. coli, and the like. The peroxidase gene reported in the literature is plant-derived and shows very large differences from codon bias in E. coli. However, genes that are not tailored to codon deflection significantly decrease expression into proteins. This is due to the amount of tRNA according to the individual codons. According to a series of reports, optimizing codons for the organisms in which the gene is to be expressed is known to significantly increase the amount of protein expressed.

Meanwhile, peroxidase is an enzyme that mediates hydrogen peroxide to oxidize other substrates and exhibits very stable and powerful activity. Based on this, it is very widely used as a labeling material for various biochemical and clinical analyses. However, most of the commercially available peroxidases are extracted from plants and have a very rare problem in mass production of genetic engineering. This is because the codons of the peroxidase gene derived from plants are very different from the preferred codons of conventional genetic engineering production systems such as E. coli.

The present invention is to improve the expression rate of peroxidase in E. coli to replace the existing agricultural peroxidase production required for cultivation conditions and land to genetic engineering production to enable a cheap and sustainable supply.

Therefore, one embodiment of the present invention is to provide a nucleic acid molecule of codon-optimized plant-derived peroxidase.

Another embodiment of the present invention is to provide a recombinant vector comprising the nucleic acid molecule and a prokaryotic cell transformed with the recombinant vector, and a method for producing a large amount of plant-derived peroxidase from prokaryotic cells using the same.

In addition, one embodiment of the present invention is to provide a method for producing a plant-derived peroxidase mutant gene, which improves the expression rate of peroxidase in E. coli.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above object, one embodiment of the present invention is a codon-optimized plant-derived peroxidase nucleic acid molecule, preferably the base sequence is modified to the base sequence shown in Table 1, codon-optimized plant-derived peroxidation Provides enzyme nucleic acid molecules.

In addition, another embodiment of the present invention provides a recombinant vector comprising the nucleic acid molecule and a prokaryotic cell transformed with the recombinant vector.

In addition, another embodiment of the present invention provides a method for producing a plant-derived peroxidase using the prokaryotic cells.

The present inventors have completed the present invention for mass production of peroxidase according to codon optimization based on the fact that there are several orders of magnitude difference in protein expression according to the optimization of the expression codon. Therefore, the codon-optimized peroxidase mutant gene can be introduced into prokaryotic cells, preferably E. coli, to produce peroxidase in large quantities by genetic engineering.

Hereinafter, the present invention will be described in detail.

One embodiment of the invention provides nucleic acid molecules of codon-optimized peroxidase. The nucleic acid molecule is a nucleic acid molecule modified with a base sequence having a codon optimized for E. coli in consideration of the codon deflection of the host cell, preferably E. coli.

In a preferred embodiment, the nucleic acid molecule is a nucleotide sequence CCT encoding proline, which is the fifth amino acid in SEQ ID NO: 1, to CCG, a base sequence ACA that encodes the sixth amino acid, threonine, to ACC; Nucleotide sequence of AGC is transformed into TCT, nucleotide sequence of 13th amino acid proline is transformed to CCG, nucleotide sequence of the 22nd amino acid threonine is transformed into ACG, and the 26th amino acid is glutamic acid The sequence GAG may be modified with GAA, and the base sequence AGA encoding the 28th amino acid arginine may be modified with CGT.

5th, 6th, 11th, 13th, 22nd, 26th, and 28th of 77 amino acids of SEQ ID NO: 1 are modified, and additionally among the base sequences encoding 1 to 70 amino acids (Table 1 below) One or more may be a modified codon optimized plant derived peroxidase nucleic acid molecule.

location
(SEQ ID NO 1) amino acid Before transformation After transformation location
(SEQ ID NO 1) amino acid Before transformation After deformation 5th Proline CCT CCG 80th Valine GTG GTT 6th Threonine ACA ACC 85th Lee Sin AAG AAA 7th Phenylalanine TTC TTT 86th Alanine GCT GCG 8th Tyrosine TAC TAT 89th Glutamic acid GAG GAA 9th Aspartic acid GAC GAT 93rd Proline CCA CCG 11th Serine AGC TCT 94th Arginine CGA CGC 13th Proline CCC CCG 95th Threonine ACA ACG 14th Asparagine AAC AAT 97th Serine AGT TCT 15th Valine GTG GTT 104th Isoleucine ATA ATT 16th Serine TCC TCA 110th Valine GTG GTA 17th Asparagine AAC AAT 115th Glycine GGA GGC 21st Aspartic acid GAC GAT 119th Arginine AGA CGT 22nd Threonine ACA ACG 120th Valine GTG GTA 25th Asparagine AAC AAT 124th Arginine CGA CGC 26th Glutamic acid GAG GAA 128th Leucine CTA CTG 27th Leucine CTC TTG 132th Leucine CTA CTT 28th Arginine AGA CGT 142 th Proline CCA CCG 31st Proline CCC CCG 150th Lee Sin AAG AAA 32nd Arginine AGG CGC 154th Arginine AGA CGT 34th Alanine GCT GCG 156th Valine GTG GTA 35th Alanine GCT GCG 162th Serine AGT TCG 37th Isoleucine ATA ATT 169 th Glycine GGA GGC 41st Histidine CAC CAT 170 th Glycine GGA GGT 42nd Phenylalanine TTC TTT 172 th Threonine ACA ACG 44th Aspartic acid GAC GAT 174th Glycine GGA GGT 46th Phenylalanine TTC TTT 175th Lee Sin AAG AAA 47th Valine GTG GTT 179th Arginine AGG CGC 52nd Alanine GCT GCG 184th Arginine AGG CGC 54th Isoleucine ATA ATT 205th Threonine ACA ACG 58th Asparagine AAC AAT 207th Arginine AGA CGT 61th Serine AGT TCT 210 th Proline CCA CCG 64th Threonine ACT ACC 217th Serine AGT TCG 66th Lee Sin AAG AAA 219th Leucine CTA CTT 68th Alanine GCA GCG 225th Arginine CGG CGT 71st Asparagine AAC AAT 238th Leucine CTA TTG 72nd Alanine GCT GCG 245 th Isoleucine ATA ATT 75th Alanine GCC GCG 265th Arginine AGA CGT 76th Arginine AGG CGC 303 th Arginine AGA CGR 79th Proline CCA CCG

Most preferred example may be a codon-optimized peroxidase nucleic acid molecule consisting of SEQ ID NO: 2, the base sequence having SEQ ID NO: 2 is shown in FIG. SEQ ID NO: 2 is the optimal codonization of 390 amino acids of Western mustard peroxidase according to the codon bias of E. coli. Therefore, nucleic acid molecules show high expression of peroxidase in host cell E. coli due to the optimal codonization.

In another embodiment of the present invention also provides a recombinant vector comprising the nucleic acid molecule. The vector typically includes a delivery DNA into which an external DNA can be inserted, and means that a nucleic acid molecule can be combined with another nucleic acid and transferred to a host cell to express a target protein. Such vectors include all conventional vectors, including plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like.

In another embodiment of the present invention provides a prokaryotic cell transformed with the recombinant vector. The recombinant vector may be introduced into prokaryotic cells, and the introduction method may include electroshock gene transfer (electroporation), calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, PEG, textlan sulfate, lipofectamine, and the like. It can be carried out by a known method of.

The prokaryotic cells may be prokaryotic cells that can be transformed with a foreign gene, for example, Escherichia coli, Rhodococcus, Pseudomonas, Streptomyces, Staphylococcus ( It may include various microorganisms such as Staphylococcus, Syfolobus, Thermoplasma, Thermoproroteus, preferably from the group consisting of E. coli and Saccharomyces cerevisiae. Can be selected.

Preferably, it may be E. coli, and may specifically include E. coli XL1-blue, E. coli BL21 (DE3), E. coli JM109, E. coli DH series, E. coli TOP10 and E. coli HB101.

In addition, another example of the present invention provides a method for producing a plant-derived peroxidase using the prokaryotic cells.

Conditions such as media components, culture temperature and culture time can be appropriately adjusted to culture the transformed prokaryotic cells. Specifically, the culture medium is a carbon source, nitrogen source, trace amount

It may contain all the nutrients necessary for the growth and survival of microorganisms such as elemental components. The pH of the medium can be adjusted appropriately and may include components such as antibiotics.

In addition, an inducer such as isopropyl beta dithiogalactopyranoside (hereinafter referred to as IPTG) can be treated to induce the expression of peroxidase. The type of inducer to be treated may be determined according to the vector system, and conditions such as the induction agent administration time and dosage may be appropriately controlled. The selection of the conditions such as the medium component, the incubation temperature and the incubation time can be appropriately determined depending on the type of prokaryotic cell used.

The expressed peroxidase is recovered and purified by conventional methods. For example, the cells recovered by the centrifugation method can be crushed using a French press, an ultrasonic crusher or the like. If superoxide is secreted into the culture, the culture supernatant can be collected. If aggregated by overexpression, it can be obtained by dissolving and denaturing and refolding the peroxidase in a suitable solution. Oxidation and reduction systems of glutathione, dithiothreitol, β-mercaptoethanol, β-mercaptomethanol, cystine and cystamine can be used, and refolding agents such as urea, guanidine, arginine and the like can be used. Some of the salts may be used with the refolding agent.

At this time, the step of heat treatment for 10 minutes to 60 minutes at 70 ℃ to 85 ℃ may be added.

The production scale of the peroxidase can be adjusted to suit the purpose.

The codon-optimized plant-derived peroxidase nucleic acid molecule of the present invention exhibits high peroxidase expression in conventional genetic engineering production systems, e.g., E. coli, and therefore, conventional agricultural peroxidase production requiring cultivation conditions and land. There is an advantage that can be replaced by genetic engineering to enable cheap and sustainable supply.

Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 3) of Western mustard-derived peroxidase.
Figure 2 shows the base sequence (SEQ ID NO: 2) and amino acid sequence (SEQ ID NO: 3) of the codon optimized peroxidase according to an embodiment of the present invention.
Figure 3 shows the peroxidase base sequence and amino acid sequence before and after codon optimization.
4 shows a cloning map of the peroxidase gene.
5 shows SDS-PAGE results according to Example 3 and Comparative Example 1. FIG.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

EXAMPLE

Example  1. Codon Optimization Design

Codon optimization of the horseradish peroxidase gene was performed. As shown in FIG. 1, horseradish peroxidase (SEQ ID NO: 1), which is started with methionine, consists of 309 amino acids ending with serine, and the last TAA is a stop codon.

Table 2 shows the amino acid name of the codons encoding the individual amino acids of the mustard radish peroxidase, the frequency of the codons used to code the amino acids, the percentage of use of the codon in E. coli, and the percentage of redistribution The new frequency of each codon was shown.

amino acid division One 2 3 4 5 6 Alanine Codon GCT GCA GCC GCG Number of preoxidation codons used before optimization 9 6 7 One Codon usage frequency of Escherichia coli (%) 19 25 22 34 Redistributed codon usage 4 5 6 8 Cysteine Codon TGC TGT Number of preoxidation codons used before optimization 5 3 Codon usage frequency of Escherichia coli (%) 57 43 Redistributed codon usage 5 3 Aspartate Codon GAC GAT Number of preoxidation codons used before optimization 12 9 Codon usage frequency of Escherichia coli (%) 41 59 Redistributed codon usage 9 12 Glutamate Codon GAA GAG Number of preoxidation codons used before optimization 3 4 Codon usage frequency of Escherichia coli (%) 70 30 Redistributed codon usage 5 2 Phenylalanine Codon TTT TTC Number of preoxidation codons used before optimization 7 13 Codon usage frequency of Escherichia coli (%) 51 49 Redistributed codon usage 10 10 Glycine Codon GGT GGC GGA GGG Number of preoxidation codons used before optimization 5 6 4 2 Codon usage frequency of Escherichia coli (%) 38 40 9 13 Redistributed codon usage 7 8 0 2 Histidine Codon CAT CAC Number of preoxidation codons used before optimization One 2 Codon usage frequency of Escherichia coli (%) 52 48 Redistributed codon usage 2 One Isoleucine Codon ATC ATT ATA Number of preoxidation codons used before optimization 7 2 4 Codon usage frequency of Escherichia coli (%) 46 47 7 Redistributed codon usage 7 6 0 Lysine Codon AAA AAG Number of preoxidation codons used before optimization One 5 Codon usage frequency of Escherichia coli (%) 76 24 Redistributed codon usage 5 One Leucine Codon CTG CTT CTC TTA TTG CTA Number of preoxidation codons used before optimization 18 2 5 4 2 4 Codon usage frequency of Escherichia coli (%) 55 10 10 11 11 3 Redistributed codon usage 19 4 4 4 4 0 Methionine Codon ATG Number of preoxidation codons used before optimization 5 Codon usage frequency of Escherichia coli (%) 100 Redistributed codon usage 5 Asparagine Codon AAC AAT Number of preoxidation codons used before optimization 21 6 Codon usage frequency of Escherichia coli (%) 61 39 Redistributed codon usage 16 11 Proline Codon CCG CCA CCT CCC Number of preoxidation codons used before optimization 2 7 4 4 Codon usage frequency of Escherichia coli (%) 55 20 16 10 Redistributed codon usage 9 3 3 2 Glutamine Codon CAG CAA Number of preoxidation codons used before optimization 9 4 Codon usage frequency of Escherichia coli (%) 69 31 Redistributed codon usage 9 4 Arginine Codon CGT CGC CGA CGG AGA AGG Number of preoxidation codons used before optimization 4 4 2 One 6 4 Codon usage frequency of Escherichia coli (%) 42 37 5 8 4 3 Redistributed codon usage 11 10 0 0 0 0 Serine Codon TCT TCC TCA TCG AGT AGC Number of preoxidation codons used before optimization 2 5 2 One 7 8 Codon usage frequency of Escherichia coli (%) 19 17 12 13 13 27 Redistributed codon usage 5 4 3 3 3 7 Throneine Codon ACC ACT ACA ACG Number of preoxidation codons used before optimization 10 7 5 3 Codon usage frequency of Escherichia coli (%) 48 24 2 26 Redistributed codon usage 12 6 0 7 Valine Codon GTT GTG GTC GTA Number of preoxidation codons used before optimization 2 12 3 0 Codon usage frequency of Escherichia coli (%) 29 34 20 17 Redistributed codon usage 5 6 3 3 Tryptophan Codon TGG Number of preoxidation codons used before optimization One Codon usage frequency of Escherichia coli (%) 100 Redistributed codon usage One Tyrosine Codon TAT TAC Number of preoxidation codons used before optimization 2 3 Codon usage frequency of Escherichia coli (%) 53 47 Redistributed codon usage 3 2

As shown in Table 2, there are 23 alanines in horseradish peroxidase 390 amino acids. Alanine uses a total of four codons, while horseradish peroxidase uses 9, 6, 7, and 1 GCT, GCA, GCC, and GCG codons, respectively. On the other hand, in the case of E. coli, the frequency of using each codon to encode alanine is known to be 19%, 22%, 23%, 32%. Based on this, 23 amino acids were redistributed and used 4, 5, 6 and 8 times, respectively. On the other hand, codons with a frequency of less than 10% were added to the highest frequency amino acid by not using it.

Codon optimization has a significant effect on gene expression, especially as it is closer to the N-terminal (Nucleic Acids Research, vol. 18, no. 6, 1465-1473,1990), which indicates that the smoothness of tRNA in the early This means that the supply is important, and based on this, the codon frequency was changed from the codon close to the N-terminal and the codons were changed from 77 codons (SEQ ID NO: 2). The mutant gene sequence of the peroxidase containing the modified codon is shown in FIG. 2.

Example  2. Codon-Optimized Peroxidase Mutation Gene Preparation

2.1 Preparation of Expression Vectors

Codon-optimized genes according to Example 1 were synthesized in a single body using Bioneer's gene synthesis service. Recognition sites of specific restriction enzymes, ie, BstBI and NotI, were inserted at both ends for easy handling of DNA fragments, and the DNA fragments were inserted into pGEM-B1 or pGEM-B2 as shown in FIG. Used after amplification and purification.

To transfer the peroxidase gene to the expression vector, the previously inserted specific restriction enzyme recognition site was cut out by treatment with the corresponding BstBI and NotI. After treatment with pET-45 (+) with the BstBI and NotI, the DNA fragments were mixed and linked using T4 DNA ligase. Whether the expression vector contained peroxidase was determined by miniprep. It was confirmed using the method.

2.2 Transduction

The expression vector containing the peroxidase mutant gene prepared in Example 2.1 is Escherichia which is an expression strain through the calcium chloride method coli BL21 (DE3) was transduced.

Example  3. Codon Optimized In E. coli  Peroxidase Expression

Escherichia with Expression Vector coli BL21 (DE3) strain (Example 2.2) was isolated from LB-agar / Amp. The medium was incubated overnight at 37 ° C. Cultured Escherichia coli BL21 (DE3) strain colonies were identified as LB / Amp. The medium was incubated overnight at 37 ° C. 200ul of cultured liquid strain was added to 4ml LB / Amp medium and incubated at 37 ° C for 4-6 hours.

When the absorbance at 600 nm wavelength of the culture reached 0.5 to 1.0, the expression was induced by adding IPTG to 1mM, and further cultured at 37 ℃ 2 hours after the addition of IPTG.

Peroxidase expression was confirmed by SDS-PAGE. Since the expressed peroxidase has a molecular weight of about 34 kD, the presence or absence of expression was found by comparing with before and after expression of IPTG in comparison with the molecular weight marker. The peroxidase band on SDS-PAGE was measured using ImageJ, and the results are shown in FIG. 5.

Comparative example  One. In E. coli  Peroxidase Expression

Escherichia not transformed in Example 3 Except for using coli BL21 (DE3) was cultured using the same method, and the expressed peroxidase was observed through SDS-PAGE, the results are shown in FIG.

As shown in FIG. 5, it was confirmed that the codon-optimized peroxidase expression amount increased by about 4.7 to 6.3 times than the codon-optimized expression amount before codon optimization for E. coli expression.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

<110> ELECTRONICS AND TELECOMMUNICATIONS RESEARCH INSTITUTE <120> MODIFIED PEROXIDASE GENE AND METHOD OF PREPARING THE SAME <130> DPP20127095KR <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 930 <212> DNA <213> Artificial Sequence <220> <223> horseradish peroxidase <400> 1 atgcagttaa cccctacatt ctacgacaat agctgtccca acgtgtccaa catcgttcgc 60 gacacaatcg tcaacgagct cagatccgat cccaggatcg ctgcttcaat attacgtctg 120 cacttccatg actgcttcgt gaatggttgc gacgctagca tattactgga caacaccacc 180 agtttccgca ctgaaaagga tgcattcggg aacgctaaca gcgccagggg ctttccagtg 240 atcgatcgca tgaaggctgc cgttgagtca gcatgcccac gaacagtcag ttgtgcagac 300 ctgctgacta tagctgcgca acagagcgtg actcttgcag gcggaccgtc ctggagagtg 360 ccgctcggtc gacgtgactc cctacaggca ttcctagatc tggccaacgc caacttgcct 420 gctccattct tcaccctgcc ccagctgaag gatagcttta gaaacgtggg tctgaatcgc 480 tcgagtgacc ttgtggctct gtccggagga cacacatttg gaaagaacca gtgtaggttc 540 atcatggata ggctctacaa tttcagcaac actgggttac ctgaccccac gctgaacact 600 acgtatctcc agacactgag aggcttgtgc ccactgaatg gcaacctcag tgcactagtg 660 gactttgatc tgcggacccc aaccatcttc gataacaagt actatgtgaa tctagaggag 720 cagaaaggcc tgatacagag tgatcaagaa ctgtttagca gtccaaacgc cactgacacc 780 atcccactgg tgagaagttt tgctaactct actcaaacct tctttaacgc cttcgtggaa 840 gccatggacc gtatgggtaa cattacccct ctgacgggta cccaaggcca gattcgtctg 900 aactgcagag tggtcaacag caactcttaa 930 <210> 2 <211> 930 <212> DNA <213> Artificial Sequence <220> <223> modified (codon-optimized) peroxidase <400> 2 atgcagttaa ccccgacctt ttatgataat tcttgtccga atgtttcaaa tatcgttcgc 60 gatacgatcg tcaatgaatt gcgttccgat ccgcgcatcg cggcgtcaat tttacgtctg 120 cattttcatg attgctttgt taatggttgc gacgcgagca ttttactgga caataccacc 180 tctttccgca ccgaaaaaga tgcgttcggg aatgcgaaca gcgcgcgcgg ctttccggtt 240 atcgatcgca tgaaagcggc cgttgaatca gcatgcccgc gcacggtctc ttgtgcagac 300 ctgctgacta ttgctgcgca acagagcgta actcttgcag gcggcccgtc ctggcgtgta 360 ccgctcggtc gccgtgactc cctgcaggca ttccttgatc tggccaacgc caacttgcct 420 gctccgttct tcaccctgcc ccagctgaaa gatagctttc gtaacgtagg tctgaatcgc 480 tcgtcggacc ttgtggctct gtccggcggt cacacgtttg gtaaaaacca gtgtcgcttc 540 atcatggatc gcctctacaa tttcagcaac actgggttac ctgaccccac gctgaacact 600 acgtatctcc agacgctgcg tggcttgtgc ccgctgaatg gcaacctctc ggcacttgtg 660 gactttgatc tgcgtacccc aaccatcttc gataacaagt actatgtgaa tttggaggag 720 cagaaaggcc tgattcagag tgatcaagaa ctgtttagca gtccaaacgc cactgacacc 780 atcccactgg tgcgtagttt tgctaactct actcaaacct tctttaacgc cttcgtggaa 840 gccatggacc gtatgggtaa cattacccct ctgacgggta cccaaggcca gattcgtctg 900 aactgccgtg tggtcaacag caactcttaa 930 <210> 3 <211> 309 <212> PRT <213> Artificial Sequence <220> <223> peroxidase <400> 3 Met Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn Val Ser   1 5 10 15 Asn Ile Val Arg Asp Thr Ile Val Asn Glu Leu Arg Ser Asp Pro Arg              20 25 30 Ile Ala Ala Ser Ile Leu Arg Leu His Phe His Asp Cys Phe Val Asn          35 40 45 Gly Cys Asp Ala Ser Ile Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr      50 55 60 Glu Lys Asp Ala Phe Gly Asn Ala Asn Ser Ala Arg Gly Phe Pro Val  65 70 75 80 Ile Asp Arg Met Lys Ala Ala Val Glu Ser Ala Cys Pro Arg Thr Val                  85 90 95 Ser Cys Ala Asp Leu Leu Thr Ile Ala Ala Gln Gln Ser Val Thr Leu             100 105 110 Ala Gly Gly Pro Ser Trp Arg Val Pro Leu Gly Arg Arg Asp Ser Leu         115 120 125 Gln Ala Phe Leu Asp Leu Ala Asn Ala Asn Leu Pro Ala Pro Phe Phe     130 135 140 Thr Leu Pro Gln Leu Lys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg 145 150 155 160 Ser Ser Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Asn                 165 170 175 Gln Cys Arg Phe Ile Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly             180 185 190 Leu Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln Thr Leu Arg Gly         195 200 205 Leu Cys Pro Leu Asn Gly Asn Leu Ser Ala Leu Val Asp Phe Asp Leu     210 215 220 Arg Thr Pro Thr Ile Phe Asp Asn Lys Tyr Tyr Val Asn Leu Glu Glu 225 230 235 240 Gln Lys Gly Leu Ile Gln Ser Asp Gln Glu Leu Phe Ser Ser Pro Asn                 245 250 255 Ala Thr Asp Thr Ile Pro Leu Val Arg Ser Phe Ala Asn Ser Thr Gln             260 265 270 Thr Phe Phe Asn Ala Phe Val Glu Ala Met Asp Arg Met Gly Asn Ile         275 280 285 Thr Pro Leu Thr Gly Thr Gln Gly Gln Ile Arg Leu Asn Cys Arg Val     290 295 300 Val Asn Ser Asn Ser 305

Claims (7)

The base sequence CCT encoding proline, the fifth amino acid of SEQ ID NO: 1, is transformed into CCG, the base sequence ACA encoding the threonine, which is the sixth amino acid, is transformed into ACC, and the base sequence AGC, encoding the eleventh amino acid, is converted to TCT. Modified, base sequence CCC encoding 13th amino acid proline to CCG, base sequence ACA encoding threonine 22nd amino acid to ACG, base sequence GAG encoding glutamic acid 26th amino acid to GAA, And a nucleic acid molecule of peroxidase wherein the base sequence AGA encoding the arginine as the 28th amino acid is modified with CGT. The method of claim 1,
The nucleic acid molecule is a nucleic acid molecule consisting of SEQ ID NO: 2. A recombinant vector comprising the nucleic acid molecule of claim 1. The method of claim 3,
Wherein said vector is selected from the group consisting of plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. E. coli transformed with the recombinant vector of claim 3, transformed E. coli. The method of claim 5,
The E. coli is selected from the group consisting of E. coli XL1-blue, E. coli BL21 (DE3), E. coli JM109, E. coli DH series, E. coli TOP10 and E. coli HB101, transformed E. coli. A method for preparing a peroxidase using the transformed Escherichia coli of claim 5.

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