JP2008253248A - Oxidation-resistant ascorbate peroxidase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an oxidation-resistant ascorbate peroxidase (ascorbate peroxidase), to provide a method for producing the same, and to provide a method for imparting oxidation resistance to the ascorbate peroxidase. <P>SOLUTION: The oxidation-resistant ascorbate peroxidase has an amino acid sequence in which cysteine of the amino acid in the position corresponding to the 25th position of at least a specific sequence is substituted with an amino acid other than the cysteine in the amino acid sequence of the un-modified ascorbate peroxidase. The method for producing the ascorbate peroxidase is provided. The method for imparting the oxidation resistance to the ascorbate peroxidase comprises a step of substituting the cysteine of the amino acid in the position corresponding to the 25th position of at least the specific sequence with the amino acid other than the cysteine in the amino acid sequence of the un-modified ascorbate peroxidase. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a novel oxidation resistant ascorbate peroxidase, a method for producing the same, and a method for imparting oxidation resistance to ascorbate peroxidase.

Ascorbate peroxidase (also referred to as “ascorbate peroxidase”; hereinafter abbreviated as “APX”) is a member of the type I heme peroxidase gene family found in eukaryotic photosynthetic organisms from algae to higher plants It is. APX reduces hydrogen peroxide (H ₂ O ₂ ) to water (H ₂ O) using ascorbic acid as an electron donor. Specifically, when APX reacts with one molecule of hydrogen peroxide, the ferric (Fe ^III ) atom of heme is oxidized to oxyferyl (Fe ^IV ═O), and the porphyrin is referred to as a cation radical intermediate (Compound I). ) This intermediate is then converted to ground-state iron (Fe ^III ) by a two-step reaction with two molecules of ascorbic acid, resulting in two molecules of monodehydroascorbic acid radicals as a by-product.

  There are multiple isoforms in APX of higher plants. Of these, chloroplast localized APX is sensitive to hydrogen peroxide, while cytoplasmic localized APX is resistant to hydrogen peroxide. In higher plant chloroplasts, there are two APX isoforms, one localized to the stroma and the other bound to the stromal side of the thylakoid membrane. All of these are very sensitive to hydrogen peroxide, and when the ascorbic acid as a reducing agent is deficient, it is deactivated within a few minutes by excess hydrogen peroxide (Non-patent Document 1). The APX (APXB) obtained by the present inventors from the red alga Gardieria partita has sequence similarity to the chloroplast APX of higher plants in the N-terminal domain (Non-patent Document 2). Like APX isoforms localized in cytosols and microbodies of higher plants, they are relatively resistant to hydrogen peroxide (Non-patent Document 3).

  Since plants cannot move, they are always exposed to stresses such as high temperature, low temperature, and drought. Under extreme environmental conditions, plants suffer from obstacles such as growth inhibition and death. One of the causes of these disorders is known to be active oxygen generated in the plant body. For the purpose of removing this active oxygen, attempts have been made to extinguish hydrogen peroxide using APX to impart stress tolerance to plants (Patent Documents 1-2 and 4-8). However, as described above, APX is easily inactivated, and a stress-tolerant plant as expected is not obtained.

  In the above APX reaction, if ascorbic acid as a reducing agent does not exist and the cation radical intermediate is not reduced, the radical is transferred to the amino acid residue of the apoprotein. It is believed that the deactivation of APX is due to such attack of the reaction intermediate by hydrogen peroxide. Thus, information on intraprotein radical transfer in APX is necessary to understand the mechanism of hydrogen peroxide-mediated deactivation and the sensitivity of APX isoforms to hydrogen peroxide.

  For example, thiol groups of cysteine residues in proteins are known to be oxidized in vivo (Non-Patent Documents 9-10), and the relationship between APX deactivation and individual amino acid residues is described in more detail. It is necessary to investigate. However, although there is a report that a mutation was introduced into a cysteine residue in APX in order to investigate the ascorbic acid binding site of APX (Non-patent Document 11), the amino acid residues involved in intra-protein radical transfer in APX are reported. There was little knowledge.

  The inventors have determined that the tryptophan residue facing the distal cavity of the catalytic site is a radical site in the chloroplast APX, and that when APX is inactivated by excess hydrogen peroxide, tryptophan. Based on the knowledge that the residue is cross-linked to heme (Patent Document 3). However, other radical sites in APX have not been fully investigated, and based on the finding that a residue that appears to be a proximal tryptophan is hydroxylated by treatment with hydrogen peroxide, bean cytosolic APX There is only a report suggesting that the radical site is a proximal tryptophan residue (Non-patent Document 12).

JP 2003-009692 A JP 2004-105136 A JP 2006-296209 A Asada, K., Annu. Rev. Plant Physiol. Plant Mol. Biol., 50: 601-639 (1999) Kitajima, S. et al., Biosic. Biotechnol. Biochem., 66: 2367-2375 (2002) Kitajima, S. et al., FEBS J., 273, 2704-2710 (2006) Kornyeyev, D. et al., Physiol. Plant, 113: 323-331 (2001) Payton, P. et al., J. Exp. Bot., 52: 2345-2354 (2001) Yabuta, Y. et al., Plant J., 32: 915-925 (2002) Murgia, I. et al., Plant J., 38: 940-953 (2004) Badawi, G.H. et al., Physiol.Plant, 121: 231-238 (2004) Ghezzi, P., Biochem. Soc. Trans., 33: 1378-1381 (2005) Spickett, C.M. et al., Biochim. Biopys. Acta, 1764: 1823-1841 (2006) Mandelman, D. et al., Biochemistry, 37: 17610-17617 (1998) Hiner, A.N. et al., Eur. J. Biochem., 268: 3091-3098 (2001)

  An object of the present invention is to provide a novel oxidation resistant ascorbate peroxidase, a method for producing the same, and a method for imparting oxidation resistance to ascorbate peroxidase.

The present invention relates to:
[1] An oxidation-resistant ascorbate peroxidase having an amino acid sequence in which at least the amino acid corresponding to position 25 of SEQ ID NO: 1 is substituted with an amino acid other than cysteine in the amino acid sequence of unmodified ascorbate peroxidase;
[2] The oxidation-resistant ascorbate peroxidase according to [1], wherein the unmodified ascorbate peroxidase is derived from Galdieria partita or tobacco;
[3] An amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, 14, and 15, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, and ascorbic acid [1] or [2] oxidation-resistant ascorbate peroxidase having peroxidase activity;
[4] The oxidation-resistant ascorbate peroxidase according to any one of [1] to [3], wherein the amino acid other than cysteine is serine;
[5] The oxidation resistant ascorbate peroxidase according to any one of [1] to [4], which exhibits oxidation resistance in the presence of a radical scavenger;
[6] Further, substitution of amino acid at position 34 corresponding to SEQ ID NO: 1 from amino acid other than tryptophan and / or amino acid at position corresponding to position 121 of SEQ ID NO: 1 to amino acid other than cysteine The oxidation-resistant ascorbate peroxidase according to any one of [1] to [5], having a substitution of:
[7] A method for producing oxidation-resistant ascorbate peroxidase, comprising a step of culturing cells that produce oxidation-resistant ascorbate peroxidase, wherein the oxidation-resistant ascorbate peroxidase is an amino acid of unmodified ascorbate peroxidase A method having an amino acid sequence in which at least an amino acid at a position corresponding to position 25 of SEQ ID NO: 1 is replaced with an amino acid other than cysteine in the sequence;
[8] The method according to [7], wherein the unmodified ascorbate peroxidase is derived from Galdieria partita or tobacco;
[9] Oxidation-resistant ascorbate peroxidase has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, 14, and 15, or one or several amino acids in the amino acid sequence have been deleted, substituted, or added The method according to [7] or [8], comprising an amino acid sequence and having ascorbate peroxidase activity;
[10] The method according to any one of [7] to [9], wherein the amino acid other than cysteine is serine;
[11] The method according to any one of [7] to [10], wherein the oxidation resistant ascorbate peroxidase exhibits oxidation resistance in the presence of a radical scavenger;
[12] Oxidation-resistant ascorbate peroxidase is further substituted with amino acids other than tryptophan at the position corresponding to position 34 of SEQ ID NO: 1 and / or amino acid at the position corresponding to position 121 of SEQ ID NO: 1. The method according to any one of [7] to [11], wherein the method has a substitution of cysteine with an amino acid other than cysteine.
[13] In the amino acid sequence of unmodified ascorbate peroxidase, the method comprises substituting an amino acid at a position corresponding to position 25 of SEQ ID NO: 1 with an amino acid other than cysteine from an amino acid other than cysteine. How to grant;
[14] The method according to [13], wherein the unmodified ascorbate peroxidase is derived from Gardieria partita or tobacco;
[15] Ascorbate peroxidase imparted with oxidation resistance is deleted or substituted with an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, 14, and 15, or one or several amino acids in the amino acid sequence Or the method according to [13] or [14], comprising an added amino acid sequence and having ascorbate peroxidase activity;
[16] The method according to any one of [13] to [15], wherein the amino acid other than cysteine is serine;
[17] The method according to any of [13] to [16], wherein oxidation resistance in the presence of a radical scavenger is imparted.
[18] A step of substituting an amino acid at position 34 corresponding to SEQ ID NO: 1 with an amino acid other than tryptophan and / or an amino acid at a position corresponding to position 121 of SEQ ID NO: 1 with an amino acid other than cysteine The method according to any one of [13] to [17], further comprising the step of:

  The present invention provides a novel oxidation resistant ascorbate peroxidase, a method for producing the same, and a method for imparting oxidation resistance to ascorbate peroxidase. The ascorbate peroxidase of the present invention exhibits oxidation resistance, and in addition to the production of stress-tolerant plants, research reagents, cosmetics (see JP-T-2004-529866), pharmaceuticals (JP-T-2002-523378, special table) Conventional ascorbate peroxidase is useful for all the purposes for which it can be used, such as JP-A-10-504304 and International Publication No. 2004/066988.

  The present invention provides an oxidation resistant ascorbate peroxidase. The oxidation-resistant ascorbate peroxidase of the present invention has an amino acid sequence in which at least an amino acid corresponding to position 25 of SEQ ID NO: 1 is substituted with an amino acid other than cysteine in the amino acid sequence of unmodified ascorbate peroxidase . The oxidation resistant ascorbate peroxidase of the present invention further comprises substitution of an amino acid at a position corresponding to position 34 of SEQ ID NO: 1 with an amino acid other than tryptophan and / or an amino acid at a position corresponding to position 121 of SEQ ID NO: 1. It may have a substitution from cysteine to an amino acid other than cysteine.

  “Ascorbic acid peroxidase” is an enzyme that reduces hydrogen peroxide using ascorbic acid as a reducing agent. Ascorbate peroxidase activity can be measured by any known method. For example, according to the method of Nakano et al. (Nakano, Y. et al., Plant Cell Physiol., 22, 867-880 (1981)), the decrease in ascorbic acid by hydrogen peroxide is measured as a decrease in absorbance. be able to.

  In this specification, “unmodified ascorbate peroxidase” refers to a protein having cysteine as an amino acid at a position corresponding to position 25 of SEQ ID NO: 1 and having ascorbate peroxidase activity. The unmodified ascorbate peroxidase may have the same amino acid sequence as that of natural ascorbate peroxidase, or may have an amino acid sequence different from that of natural ascorbate peroxidase.

  Unmodified ascorbate peroxidase can be derived from any organism. For example, ascorbate peroxidase derived from Galdieria partita isolated by the present inventors can be used. The amino acid sequence of the enzyme is shown in SEQ ID NO: 1. Tobacco-derived ascorbate peroxidase can also be preferably used. The amino acid sequence of the enzyme is shown in SEQ ID NO: 9.

  In the present specification, “oxidation-resistant ascorbate peroxidase” is an amino acid sequence of unmodified ascorbate peroxidase in which at least the amino acid corresponding to position 25 of SEQ ID NO: 1 is substituted with an amino acid other than cysteine from cysteine. A protein having an amino acid sequence and having ascorbate peroxidase activity, which has improved resistance to oxidation compared to unmodified ascorbate peroxidase. It can be confirmed that the oxidation-resistant ascorbate peroxidase retains the same enzyme activity as that of the unmodified ascorbate peroxidase, for example, by examining the values of Km and kcat, the absorption spectrum, and the like.

  In the present specification, “the amino acid at the position corresponding to position 25 of SEQ ID NO: 1” means the amino acid sequence of the ascorbate peroxidase derived from the red alga Gardielia perchita shown in SEQ ID NO: 1 and the target ascorbate peroxidase. An amino acid present at a position in the subject sequence corresponding to the cysteine at position 25 in SEQ ID NO: 1 when compared with the amino acid sequence. Alternatively, the amino acid at this position can also be identified as a cysteine residue that is proximal to the heme propionate side chain. Similarly, “the amino acid at the position corresponding to position 34 of SEQ ID NO: 1” and “the amino acid at the position corresponding to position 121 of SEQ ID NO: 1” are ascorbic acid percutane derived from the red alga Gardieria perchita shown in SEQ ID NO: 1. When comparing the amino acid sequence of the oxidase with the amino acid sequence of the target ascorbate peroxidase, the amino acids present at the positions in the target sequence corresponding to tryptophan at position 34 and cysteine at position 121 of SEQ ID NO: 1, respectively. Say.

  Comparison of amino acid sequences can be performed using a known sequence analysis program or algorithm (for example, BLAST). An example of alignment results comparing the amino acid sequences of some known ascorbate peroxidases is shown in Table 1. In the table, “Pea_Cytosolic_P48534” indicates the amino acid sequence of pea cytoplasmic localized ascorbate peroxidase registered in UniProt accession number P48534. The amino acid sequence of the native ascorbate peroxidase is shown. “Galdieria_Cytosolid_AB037537” is the amino acid sequence of the ascorbate peroxidase of red algae Gardiarea pertita registered in GenBank accession number AB037537 (ie, the amino acid sequence of SEQ ID NO: 1). "Tobacco_Stromal_BAA7855 "Indicates the chloroplast stroma full length amino acid sequence of localized ascorbate peroxidase tobacco registered in GenBank Protein ID No. BAA78553 (including transit peptide).

  The position corresponding to position 25 of SEQ ID NO: 1 corresponds to the 15th position in the third row in Table 1-1 (indicated by “C25”). As is apparent from the fact that cysteine residue (C) is found at the same position in all the sequences shown in the table (the symbol “*” indicating perfect match is displayed), there are various cysteine residues at this position. It is highly conserved among ascorbate peroxidase. This position corresponds to positions 32, 30, and 115 of the amino acid sequences of pea cytoplasmic localization type, cotton microbody localization type, and tobacco chloroplast stromal localization type ascorbate peroxidase, respectively. . Therefore, those skilled in the art can easily identify the amino acid at the position corresponding to position 25 of SEQ ID NO: 1 in the amino acid sequence of the target ascorbate peroxidase. Similarly, a position corresponding to position 34 of SEQ ID NO: 1 (indicated by “W34” in the table) and a position corresponding to position 121 of SEQ ID NO: 1 (indicated by “C121” in the table) are easily identified. For example, positions 26, 35, and 126 of the amino acid sequence of tobacco-derived ascorbate peroxidase shown in SEQ ID NO: 9 correspond to positions 25, 34, and 121 of SEQ ID NO: 1, respectively.

  Oxidation resistant ascorbate peroxidase consists of the amino acid sequence of SEQ ID NO: 3, for example. Here, the amino acid sequence of SEQ ID NO: 3 is obtained by substituting the amino acid at position 25 of SEQ ID NO: 1 from cysteine to serine. Alternatively, the oxidation resistant ascorbate peroxidase may comprise an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 3 as long as it has ascorbate peroxidase activity. In addition, for example, the oxidation-resistant ascorbate peroxidase comprises an amino acid sequence selected from SEQ ID NOs: 11, 14 and 15, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence. And a protein having ascorbate peroxidase activity can be used. The amino acid sequences of SEQ ID NOs: 11 to 15 have the following substitutions in the amino acid sequence of SEQ ID NO: 9: SEQ ID NO: 11: Cysteine to serine at position 26; SEQ ID NO: 12: Tryptophan from position 35 to phenylalanine; SEQ ID NO: 13: Cysteine from position 126 to alanine; SEQ ID NO: 14: Cysteine from position 26 to serine and cysteine from position 126; SEQ ID NO: 15: Cysteine from position 26 to serine, Tryptophan at position 35 to Phenylalanine and Position 126 Cysteine to alanine.

  As the amino acid other than cysteine arranged at the position corresponding to position 25 of SEQ ID NO: 1, any natural amino acid other than cysteine can be used as long as the ascorbate peroxidase activity is retained after the substitution. . For example, by using serine having a molecular structure similar to that of cysteine, changes in the three-dimensional structure of the protein due to substitution can be reduced. Alanine can also be suitably used as an amino acid other than cysteine arranged at a position corresponding to position 25 of SEQ ID NO: 1. Similarly, those skilled in the art can appropriately select an amino acid other than tryptophan located at a position corresponding to position 34 of SEQ ID NO: 1, for example, phenylalanine can be used.

  The ascorbate peroxidase of the present invention may have other substituted amino acid residues in addition to the position corresponding to position 25 of SEQ ID NO: 1. Examples of other substitutions include substitution of tryptophan at a position corresponding to position 34 of SEQ ID NO: 1 with another amino acid, cysteine residue at a position corresponding to position 121 of SEQ ID NO: 1 to another amino acid residue Substitution may be mentioned.

In one embodiment, the ascorbate peroxidase of the present invention exhibits oxidation resistance in the presence of a radical scavenger. A radical scavenger (also referred to as a radical scavenger) is a compound used to capture radicals generated during the reaction. Any radical scavenger can be used in the present invention. For example, 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO) can be used. TEMPO is known to bind to amino acid radicals to form stable products. Incidentally, TEMPO thiol (-SH) as well radicals and chain react with TEMPO adduct, oxide and is sulfinic acid thiol group _(-SO 2 H), sulfonic acid _(-SO 3 H) also occurs that Borisenko, GG et al., J. Am. Chem. Soc., 126, suggesting that this irreversible oxidation reaction is caused by the action of a nitroxide radical that is a functional group of TEMPO. : 9221-9232 (2004)). Therefore, even if the compound is not generally called a radical scavenger, the radical scavenger includes a nitroxide radical that can cause an irreversible oxidation reaction of a cysteine radical, like TEMPO.

  Resistance to oxidation can be confirmed, for example, as follows. That is, a radical scavenger is added at a final concentration of 0.2 or 0 mM to 1.0 mL of an ascorbate peroxidase-containing enzyme solution substituted with anaerobic 50 mM sodium phosphate buffer, pH 7.0, followed by 5 mol of hydrogen peroxide or Add to a molar equivalent of 0. A part is taken out every fixed time and immediately mixed with the reaction solution (0.5 mM ascorbic acid, 50 mM sodium phosphate, pH 7.0, 0.01 mg / ml BSA) to stop the inactivation reaction, Measure activity. Here, the activity is determined by measuring hydrogen peroxide-dependent ascorbic acid oxidation at 25 ° C. when hydrogen peroxide having a final concentration of 100 μM is added to 1.0 mL of the reaction solution. In the present specification, the term “molar equivalent” represents the ratio of the hydrogen peroxide to the ascorbate peroxidase in terms of moles. When the reaction is performed for 10 minutes in the presence of TEMPO as a radical scavenger under the above conditions, the ascorbate peroxidase of the present invention is, for example, 60 to 100%, preferably 80 to 100%, more preferably 90 to 100. % Residual activity.

  The present invention provides a method for producing an oxidation resistant ascorbate peroxidase, comprising a step of culturing cells that produce the oxidation resistant ascorbate peroxidase.

  As a cell that produces an oxidation resistant ascorbate peroxidase, for example, a host cell transformed with a recombinant DNA containing a nucleic acid encoding an oxidation resistant ascorbate peroxidase can be used. Therefore, according to the present invention, a nucleic acid encoding the ascorbate peroxidase of the present invention, a recombinant DNA containing the nucleic acid, and a transformant containing the recombinant DNA are also provided.

  The nucleic acid encoding the oxidation-resistant ascorbate peroxidase comprises, for example, the amino acid sequence of SEQ ID NO: 3, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 3, And a nucleic acid encoding a protein having ascorbate peroxidase activity. An example of such a nucleic acid includes a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 4. In addition, the sequence of SEQ ID NO: 4 is the sequence number of 198 to 941 (SEQ ID NO: 2) corresponding to the coding region of the nucleotide sequence of ascorbate peroxidase of red alga Gardiarea perchita registered under GenBank accession number AB037537. The cysteine codon (TGT) (positions 73 to 75 of SEQ ID NO: 2) at the position corresponding to position 25 of 1 is changed to the serine codon (TCT) (positions 73 to 75 of SEQ ID NO: 4). Alternatively, the nucleic acid may be a nucleic acid that hybridizes to a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 4 under stringent conditions and encodes a protein having ascorbate peroxidase activity. Examples of stringent hybridization conditions include those described in J. Sambrook et al., “Molecular Cloning: A Laboratory Manual, Second Edition”, 1989, Cold Spring Harbor Laboratory Press. For example, after hybridization with a probe at 68 ° C. in a solution containing 6 × SSC, 5 × Denhardt's solution, 0.5% SDS, 100 μg / ml denatured salmon sperm DNA, the washing conditions are 2 × SSC, Examples include conditions in which the temperature is changed from room temperature in 0.1% SDS to 68 ° C. in 0.1 × SSC and 0.5% SDS. An amino acid sequence selected from SEQ ID NOs: 11, 14, and 15, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, and encodes a protein having ascorbate peroxidase activity Nucleic acids can be used as well.

  Host cells should be selected from any prokaryotic cells such as bacteria, eukaryotic microbial cells such as filamentous fungi, yeast, animal cells, insect cells, plant cells, etc., as long as they can express the target gene. Can do. In addition to the nucleic acid encoding the oxidation resistant ascorbate peroxidase, the recombinant DNA may contain any nucleic acid sequence necessary for transcription, translation, selection of transformants, and the like. The cells are cultured under conditions suitable for the cells to be used. Moreover, an animal individual (transgenic animal) can also be produced using the cells into which the recombinant DNA has been introduced. For example, transgenic animals that produce oxidation-resistant ascorbate peroxidase in milk can be made. Such recombinants can be prepared according to known methods.

Alternatively, a plant individual (transgenic plant) can be produced using cells into which the recombinant DNA has been introduced. Such plant individuals are useful as stress-resistant crops in addition to the production of ascorbate peroxidase. Any plant can be used as the plant into which the recombinant DNA is introduced. Such plants include both monocotyledonous plants (rice, wheat, barley, corn, etc.) and dicotyledonous plants (cotton, tomatoes, tobacco, etc.). Such plant individual production methods are known in the art, and are described, for example, in Patent Document 1-2 and Non-Patent Document 4-8. Such plants resistant to oxidative poison stress, desert greening, CO ₂ reduction is useful for such crop production in agriculture.

  The present invention relates to oxidation resistance to ascorbate peroxidase, which comprises the step of substituting at least the amino acid corresponding to position 25 of SEQ ID NO: 1 with an amino acid other than cysteine in the amino acid sequence of unmodified ascorbate peroxidase. Provide a method of granting. As described above, the ascorbate peroxidase is imparted with oxidation resistance by the method of the present invention.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the scope of the present invention is not limited to these examples.

Mass Spectrometry of APXB Treated with Hydrogen Peroxide in the Presence of TEMPO Ascorbic acid peroxidase from Galdieria partita, 50 mM sodium phosphate buffer, anaerobic with 5 molar equivalents of hydrogen peroxide, in pH 7.0 Incubation with the polymer formed a polymer, but in the presence of 0.2 mM radical scavenger 2,2,6,6-tetramethyl-1-piperidinyloxyl (TEMPO), such a polymer was formed. There wasn't. This indicates that polymerization is due to radical formation on the protein surface, and TEMPO has captured the radical site. Therefore, in order to identify the radical site, the tryptic digest of APXB treated with hydrogen peroxide in the presence of untreated and TEMPO was analyzed by MALDI-TOF MS and MS / MS. APXB enzyme solution was prepared as described in Example 3 below.

The purified APXB was substituted with 50 mM sodium phosphate buffer, pH 7.0, anaerobized with nitrogen gas using two NAP5 gel filtration columns to obtain 1.0 mL of enzyme solution. While maintaining the anaerobic condition, the Soret peak was measured with an ultraviolet-visible spectrophotometer UV-1700 PharmaSpec to determine the enzyme concentration. The molar extinction coefficient of APXB under this condition is 102 mM ⁻¹ · cm ⁻¹ . In addition, the molar extinction coefficient of the following APXB variant APXBC25S is 93 mM ⁻¹ · cm ⁻¹ . The heme concentration was determined by the Pyridine hemochromogen method (Non-patent Document 2) using horseradish peroxidase (molar extinction coefficient 100 mM ⁻¹ · cm ⁻¹ , Nacalai Tesque) as a standard product.

  To 1.0 mL of APXB enzyme solution substituted with anaerobic 50 mM sodium phosphate buffer, pH 7.0, a final concentration of 0.2 or 0 mM radical scavenger TEMPO (Wako Pure Chemical Industries) was added, followed by 5 mol of hydrogen peroxide. Or it added so that it might become 0 molar equivalent. After a certain time, it was applied to a NAP10 gel filtration column and eluted with anaerobic 50 mM ammonium bicarbonate buffer, pH 8.0 to remove unreacted TEMPO or glutathione and hydrogen peroxide to obtain 0.5 mL of enzyme solution. The enzyme solution was dispensed 100 μl at a time into a sample tube, frozen with liquid nitrogen and stored at −80 ° C.

  100 μl of these enzyme solutions were mixed with 33 μl of 8M urea, 1 μl of trypsin (Sequencing Grade Modified Trypsin, Frozen, Promega) was added and digested overnight. APX digested with trypsin was applied to ZipTip (registered trademark) C18 (Millipore) and eluted according to the instructions using a solvent for measurement sample (75% acetonitrile, 0.1% TFA). MALDI-TOF MS (VoyagerDE STR, Applied biosystems) and MS / MS using α-cyano-4-hydroxycinnamic acid (CHCA, Bruker Daltonics) as matrix and peptide calibration standard II (Bruker Daltonics) as standard sample Mass spectrometry of APXB trypsin digest was performed with (ultraflex TOF / TOF, Bruker Daltonics). The measuring method followed the instruction manual of the apparatus. For the analysis, Mascot Search (http://www.matrixscience.com/search_form_select.html) was used.

  The results of MALDI-TOF MS of APXB treated with hydrogen peroxide in the presence of TEMO are shown in FIG.

  From the results of MALDI-TOF MS, a peak of mass 1565 m / z corresponding to amino acid sequence 19 to 31 (LFEQTPCPMIMVR, calculated molecular weight: 1563.711) confirmed with untreated APXB was observed with TEMPO and hydrogen peroxide. It was clarified that some peaks appeared along with this process.

Further, when MS / MS analysis was performed on the peak at 1597 m / z in FIG. 1, the increase in mass of the cysteine residue at position 25 (Cys-25) was 32 m / z, and Cys-25 had two oxygen atoms (atomic weight). 16) was confirmed to be linked _(-SO 2 H). When MS / MS analysis was performed on the peak at 1736 m / z in FIG. 1, the mass increase of Cys-25 was 171 m / z, and one molecule of TEMPO (molecular weight 156.3) and one atom of oxygen in Cys-25. (-SO-TEMPO) was confirmed.

TEMPO thiol (-SH) as well radicals and chain react with TEMPO adduct, sulfinic acid is an oxide of a thiol group _(-SO 2 H), sulfonic acid _(-SO 3 H) also occurs it is reported (Borisenko, GG et al., J. Am. Chem. Soc., 126: 9221-9232 (2004)), which was consistent with the present results. From the above, it was revealed that Cys-25 is a radicalization site. In the following examples, Cys-25 is mainly described, but it is suggested that the cysteine residue at position 121 (Cys-121) is also a radicalization site, and this cysteine residue is inactivated with APX. The relationship is described in Example 6.

In addition, when analyzed by MALDI-TOF MS without digestion with trypsin, APXB shows a mass increase of about 50 Da when treated with hydrogen peroxide in the presence of TEMPO as compared to untreated (FIG. 2). From this result, there is actually almost no TEMPO adduct in the cysteine residue, most of which is sulfinic acid (—SO ₂ H) or sulfonic acid (—SO ₃ H), which is an oxide of the cysteine residue. It was shown that.

2. Effect of glutathione on radicalization of APXB 2-1. SDS-PAGE of APXB treated with hydrogen peroxide in the presence of glutathione
Although TEMPO does not exist in living bodies such as plants, oxidation of the thiol group of a cysteine residue can occur in vivo (Non-patent Documents 9-10). However, there is a possibility that such oxidation is prevented by binding glutathione to a radicalized cysteine residue in vivo. Below, the influence of glutathione on the radicalization of APXB was examined.

  The final concentration of glutathione is 0, 0.1, 0.2, 0.5, 1.0, and 2.0 mM in 0.3 mL of an APXB enzyme solution substituted with anaerobic 50 mM sodium phosphate buffer, pH 7.0. Subsequently, hydrogen peroxide was injected with a microsyringe to 5 mol or 0 molar equivalent. Unreacted glutathione and hydrogen peroxide are removed by applying the enzyme solution containing glutathione and hydrogen peroxide to a NAP10 (Pharmacia) gel filtration column after a certain time and eluting with anaerobic 50 mM sodium phosphate buffer, pH 7.0. As a result, 0.5 mL of the enzyme solution was obtained. 100 μl of the obtained enzyme solution was dispensed into a sample tube, frozen with liquid nitrogen and stored at −80 ° C.

  Add 100 µl of the enzyme solution from which glutathione and hydrogen peroxide have been removed to the equivalent sample buffer for SDS-PAGE (without 2-mercaptoethanol), mix, and dispense 50 µl each into a sample tube. And frozen at −80 ° C. 2-mercaptoethanol was added immediately before electrophoresis so that the final concentration was 5%. SDS-PAGE was performed using 12.5% polyacrylamide gel and stained with Coomassie Brilliant Blue (CBB) staining.

The results are shown in FIG. In FIG. 3, each lane represents the following from the left: marker (NEB BroadRangeP7702); untreated APXB; APXB + 5 molar equivalent H ₂ O ₂ ; APXB + 5 molar equivalent H ₂ O ₂ +0.1 mM glutathione; APXB + 5 molar equivalent H ₂ O ₂ + 0.2 mM glutathione; APXB + 5 molar equivalent H ₂ O ₂ +0.5 mM glutathione; APXB + 5 molar equivalent H ₂ O ₂ +1.0 mM glutathione; APXB + 5 molar equivalent H ₂ O ₂ +2.0 mM glutathione. For untreated APXB, a single band can be confirmed around 28 kDa, whereas when hydrogen peroxide is added, multiple bands that are considered to be APXB monomers and polymers are confirmed around 28, 60, and 120 kDa. It was done. On the other hand, when hydrogen peroxide treatment was performed in the presence of glutathione, these bands became thinner as the glutathione concentration increased, and when hydrogen peroxide treatment was performed in the presence of 2.0 mM glutathione, 28 kDa corresponding to the monomer of APXB. Other bands were hardly confirmed. That is, it was suggested that glutathione binds to a radicalized cysteine residue on the surface of the APXB protein and prevents polymerization of proteins by hydrogen peroxide.

2-2. Mass Spectrometry of APXB Treated with Hydrogen Peroxide in the Presence of Glutathione To clarify whether glutathione binds to the cysteine residue that is the radicalization site, tryptic digest of APXB treated with hydrogen peroxide in the presence of glutathione was analyzed. Analysis was performed by MALDI-TOF MS and MS / MS. The experiment was performed in the same manner as in Example 1 except that 2.0 mM or 0 mM glutathione (Wako Pure Chemical Industries) was used instead of TEMPO. The results are shown in FIG.

  From the results of MALDI-TOF MS, the peak of mass 1565 m / z corresponding to amino acid sequence 19 to 31 confirmed with untreated APXB was significantly reduced by treatment with glutathione and hydrogen peroxide. Along with this, it was clarified that one peak of glutathione molecule (molecular weight 307.33) was bonded to this peptide fragment, and a peak thought to be desorbed from two hydrogen atoms (atomic weight 1.01) was revealed (- S-S-G).

  Further, when MS / MS analysis was performed on the peak at 1870 m / z in FIG. 4, it was confirmed that glutathione was bonded to Cys-25 and two hydrogen atoms were eliminated. This indicates that APXB reacts with hydrogen peroxide to radicalize cysteine residues on the protein surface and bind to glutathione.

  The residual activity of the peroxidized APXB was measured in the same manner as in Example 5 below. The residual activity was about 50% of that in the untreated state, but the APXB in the presence of glutathione was treated with hydrogen peroxide. The residual activity was 80% when untreated, indicating that there was no decrease in the catalytic activity of APXB due to glutathione binding to Cys-25.

  It is said that 2.0 to 5.0 mM glutathione is present in plant cells. Considering the above results, it is usually possible that the polymerization between proteins and the irreversible oxidation of cysteine residues are prevented by the binding of APX and glutathione even in vivo. However, the concentration of reduced glutathione is said to decrease when cells are exposed to severe oxidative stress. In such a case, the cysteine residue of APX is oxidized without being protected by glutathione, and as a result, the catalytic activity of APXB may be lost. Therefore, it was suggested that depending on the state of plant cells, oxidation of the cysteine residue (Cys-25) at position 25 similar to that caused by hydrogen peroxide and TEMPO shown in Example 1 may occur in vivo. .

Construction, expression and purification of APXB and APXBC25S In Example 1, it was revealed that by reacting with hydrogen peroxide in the presence of TEMPO, the cysteine residue at position 25 of APXB was radicalized and further oxidized. Further, in Example 2, it was suggested that glutathione binds to this cysteine residue and prevents polymerization of APXB by hydrogen peroxide. Therefore, in order to verify the effect of oxidation of cysteine residues on the catalytic activity of APXB, a mutant APXB lacking the cysteine residues was prepared.

3-1. Construction of pETAPXBC25S For Escherichia coli expressing APX (named APXBC25S) in which the cysteine residue (Cys-25) at position 25 of cytoplasmic localized APX (APXB) of red alga Gardiarea pertita is replaced with serine (Ser) The expression vector pET16bAPXBC25S was prepared as follows.

  In order to amplify the 5 'terminal fragment, a primer APXB29R (5'-AGGAGTCTGTTCAAATACTTTTAC-3') (SEQ ID NO: 5) was designed, and its 5 'end was phosphorylated using T4 Polynucleotide Kinase (Toyobo). The protocol followed the instructions in the catalog TOYOBO BIOCHEMICALS FOR LIFE SCIENCE (2004/2005).

  Primer APXB30 (5'-TCTATGCCCTATTAGGGTGCGATAGAG-3 ') (SEQ ID NO: 6) was designed for amplification and mutagenesis of the 3' terminal fragment. This primer is a base (TCT) corresponding to Ser by substituting one base in the base (TGT) corresponding to Cys-25.

  Using the plasmid pET16bAPXB (Non-patent Document 2) containing the full-length APXB gene as a template, the primer T7 promoter (5′-TAATACGACTCACTATAGG-3 ′) (SEQ ID NO: 7) and 3A phosphorylated primer APXB29R were used as the 5 ′ terminal fragment The primer fragment APXB30 and primer T7 terminator (5′-ACCGCTGGACAATAACTAGC-3 ′) (SEQ ID NO: 8) were used for the terminal fragment, and the first-stage PCR reaction was performed. Pfu Turbo DNA Polymerase (Strategene) was used as the DNA polymerase. The reaction conditions and composition of the reaction solution were in accordance with the recommended protocol of the enzyme, and Mini Cycler (MJ Research) was used as the thermal cycler.

  The 5 'terminal fragment and 3' terminal fragment obtained by PCR were purified and ligated. The ligation is carried out according to Takara Ligation kit ver. 2 (Takara Bio). However, the DNA solution was heated at 42 ° C. for 30 seconds before adding the kit I solution. PCR products were purified using Wizard (registered trademark) SV Gel and PCR Clean-Up System according to the instructions unless otherwise specified.

  Using the ligation product as a template, PCR at the second stage was performed using primer T7 promoter and primer T7 terminator. The PCR product was purified, digested with restriction enzymes NcoI (Toyobo) and XhoI (Takara Bio) and purified again. The pET16-b vector (Novagen) was also digested with NcoI and XhoI and purified.

  The PCR product digested with NcoI and XhoI was inserted into the NcoI / XhoI site of pET16-b. The obtained pET16bAPXBC25S was used to transform E. coli XL1-blue strain.

  Colony PCR was performed using the primer T7 promoter and the primer T7 terminator, using Escherichia coli taken from the LB plate colony with a toothpick as a template. The PCR product was electrophoresed on a 1.5% agarose gel, colonies giving the desired PCR product of about 1000 bp were detected, and the PCR product was purified. Taq Polymerase (NEB) was used as the DNA polymerase, and Ex Taq Polymerase buffer was used as the buffer. The reaction conditions and composition of the reaction solution were in accordance with the recommended protocol for the enzyme, and Mini Cycler was used as the thermal cycler.

  Using the purified PCR product as a template, Big Dye Terminator ver. 3.1 A sequence reaction solution was prepared using Cycle Sequencing Kit (Applied Biosystems) according to the instructions. Samples prepared using a sample injection buffer TSR (Template Suppression Reagent, Applied Biosystems) were subjected to DNA sequencer ABI PRISM 310 (Applied Biosystems) to analyze the sequence.

3-2. Purification of APXB and APXBC25S APXB and APXBC25S were purified by the method described in Non-Patent Document 3. Specifically, introduction of pET16bAPXB (encoding wild-type APXB) and pET16bAPXBC25S (encoding APXB in which Cys-25 is replaced with Ser) into a competent cell (E. coli: BL21 (DE3) strain) is a conventional method. Went according to. Escherichia coli carrying pET16bAPXB or pET16bAPXBC25S was cultured with shaking at 120 rpm and 37 ° C. in 1 L of LB medium containing ampicillin having a final concentration of 50 mg / L using a shaking culture apparatus IC-43 (Simadzu). In the culture, the turbidity of the medium was measured with an ultraviolet-visible spectrophotometer UV-1700 PharmaSpec (Shimadzu). Culturing for about 12 hours until OD ₆₀₀ = 0.6, final concentration of 50 mg / L at the same time as isopropyl-β-D (−)-thiogalactopyranoside (IPTG, Wako Pure Chemical Industries) with a final concentration of 0.5 mM. Of ampicillin was added. O. D. _The culture was continued for further 5 hours until ₆₀₀ = 1.0, and the cells were collected. The liquid medium is collected by centrifugation (2800 × g, 4 ° C., 20 minutes), and after washing the cells with buffer A (50 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA, 1 mM ascorbic acid), liquid Frozen with nitrogen and stored at -80 ° C.

  E. coli for 4 L of the stored LB medium was suspended in 40 mL of buffer A, and the cells were disrupted using an ultrasonic disrupter VP-15S and a standard horn HNN-0200 (Taitec). The crushing was repeated twice under conditions of output 4, cycle 20%, and 5 minutes, and then repeated twice under conditions of output 5, cycle 20%, and 5 minutes. This crushed material was centrifuged (13,000 × g, 4 ° C., 30 minutes), and an ammonium sulfate precipitation fraction was applied to the supernatant. Method is “Protein Experiment Note” (Masato Okada, Kaoru Miyazaki (1999) Bradford Method and Ammonium Sulfate Fractionation: Unbeatable Biotechnical Series Revised Protein Experiment Note Up Extraction and Separation Purification (Yodosha), pp33-34, 72-74 ), The fraction that does not precipitate in 40% saturated ammonium sulfate but precipitates in 80% saturated ammonium sulfate is added to buffer B (10 mM potassium phosphate buffer, pH 7.0, 1 mM EDTA, 1 mM ascorbic acid) containing 40% saturated ammonium sulfate. Suspended.

  The sample obtained by the ammonium sulfate precipitation was filtered (DISMIC-25CS 0.45 μm, Advantec) and subjected to a hydrophobic column HiLoad 16/10 mm Phenylsepharose (Pharmacia). All chromatography was performed at 4 ° C. using BioLogic Duo Flow (Bio-Rad) in the chromatography system. Non-adsorbed protein was eluted from the column with buffer B + 40% saturated ammonium sulfate over 40 minutes, and then a concentration gradient of ammonium sulfate was formed from 40% to 0% over 100 minutes, followed by elution. The flow rate was 1.0 mL / min, and the fraction fraction was 1.5 mL / tube. Fractions containing APX were selected by SDS-PAGE. SDS-PAGE was performed according to a conventional method using 12.5% polyacrylamide gel.

  Fractions fractionated by hydrophobic chromatography were concentrated to 5 mL by ultrafiltration Centriprep YM-10 (Millipore) and subjected to gel filtration column Hiload 16/60 mm Superdex 75 prep grade (Pharmacia). Elute with buffer B containing 0.15M potassium chloride. The flow rate was 0.5 mL / min, and the fraction fraction was 2.0 mL / tube. Fractions containing APXB were selected by SDS-PAGE.

APXB and APXBC25S were diluted with buffer B containing 0.15M potassium chloride so that the Soret peak of the recovered APXB enzyme solution was about 4, and dispensed in 200 μl aliquots into sample tubes, frozen in liquid nitrogen at −80 ° C. Saved with. Protein quantification of purified APXB and APXBC25S was performed by a conventional method.

  By the above operation, a purified sample giving a single band by SDS-PAGE was obtained.

K _m , k _cat and absorption spectrum of APXBC25S The effect of the mutation of the produced mutant APXB on the catalytic activity and structure was verified by the enzyme chemical parameters and the absorption spectrum obtained according to the method described in Non-Patent Document 3.

4-1. K _m and k _{cat of} APXBC25S
The purified preparation of APXBC25S is diluted 1/100 with a buffer (50 mM sodium phosphate buffer, pH 7.0, 0.5 mM ascorbic acid, 0.01 mg / mL BSA) to a final concentration of 0.05, 0.1, 0. Mixing with a reaction solution (50 mM sodium phosphate buffer, pH 7.0) containing .15, 0.2, 0.3, 0.4, 0.5 mM ascorbic acid, the activity was measured. Hydrogen peroxide-dependent oxidation of ascorbic acid was measured when hydrogen peroxide with a final concentration of 100 μM was added to a 1.0 mL reaction solution (Nakano, Y. et al., Plant Cell Physiol., 22: 867-880 ( 1981)). The measurement was performed at 25 ° C., and the decrease in absorption at 290 nm (molar extinction coefficient 2.8 mM ⁻¹ cm ⁻¹ ) was observed with an ultraviolet-visible spectrophotometer UV-1700 PharmaSpec. K _m and k _cat for ascorbic acid were determined from the activity of APXBC25S at each ascorbic acid concentration.

Next, the purified preparation of APXBC25S was diluted 1/100 with a buffer (50 mM sodium phosphate buffer, pH 7.0, 0.5 mM ascorbic acid, 0.01 mg / mL BSA), and the reaction solution (50 mM sodium phosphate buffer) was diluted. Buffer, pH 7.0, 0.5 mM ascorbic acid) and the activity was measured. Hydrogen peroxide-dependent ascorbic acid oxidation was measured when hydrogen peroxide having a final concentration of 10, 20, 30, 40, 60, 80, 100 μM was added to 1.0 mL of the reaction solution. It was determined _{K m} for hydrogen peroxide than the activity of APXBC25S at each concentration of hydrogen peroxide. Wild type APXB was used as a control. The results are shown in Table 3.

The Km of APXBC25S with respect to hydrogen peroxide was comparable to the value of APXB. Further, the K _m of APXBC25S with respect to ascorbic acid was about 4 to 5 times the value of APXB, and k _cat was 70% or more. From this result, it was shown that the substitution of Cys-25 with Ser had little effect on the catalytic activity, although the affinity for ascorbic acid was lowered.

4-2. Absorption spectrum of APXBC25S The buffer was replaced with 50 mM sodium phosphate buffer, pH 7.0, anaerobic with nitrogen gas. About the buffer-substituted APX, when not treated, when 0.5 mM potassium cyanide was added, and when a small amount of dithionite was added, respective spectra were measured. The measurement was performed at 25 ° C. using an ultraviolet-visible spectrophotometer UV-1700 PharmaSpec.

  As a result, the absorption spectrum of the mutant APX with no treatment and the addition of potassium cyanide or dithionite is very similar to that of APXB, indicating that substitution of Cys with Ser has little effect on the structure of the catalytic site. It was done. Based on this, it was determined that these mutant APXBs could be used for the following experiments.

Residual activity of APXB and APXBC25S in the presence of TEMPO To examine how the catalytic activity of APXB is affected by the oxidation of cysteine residues by reaction with hydrogen peroxide in the presence of TEMPO. Residual activities of APXB and APXBC25S were measured.

  To 1.0 mL of enzyme solution substituted with anaerobic 50 mM sodium phosphate buffer, pH 7.0, a final concentration of 0.2 or 0 mM TEMPO as a radical scavenger is added, followed by 5 or 0 molar equivalents of hydrogen peroxide. It added so that it might become. A part is taken out every fixed time and immediately mixed with the reaction solution (0.5 mM ascorbic acid, 50 mM sodium phosphate, pH 7.0, 0.01 mg / ml BSA) to stop the inactivation reaction, Activity measurement was performed. The activity was determined by measuring hydrogen peroxide-dependent ascorbic acid oxidation at 25 ° C. when hydrogen peroxide having a final concentration of 100 μM was added to 1.0 mL of the reaction solution.

  The results are shown in FIG. In FIG. 5, A is when wild-type APXB is treated with hydrogen peroxide alone, B is when mutant APXBC25S is treated with hydrogen peroxide alone, C is when APXB is treated with hydrogen peroxide in the presence of TEMPO, D shows the case where APXBC25S is treated with hydrogen peroxide in the presence of TEMPO. Further, □ shows a control not containing hydrogen peroxide, and ◆ shows a case containing hydrogen peroxide.

  The residual activity when treated with hydrogen peroxide in the absence of TEMPO was about 50% of both APXB and APXBC25S. However, the remaining activity when treated with hydrogen peroxide in the presence of TEMPO was almost completely lost in APXB, whereas in APXBC25S, the remaining activity decreased only by about 10%. From this, it was shown that the catalytic activity of APXB was significantly reduced due to the accelerated oxidation of the cysteine residue at position 25 depending on TEMPO and hydrogen peroxide. The reason why the APXB mutant was less likely to be deactivated by hydrogen peroxide in the presence of TEMPO than in the absence of TEMPO was presumed that TEMPO reduced heme at the catalytic site instead of ascorbic acid.

  In this example, based on tobacco APX described in Patent Document 3, in addition to cysteine at the position corresponding to position 25 of SEQ ID NO: 1, tryptophan at the position corresponding to position 34 of SEQ ID NO: 1, SEQ ID NO: Double mutants and triple mutants were prepared by substituting cysteine at the position corresponding to position 121 of 1 with another amino acid, and the effect of these mutations on oxidation resistance was examined. The amino acid sequence of tobacco APX is shown in SEQ ID NO: 9, and the nucleotide sequence of the nucleic acid encoding it is shown in SEQ ID NO: 10. Position 25 of SEQ ID NO: 1 corresponds to position 26 of SEQ ID NO: 9, position 34 corresponds to position 35, and position 121 corresponds to position 126.

  Wild-type tobacco APX (tsAPX) having the amino acid sequence of SEQ ID NO: 9 and tsAPXC26S (sequence of cysteine at position 26 of SEQ ID NO: 9 substituted with serine by a method similar to the method described in Example 3 and Patent Document 3 No. 11), tsAPXW35F (SEQ ID NO: 12) in which tryptophan at position 35 in SEQ ID NO: 9 is substituted with phenylalanine, tsAPXC126A (SEQ ID NO: 13) in which cysteine at position 126 in SEQ ID NO: 9 is substituted with alanine, position 26 in SEQ ID NO: 9 TsAPXC26SC126A (double mutant) (SEQ ID NO: 14) in which cysteine at position 126 was substituted with serine at position 126, serine at position 26 in serine, tryptophan at position 35 as phenylalanine, 126 Place serine in alanine To give the TsAPXC26SW35FC126A (3 double mutant) (SEQ ID NO: 15).

The extinction coefficient (ε _soret ) of each purified enzyme preparation was measured, and the steady-state kinetic parameters (Km, kcat) for ascorbic acid and hydrogen peroxide (H ₂ O ₂ ) were determined in the same manner as described in Example 4. ) Was measured. Various concentrations of ascorbic acid (0-0.5 mM) and a fixed concentration of H ₂ O ₂ (0.1 mM) were used to determine the Km value for ascorbic acid, and the Km value for H ₂ O ₂ was determined Various concentrations of H ₂ O ₂ (0-0.1 mM) and a fixed concentration of ascorbic acid (0.5 mM) were used. The results are shown in Tables 4-1 and 4-2.

  From these results, it was shown that each mutant APX has an enzymatic performance comparable to that of wild-type APX.

  Next, by the same method as described in Example 5, each enzyme preparation was treated with hydrogen peroxide in the presence or absence of TEMPO, and the residual activity was measured. FIG. 6 shows the results of treatment with 5 molar equivalents of hydrogen peroxide in the presence of 0.2 mM TEMPO, and FIG. 7 shows the results of treatment with 20 molar equivalents of hydrogen peroxide in the absence of TEMPO.

  TEMPO promotes oxidative damage of cysteine residues by hydrogen peroxide. As shown in FIG. 6, when hydrogen peroxide treatment is performed in the presence of TEMPO, in the case of a double mutation in which two cysteine residues are mutated to other residues (tsAPXC26SC126A), oxidation is higher than that in the wild type (tsAPX). Tolerance was shown. This double mutation was more effective than the single mutation of each cysteine residue (tsAPXC26S, tsAPXC126A). Further, in the case of a triple mutation mutated to tryptophan at position 35 (tsAPXC26SW35FC126A), the highest oxidation resistance was shown.

  Treatment with hydrogen peroxide alone causes complex oxidative damage such as cross-linking of heme and tryptophan at position 35 and polymerization between apoproteins. As shown in FIG. 7, when treated with hydrogen peroxide alone, oxidation resistance equivalent to that of the wild type (tsAPX) is obtained in both cases of single mutation of two cysteine residues (tsAPXC26S, tsAPXC126A) and double mutation (tsAPXC26SC126A). Only showed. On the other hand, when a mutation in tryptophan at position 35 was introduced (tsAPXW35F), oxidation resistance higher than that of the wild type was shown, and in the case of triple mutation (tsAPXC26SW35FC126A), higher oxidation resistance was shown.

  From the above results, substitution of cysteine at the position corresponding to position 25 of SEQ ID NO: 1, substitution of tryptophan at the position corresponding to position 34 of SEQ ID NO: 1, substitution of cysteine at the position corresponding to position 121 of SEQ ID NO: 1 It was shown that the oxidation resistance was further improved by using together.

    The present invention provides a novel oxidation resistant ascorbate peroxidase, a method for producing the same, and a method for imparting oxidation resistance to ascorbate peroxidase.

It is a figure which shows the result of MALDI-TOF MS of the APXB trypsin digest obtained by hydrogen peroxide treatment in the presence of TEMPO. It is a figure which shows the result of MALDI-TOF MS of APXB which carried out hydrogen peroxide treatment in the presence of TEMPO. It is a figure which shows the SDS-PAGE analysis of APXB which processed hydrogen peroxide in presence of glutathione. It is a figure which shows the result of MALDI-TOF MS of the APXB trypsin digest obtained by hydrogen peroxide treatment in the presence of glutathione. It is a figure which shows the influence on the catalytic activity of APXB and APXBC25S by the hydrogen peroxide process in the presence of TEMPO. It is a figure which shows the influence on the catalyst activity of various APX by the hydrogen peroxide process in TEMPO presence. It is a figure which shows the influence on the catalytic activity of various APX by the hydrogen peroxide process in the absence of TEMPO.

SEQ ID NO: 3: Cys at position 25 of ascorbate peroxidase from G. partita is replaced by Ser
SEQ ID NO: 4: g at position 74 of ascorbate peroxidase-encoding sequence from G. partita is replaced by c
SEQ ID NO: 5: A primer APXB29R
SEQ ID NO: 6: A primer APXB30
SEQ ID NO: 7: A primer T7 promoter
SEQ ID NO: 8: A primer T7 terminator
SEQ ID NO: 11: Cys at position 26 of ascorbate peroxidase from tobacco is replaced by Ser
SEQ ID NO: 12: Trp at position 35 of ascorbate peroxidase from tobacco is replaced by Phe
SEQ ID NO: 13: Cys at position 126 of ascorbate peroxidase from tobacco is replaced by Ala
SEQ ID NO: 14: Cys at position 26 of ascorbate peroxidase from tobacco is replaced by Ser; Cys at position 126 of ascorbate peroxidase from tobacco is replaced by Ala
SEQ ID NO: 15: Cys at position 26 of ascorbate peroxidase from tobacco is replaced by Ser; Trp at position 35 of ascorbate peroxidase from tobacco is replaced by Phe; Cys at position 126 of ascorbate peroxidase from tobacco is replaced by Ala

Claims (18)

  An oxidation-resistant ascorbate peroxidase having an amino acid sequence in which at least the amino acid corresponding to position 25 of SEQ ID NO: 1 is substituted with an amino acid other than cysteine in the amino acid sequence of unmodified ascorbate peroxidase.   The oxidation-resistant ascorbate peroxidase according to claim 1, wherein the unmodified ascorbate peroxidase is derived from Galdieria partita or tobacco.   An amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, 14, and 15, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence, and ascorbate peroxidase activity The oxidation-resistant ascorbate peroxidase according to claim 1 or 2, wherein   The oxidation resistant ascorbate peroxidase according to any one of claims 1 to 3, wherein the amino acid other than cysteine is serine.   The oxidation resistant ascorbate peroxidase according to any one of claims 1 to 4, which exhibits oxidation resistance in the presence of a radical scavenger.   Further, substitution of the amino acid at the position corresponding to position 34 of SEQ ID NO: 1 with an amino acid other than tryptophan and / or substitution of the amino acid at the position corresponding to position 121 of SEQ ID NO: 1 with an amino acid other than cysteine. The oxidation resistant ascorbate peroxidase according to any one of claims 1 to 5.   A method for producing oxidation-resistant ascorbate peroxidase, comprising a step of culturing cells that produce oxidation-resistant ascorbate peroxidase, wherein the oxidation-resistant ascorbate peroxidase is an amino acid sequence of unmodified ascorbate peroxidase, A method having an amino acid sequence in which at least an amino acid at a position corresponding to position 25 of SEQ ID NO: 1 is substituted with an amino acid other than cysteine from cysteine.   8. The method according to claim 7, wherein the unmodified ascorbate peroxidase is derived from Galdieria partita or tobacco.   The oxidation resistant ascorbate peroxidase is selected from the group consisting of SEQ ID NOs: 3, 11, 14, and 15, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence. The method according to claim 7 or 8, wherein the method comprises and has ascorbate peroxidase activity.   The method according to any one of claims 7 to 9, wherein the amino acid other than cysteine is serine.   The method according to any one of claims 7 to 10, wherein the oxidation resistant ascorbate peroxidase exhibits oxidation resistance in the presence of a radical scavenger.   Oxidation-resistant ascorbate peroxidase is further substituted from amino acid other than tryptophan at the position corresponding to position 34 of SEQ ID NO: 1 and / or amino acid cysteine at the position corresponding to position 121 of SEQ ID NO: 1. The method according to any one of claims 7 to 11, which has substitution with an amino acid other than cysteine.   A method of imparting oxidation resistance to ascorbate peroxidase, comprising a step of substituting an amino acid at a position corresponding to position 25 of SEQ ID NO: 1 with an amino acid other than cysteine in the amino acid sequence of unmodified ascorbate peroxidase .   14. The method according to claim 13, wherein the unmodified ascorbate peroxidase is derived from Galdieria partita or tobacco.   Ascorbate peroxidase imparted with oxidation resistance is deleted, substituted or added in an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, 14, and 15, or in the amino acid sequence The method according to claim 13 or 14, which comprises an amino acid sequence and has ascorbate peroxidase activity.   The method according to any one of claims 13 to 15, wherein the amino acid other than cysteine is serine.   The method according to any one of claims 13 to 16, wherein oxidation resistance in the presence of a radical scavenger is imparted.   Substituting an amino acid at a position corresponding to position 34 of SEQ ID NO: 1 with an amino acid other than tryptophan from the tryptophan and / or a process of substituting an amino acid at a position corresponding to position 121 of SEQ ID NO: 1 with an amino acid other than cysteine. 18. A method according to any one of claims 13 to 17, further comprising.

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