JP7143985B2 - novel peroxidase - Google Patents

Description

NPMD NITE P-02670

The present invention relates to the identification of novel peroxidases and their uses. More specifically, the present invention provides a reagent for detecting hydroperoxide, a composition for reducing hydroperoxide, and a color reaction using an oxidative color-forming chromogen, which contains a novel peroxidase (or a cell containing a nucleic acid encoding the novel peroxidase). It relates to an inducer or a lignin decomposing agent.

Peroxidase plays a physiological role of removing hydrogen peroxide generated in the process of in vivo redox reaction. Peroxidase is widely distributed in the living world, from higher organisms to microorganisms, and many types are known. is also known (Non-Patent Document 1).

However, peroxidases that can use lipid peroxides and fatty acid peroxides as substrates are heme-free and use thiol as a reducing agent. Therefore, heme-free peroxidase can use phenols as a reducing agent like heme-containing peroxidase, but cannot catalyze useful reactions involving color development. In view of the above, a novel enzyme capable of catalyzing a reaction accompanied by coloration was required for the purpose of detecting peroxidized lipids and peroxidized fatty acids.

In addition, conventional heme-containing peroxidases can decompose lignin, which is a difficult molecule to decompose, but expensive hydrogen peroxide is used for the reaction. Furthermore, white rot fungi, which have been extensively studied so far, have a gene encoding a heme-containing peroxidase capable of degrading lignin, but they grow slowly and there is a problem in obtaining a large amount of related enzymes. Therefore, none of the decomposition, denaturation, and removal of lignin using biotechnology has been put to practical use.

Hayes et al., Free Rad. Res., 31: 273-300 (1999)

An object of the present invention is to provide a novel peroxidase that can catalyze the reduction reaction of not only hydrogen peroxide but also lipid peroxide and fatty acid peroxide with an oxidative chromogenic chromogen, and a hydroperoxide containing the novel peroxidase , a composition for hydroperoxide reduction, and an agent for inducing a coloration reaction by an oxidative color-developing chromogen. Another object of the present invention is to search for a novel peroxidase capable of degrading lignin, isolate the relevant gene, and provide it as a novel lignin-degrading agent.

The present inventors have conducted intensive studies to achieve the above objects, and found that Bacillus licheniformis (acceptance number NITE AP-02670) independently isolated from soil converts linoleic acid peroxide into an electron acceptor (oxidant ), and phenols such as guaiacol and syringol, which are model compounds of lignin, or tetramethylbenzidine and diaminobenzidine as electron donors (reducing agents). was found to express Furthermore, from the estimated molecular weight of the protein and the results of genome analysis, it was speculated that a protein having high sequence identity with the protein YjbI of unknown function in the genus Bacillus had the activity, and the gene encoding the protein was identified. Isolated. Recombinant E. coli expressing the isolated gene was also confirmed to have activity to catalyze the reduction of linoleic acid peroxide. Based on the above, the above proteins are not thiols as in the past, but hydrogen peroxide, lipid peroxide, or peroxidation by oxidative chromogenic chromogens such as guaiacol, syringol, or tetramethylbenzidine and diaminobenzidine. It has been found that they can catalyze the reduction of hydroperoxides such as fatty acids.
The present inventors have completed the present invention as a result of further studies based on these findings.

That is, the present invention
[1] A reagent for detecting hydroperoxide, including the following (a) or (b)
(a) YjbI,
(b) a cell containing a nucleic acid encoding YjbI;
[2-1] The reagent of [1], wherein YjbI contains (i) or (ii) below
(i) an amino acid sequence represented by SEQ ID NO: 2;
(ii) an amino acid sequence that is substantially identical to the amino acid sequence represented by SEQ ID NO: 2 and has activity to catalyze reduction of hydroperoxide and/or activity to catalyze oxidation of oxidative chromogenic chromogen; ;
[2-2] The reagent of [2-1], wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 is (i) or (ii) below
(i) an amino acid sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 2;
(ii) an amino acid sequence having 1 to 14 amino acid substitutions, deletions, insertions or additions to the amino acid sequence represented by SEQ ID NO:2;
[3-1] The reagent of any one of [1] to [2-2], wherein the cell containing a YjbI-encoding nucleic acid is Bacillus licheniformis deposited under accession number NITE AP-02670;
[3-2] The reagent according to any one of [1] to [3-1], further comprising heme;
[3-3] The reagent according to any one of [1] to [3-2], further comprising an oxidative color-forming chromogen;
[3-4] The reagent of any one of [1] to [3-3], wherein the oxidative chromogenic chromogen is tetramethylbenzidine, diaminobenzidine, guaiacol or syringol;
[3-5] The reagent according to any one of [1] to [3-4], wherein the hydroperoxide is hydrogen peroxide, lipid peroxide or fatty acid peroxide;
[3-6] The reagent of [3-5], wherein the fatty acid peroxide is linoleic acid peroxide;
[4] A hydroperoxide-reducing composition containing the following (a) or (b)
(a) YjbI,
(b) a cell containing a nucleic acid encoding YjbI;
[5-1] The composition according to [4], wherein YjbI comprises (i) or (ii) below
(i) an amino acid sequence represented by SEQ ID NO: 2;
(ii) an amino acid sequence that is substantially identical to the amino acid sequence represented by SEQ ID NO: 2 and has activity to catalyze reduction of hydroperoxide and/or activity to catalyze oxidation of oxidative chromogenic chromogen; ;
[5-2] The composition of [5-1], wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 is (i) or (ii) below
(i) an amino acid sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 2;
(ii) an amino acid sequence having 1 to 14 amino acid substitutions, deletions, insertions or additions to the amino acid sequence represented by SEQ ID NO:2;
[6-1] The composition of any one of [4] to [5-2], wherein the cell containing a YjbI-encoding nucleic acid is Bacillus licheniformis deposited under accession number NITE AP-02670;
[6-2] The composition of any one of [4] to [6-1], further comprising heme;
[6-3] The composition according to any one of [4] to [6-2], further comprising a reducing agent;
[6-4] The reducing agent is genistein, genistein-7-glucoside, genistein-acetylglycoside, genistein-malonyl glycoside, daidzein, daidzein-7-glucoside, daidzein-acetylglycoside, daidzein-malonyl glycoside, glycitein, glycitein-7 -glucoside, glycitein-malonyl glycoside, hesperetin, equol, quercetin, catechin, epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, tannic acid, theaflavin, thearubigin, azaleatin, fisetin, galangin, gossipetin, kaemperide , kaempferol, morin, myricetin, natudaidyne, pachypodol, rhamnadin, rhamnetin, tamarixetine, astragalin, azalein, hyperoside, isoquercitrin, quercitrin, robinin, rutin, spireoside, resveratrol, anthocyanin, chlorogenic acid, ellagic acid, lignan, Curcumin, guaiacol, syringol, vanillin, ethyl vanillin, eugenol, canolol, caffeic acid, sinapic acid, coumaric acid, ferulic acid, protocatechuic acid, 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde, 4-hydroxyphenylacetic acid, vanillic acid , syringic acid, naringenin, eriodictyol-7-rutinoside, hesperetin-7-rutinoside, kaempferol-3-rutinoside, kaempferol-3-glucoside, eriodictyol glycoside, naringenin-7-neohesperiodside, resveratrol glucoside and the composition of [6-3], which is selected from the group consisting of tocopherol;
[6-5] The composition according to any one of [4] to [6-4], wherein the hydroperoxide is hydrogen peroxide, lipid peroxide or fatty acid peroxide;
[6-6] The composition of [6-5], wherein the fatty acid peroxide is linoleic acid peroxide;
[7] The composition according to any one of [4] to [6-6], which is a pharmaceutical composition, food composition, feed composition or cosmetic composition;
[8] A color reaction inducer by an oxidative color-forming chromogen, including (a) or (b) below
(a) YjbI,
(b) a cell containing a nucleic acid encoding YjbI;
[9-1] The inducer of [8], wherein YjbI comprises (i) or (ii) below
(i) an amino acid sequence represented by SEQ ID NO: 2;
(ii) an amino acid sequence that is substantially identical to the amino acid sequence represented by SEQ ID NO: 2 and has activity to catalyze reduction of hydroperoxide and/or activity to catalyze oxidation of oxidative chromogenic chromogen; ;
[9-2] The inducer of [9-1], wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 is (i) or (ii) below
(i) an amino acid sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 2;
(ii) an amino acid sequence having 1 to 14 amino acid substitutions, deletions, insertions or additions to the amino acid sequence represented by SEQ ID NO:2;
[10-1] The inducer of any one of [8] to [9-2], wherein the cell containing a nucleic acid encoding YjbI is Bacillus licheniformis deposited under accession number NITE AP-02670;
[10-2] The inducer according to any one of [8] to [10-1], further comprising heme;
[10-3] The inducer according to any one of [8] to [10-2], further comprising a hydroperoxide;
[10-4] The inducer of [10-3], wherein the hydroperoxide is hydrogen peroxide, lipid peroxide or fatty acid peroxide;
[10-5] The inducer of any one of [8] to [10-4], wherein the oxidative chromogenic chromogen is tetramethylbenzidine, diaminobenzidine, guaiacol or syringol;
[11-1] A lignin decomposing agent including the following (a) or (b)
(a) YjbI,
(b) a cell containing a nucleic acid encoding YjbI;
[11-2] The decomposing agent according to [11-2], wherein YjbI comprises (i) or (ii) below
(i) an amino acid sequence represented by SEQ ID NO: 2;
(ii) an amino acid sequence that is substantially identical to the amino acid sequence represented by SEQ ID NO: 2 and has activity to catalyze reduction of hydroperoxide and/or activity to catalyze oxidation of oxidative chromogenic chromogen; ;
[11-3] The degrading agent of [11-2], wherein the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 is (i) or (ii) below;
(i) an amino acid sequence having 90% or more homology with the amino acid sequence represented by SEQ ID NO: 2;
(ii) an amino acid sequence having 1 to 14 amino acid substitutions, deletions, insertions or additions to the amino acid sequence represented by SEQ ID NO:2;
[11-4] Decomposition according to any one of [11-1] to [11-3], wherein the cell containing a YjbI-encoding nucleic acid is Bacillus licheniformis deposited under accession number NITE AP-02670 agent;
[11-5] The decomposing agent according to any one of [11-1] to [11-4], further comprising heme;
[11-6] The decomposing agent according to any one of [11-1] to [11-5], further comprising a hydroperoxide;
[11-7] The decomposing agent according to [11-6], wherein the hydroperoxide is hydrogen peroxide, lipid peroxide or fatty acid peroxide;
etc.

A cell containing YjbI or a nucleic acid encoding YjbI can be used as a reagent for simple and rapid detection of hydroperoxide in a test sample by coloration with an oxidative chromogenic chromogen. In particular, unlike horseradish peroxidase (HRP), which is a plant-derived protein, YjbI is a microbial protein, so it is possible to prepare large amounts at low cost, and it is possible to use peroxide fatty acids and lipid peroxides, which are not available for HRP, as substrates. It is excellent in that it can detect only fatty acid peroxides and lipid peroxides under basic conditions. In addition, since cells containing YjbI or a nucleic acid encoding YjbI can induce a color reaction by catalyzing the oxidation of an oxidative chromogenic chromogen by hydroperoxide, HRP, a conventional catalyst for said reaction, is capable of inducing a color reaction. can be substituted for Furthermore, cells containing YjbI or YjbI-encoding nucleic acids can be used in compositions such as pharmaceutical compositions, food compositions, feed compositions, cosmetic compositions, etc. Active oxygen that is involved in damage to DNA and the like can be reduced in the subject to which it is applied. In recent years, it has been reported that hydroxylated fatty acids obtained by reducing fatty acid peroxides, which are a type of hydroperoxide, have physiological effects such as anti-inflammatory effects and metabolic regulation. expected to be effective. In addition, cells containing YjbI or YjbI-encoding nucleic acids can be used as lignin degrading agents because they can catalyze the oxidation of lignin model compounds guaiacol and syringol by hydroperoxides. In particular, lipoxidase released from cells by crushing plants produces peroxidized fatty acids by oxidizing fatty acids, which is convenient in that it is not necessary to newly add peroxidized fatty acids from the outside.

FIG. 4 shows the results of CBB staining and activity staining. M: CBB-stained image of molecular weight markers, TKS3: CBB-stained image of TKS3-derived extracted proteins, M': Activity-stained image of molecular weight markers, TKS3': Activity-stained image of TKS3-derived extracted proteins. The arrow indicates a protein band presumed to be YjbI. FIG. 2 shows the base sequence of yjbI of TKS3 (SEQ ID NO: 1) and the amino acid sequence (SEQ ID NO: 2) encoded by the base sequence. FIG. 4 shows a Lineweaver-Burk plot of TKS3 against linoleic acid peroxide. FIG. 4 is a diagram showing the reduction activity of linoleic acid peroxide by TKS3 at each pH. Fig. 2 shows comparisons of (a) the hydrogen peroxide reduction activity by TKS3 at each pH and (b) the hydrogen peroxide reduction activity by TKS3 and the reduction activity of linoleic acid peroxide (peroxidation by TKS3 at pH 8.0). The reduction activity of linoleic acid was set to 1.0). FIG. 4 is a diagram showing the reduction activity of linoleic acid peroxide by TKS3 at each temperature. (a) Activities of hemin and TKS3 before and after heating (each activity before heating was taken as 100%), (b) Comparison of hydrogen peroxide reduction activity and linoleic acid peroxide reduction activity by hemin (hemin at pH 8.0 and (C) the effect of 2% hydrogen peroxide treatment on the reduction activity of linoleic acid peroxide by hemin or TKS3 (distilled hydrogen peroxide instead of hydrogen peroxide). The linoleic peroxide reduction activity when water was added was taken as 100%.).

YjbI of the present invention is a protein that is widely distributed in bacteria such as Bacillus subtilis, root nodule bacteria, Escherichia coli, and cyanobacteria, and is a protein containing an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO:2. be.
YjbI may be, for example, a protein isolated and purified from bacteria such as the above-mentioned Bacillus subtilis. It may also be a protein chemically synthesized or biochemically synthesized in a cell-free translation system, or a recombinant protein produced from a transformant introduced with a nucleic acid having a nucleotide sequence encoding the above amino acid sequence. It may be a protein.

The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 is about 90% or more, preferably about 93% or more, more preferably about 95% of the amino acid sequence represented by SEQ ID NO: 2. The amino acid sequences mentioned above, particularly preferably having about 98% or more homology, are included. Here, "homology" refers to the optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm aligns the sequences for optimal alignment). It means the ratio (%) of identical and similar amino acid residues to all overlapping amino acid residues, in which the introduction of gaps in one or both may be considered. "Similar amino acids" means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn ), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Amino acids classified into groups are included. Substitutions with such similar amino acids are expected not to change the protein phenotype (ie, conservative amino acid substitutions). Examples of conservative amino acid substitutions are well known in the art and have been described in various publications (see, eg, Bowie et al., Science, 247:1306-1310 (1990)).
The homology of the amino acid sequences herein is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gaps allowed; matrix = BLOSUM62; filtering = OFF). Other algorithms for determining amino acid sequence homology include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 (1970). [the algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [the algorithm is part of the CGC sequence alignment software package. is incorporated into the ALIGN program (version 2.0)], Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988). embedded in the program], etc., which can also be preferably used.
More preferably, the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 is about 90% or more, preferably about 93% or more, more preferably about 93% or more of the amino acid sequence represented by SEQ ID NO: 2. is an amino acid sequence having about 95% or more identity, particularly preferably about 98% or more identity.

Examples of proteins containing an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 include, for example, the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2, and the sequence A protein having substantially the same activity as the protein containing the amino acid sequence represented by No. 2 is preferred.

Substantially homogeneous activities include, for example, activities that catalyze the reduction of hydroperoxides (e.g., hydrogen peroxide, lipid peroxides, fatty acid peroxides, etc.) as electron acceptors, and oxidative coloring-type colors as electron donors. Activity to catalyze the oxidation of raw materials (eg, tetramethylbenzidine, diaminobenzidine, guaiacol, syringol, etc.). "Substantially the same" indicates that their activities are qualitatively (eg, physiologically or pharmacologically) the same. Therefore, it is preferable that the activities such as the activity to catalyze the reduction of hydroperoxide and the activity to catalyze the oxidation of the oxidative chromogenic chromogen are equivalent (for example, about 0.5 to about 2 times), but the extent of these activities Quantitative factors such as the molecular weight of the protein and the molecular weight of the protein may be different.
The activity to catalyze the reduction of hydroperoxide and the activity to catalyze the oxidation of the oxidative color-forming chromogen can be measured according to a method known per se. Activity can be measured using a color reaction.

The hydroperoxide is not particularly limited as long as it is a hydroperoxide whose reduction is catalyzed by YjbI of the present invention, and examples thereof include hydrogen peroxide, lipid peroxide, and fatty acid peroxide. Examples of lipid peroxides include 1-palmitoyl-2-(13-hydroperoxyoctadecadienoyl)-phosphatidylcholine, 1-palmitoyl-2-(9-hydroperoxyoctadecadienoyl)-phosphatidylcholine, 1-palmitoyl- 2-hydroperoxyeicosatetraenoyl-phosphatidylcholine, 1-palmitoyl-2-hydroperoxycosahexaenoyl-phosphatidylcholine and the like. Examples of fatty acid peroxides include linoleic acid peroxide, oleic acid peroxide, linolenic acid peroxide, arachidonic acid peroxide, eicosapentaenoic acid peroxide, and docosahexaenoic acid peroxide.

The oxidative color-forming chromogen is not particularly limited as long as it is an oxidative color-forming chromogen whose oxidation is catalyzed by YjbI of the present invention. mentioned.
Leuco-type chromogens are substances that are converted into dyes alone in the presence of hydroperoxides and YjbI of the present invention, and include tetramethylbenzidine (TMB), diaminobenzidine (DAB), o-phenylenediamine, 10-N -carboxymethylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (CCAP), 10-N-methylcarbamoyl-3,7-bis(dimethylamino)-10H-phenothiazine (MCDP), N-(carboxy methylaminocarbonyl)-4,4'-bis(dimethylamino)diphenylamine sodium salt (DA-64), 10-N-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)-10H-phenothiazine sodium salt (DA-67), 4,4'-bis(dimethylamino)diphenylamine, bis[3-bis(4-chlorophenyl)methyl-4-dimethylaminophenyl]amine (BCMA) and the like, preferably tetramethyl benzidine and diaminobenzidine;
An oxidative-coupling chromogenic chromogen is a substance that produces a dye by oxidative coupling of two compounds in the presence of a hydroperoxide and YjbI of the present invention. Combinations of two compounds include a combination of couplers and anilines, a combination of couplers and phenols, and the like.
Examples of couplers include 4-aminoantipyrine (4-AA), 3-methyl-2-benzothiazolinone hydrazone and the like.
Anilines include N-(3-sulfopropyl)aniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline (TOOS), N-ethyl-N-(2-hydroxy -3-sulfopropyl)-3,5-dimethylaniline (MAOS), N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (DAOS), N-ethyl-N- (3-sulfopropyl)-3-methylaniline (TOPS), N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline (HDAOS), N,N-dimethyl-3-methylaniline, N ,N-di(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(3-sulfopropyl)aniline, N-ethyl-N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-(3-sulfopropyl)-3,5-dimethoxyaniline, N-ethyl-N-(3-sulfopropyl)-3 ,5-dimethylaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)aniline, N-ethyl- N-(3-methylphenyl)-N'-succinylethylenediamine (EMSE), N-ethyl-N-(3-methylphenyl)-N'-acetylethylenediamine, N-ethyl-N-(2-hydroxy-3- sulfopropyl)-4-fluoro-3,5-dimethoxyaniline (F-DAOS) and the like.
Phenols include phenol, 4-chlorophenol, 3-methylphenol, 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB) and the like.

Further, the amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 includes, for example, (1) 1 or 2 or more (preferably 1 to 1) amino acid sequences represented by SEQ ID NO: 2 (2) an amino acid sequence having about 14, more preferably about 1 to 10, more preferably 1 to several (2, 3, 4 or 5) amino acid substitutions, represented by SEQ ID NO: 2 Deletion of 1 or 2 or more (preferably about 1 to 14, more preferably about 1 to 10, still more preferably 1 to several (2, 3, 4 or 5)) amino acids in the amino acid sequence (3) SEQ ID NO: 1 or 2 or more in the amino acid sequence represented by 2 (preferably about 1 to 14, more preferably about 1 to 10, more preferably 1 to several (2 (4) SEQ ID NO: 1 or 2 or more (preferably about 1 to 14, more preferably about 1 to 14) in the amino acid sequence represented by 2 Also included are proteins containing amino acid sequences having about 1 to 10, more preferably 1 to several (2, 3, 4 or 5) amino acids added, or (5) amino acid sequences combining them. .
When the amino acid sequence is substituted, deleted, inserted or added as described above, the position is not particularly limited as long as the activity of the protein is maintained.

YjbI of the present invention preferably contains the amino acid sequence represented by SEQ ID NO: 2 (Bacillus licheniformis YjbI (GenBank Accession No.: ATI75479), for example, Bacillus paralicheniformis YjbI (GenBank Accession No.: CP005965)). proteins, proteins containing homologues in other species belonging to the genus Bacillus [for example, homologues in Bacillus paralicheniformis registered in GenBank under accession number PAD00377 (having about 99% identity to Bacillus licheniformis YjbI), etc.], Alternatively, it is a protein containing a homologue in bacteria other than the genus Bacillus [eg, a homologue in Anoxybacillus flavithermus registered in GenBank under accession number ACJ34478 (having about 73% identity with Bacillus licheniformis YjbI)]. More preferably, YjbI of the present invention is a protein consisting of the amino acid sequence represented by SEQ ID NO:2.

YjbI can be produced from bacteria such as the aforementioned Bacillus subtilis by a known protein purification method. Specifically, bacteria such as Bacillus subtilis are cultured, the cells collected from the culture by a known method are suspended in an appropriate buffer, and the cells are disrupted by ultrasonic waves, lysozyme and/or freeze-thaw. After that, a method of obtaining a crude extract of soluble protein by centrifugation or filtration is appropriately used. The buffer may contain protein denaturants such as urea and guanidine hydrochloride, and surfactants such as Triton X-100 ^™ . When YjbI is extracellularly secreted, methods such as separating the culture supernatant from the culture by centrifugation, filtration, or the like are used.
YjbI contained in the soluble fraction thus obtained or the culture supernatant can be isolated and purified according to a method known per se. Examples of such methods include methods utilizing solubility such as salting out and solvent precipitation; dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis primarily utilizing differences in molecular weight. method using charge difference such as ion exchange chromatography; method utilizing specific affinity such as affinity chromatography; method utilizing hydrophobicity difference such as reversed-phase high performance liquid chromatography; isoelectric A method using a difference in isoelectric points, such as point electrophoresis, is used. These methods can be combined as appropriate.

YjbI can also be produced according to known peptide synthesis methods.
The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. An amino acid derivative with protective groups attached to the carboxy group and side chain functional groups is condensed with an amino acid derivative with protected amino groups and side chain functional groups, and the protective groups are removed to produce the target protein. can do.
Here, the condensation and elimination of the protective group are carried out according to methods known per se, for example, the methods described in (1) to (5) below.
(1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers,
New York (1966)
(2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(3) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(4) Haruaki Yajima and Shunpei Sakakibara, Biochemical Experiment Course 1, Protein Chemistry IV 205 (1977)
(5) Supervised by Haruaki Yajima, Development of Sequel Drugs, Volume 14, Peptide Synthesis, Hirokawa Shoten

YjbI thus obtained can be purified and isolated by a known purification method. Here, examples of purification methods include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
When YjbI obtained by the above method is a free form, the free form can be converted into a suitable salt by a known method or a method analogous thereto. Conversely, when YjbI is obtained as a salt, , the salt can be converted into the free form or other salt by known methods or analogous methods.

YjbI of the present invention may be provided as a cell containing nucleic acid encoding it. A YjbI-encoding nucleic acid may be DNA or RNA, or may be a DNA/RNA chimera. DNA is preferred. Also, the nucleic acid may be double-stranded or single-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA or a DNA:RNA hybrid. If single stranded, it may be the sense strand (ie, the coding strand) or the antisense strand (ie, the non-coding strand).
DNAs encoding YjbI include genomic DNAs derived from bacteria such as Bacillus subtilis. Genomic DNA encoding YjbI can also be directly amplified by Polymerase Chain Reaction (hereinafter abbreviated as "PCR method") using a genomic DNA fraction prepared from bacteria such as Bacillus subtilis as a template.

The YjbI-encoding DNA includes, for example, a DNA containing the nucleotide sequence represented by SEQ ID NO: 1, or a nucleotide sequence that hybridizes with the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions. However, it also includes a DNA encoding a protein having substantially the same activity as YjbI described above (eg, hydroperoxide reduction activity, oxidation activity of chromogenic chromogen, etc.).
DNAs that can hybridize with the nucleotide sequence represented by SEQ ID NO: 1 under stringent conditions include, for example, about 80% or more, preferably about 85% or more, of the nucleotide sequence represented by SEQ ID NO: 1, and further A DNA containing a base sequence having a homology of preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 98% or more is used.
The homology of the nucleotide sequences in the present specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gaps allowed; filtering = ON; match Score = 1; mismatch score = -3). Other algorithms for determining nucleotide sequence homology are similarly preferably exemplified by the above-described amino acid sequence homology calculation algorithms.

Hybridization should be carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning, Second Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). can be done. Moreover, when using a commercially available library, hybridization can be performed according to the method described in the attached instruction manual. Hybridization is preferably performed under stringent conditions.
Examples of highly stringent conditions include hybridization in 6x SSC (sodium chloride/sodium citrate) at 45°C, followed by one or more washes in 0.2x SSC/0.1% SDS at 65°C. mentioned. Those skilled in the art can appropriately change the salt concentration of the hybridization solution, hybridization reaction temperature, probe concentration, probe length, number of mismatches, hybridization reaction time, washing solution salt concentration, washing temperature, etc. can be easily adjusted to the desired stringency.

The DNA encoding YjbI is preferably a nucleotide sequence represented by SEQ ID NO: 1 (nucleotide sequence encoding Bacillus licheniformis YjbI (GenBank Accession No.: CP023729), for example, Bacillus paralicheniformis YjbI (GenBank Accession No.: CP005965)), a DNA containing a nucleotide sequence encoding a homologue in another species belonging to the genus Bacillus [for example, a nucleotide sequence encoding a homologue in Bacillus paralicheniformis registered in GenBank under accession number NPCO01000007], or A DNA containing a nucleotide sequence encoding a homolog in bacteria other than the genus Bacillus [eg, a nucleotide sequence encoding a homolog in Anoxybacillus flavithermus registered in GenBank under accession number CP000922]. More preferably, the YjbI-encoding DNA of the present invention is a base sequence consisting of the base sequence represented by SEQ ID NO:1.

The YjbI-encoding DNA was amplified by PCR using synthetic DNA primers (for example, SEQ ID NO: 3 and SEQ ID NO: 4) having a portion of the base sequence encoding YjbI, or incorporated into an appropriate expression vector. DNA can be cloned by hybridization with labeled DNA fragments or synthetic DNA encoding part or all of YjbI. Hybridization can be performed, for example, according to the method described in Molecular Cloning, 2nd edition (mentioned above). Moreover, when using a commercially available library, hybridization can be performed according to the method described in the instruction manual attached to the library.

Cells containing a nucleic acid encoding YjbI of the present invention (hereinafter also referred to as cells of the present invention) include cells containing a genome containing a nucleic acid encoding YjbI of the present invention, and cells containing a nucleic acid encoding YjbI of the present invention. Examples include cells containing vectors.

Cells containing a genome containing a nucleic acid encoding YjbI of the present invention include bacteria such as the Bacillus subtilis described above. Cells containing a genome containing a nucleic acid encoding YjbI of the present invention may be known bacteria, or novel bacteria may be isolated and used. The inventors identified Bacillus licheniformis (TKS3) as a cell containing a genome containing the YjbI-encoding nucleic acid of the present invention, as described in the examples below, and received this TKS3 on March 23, 2018. It was deposited under the number NIT EP- 02670 at the National Institute of Technology and Evaluation Patent Microorganism Depositary Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture).

A cell containing an expression vector containing a YjbI-encoding nucleic acid of the present invention can be obtained by known means. For example, it can be obtained by ligating the YjbI-encoding DNA of the present invention cloned as described above to an expression vector in such a manner as to allow expression, and transforming cells with the expression vector.

An expression vector containing the YjbI-encoding DNA of the present invention can be produced, for example, by excising a YjbI-encoding DNA fragment and ligating the DNA fragment downstream of the promoter in an appropriate expression vector.
Examples of expression vectors include E. coli-derived plasmids (eg, pBR322, pBR325, pUC12, pUC13); Bacillus subtilis-derived plasmids (eg, pUB110, pTP5, pC194); and yeast-derived plasmids (eg, pSH19, pSH15). .
Any promoter may be used as long as it is suitable for the host used for gene expression.
For example, when the host is Escherichia, trp promoter, lac promoter, recA promoter, _λPL promoter, lpp promoter, T7 promoter and the like are preferred.
When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferred.

As the expression vector, in addition to the above, those containing enhancers, selection markers and the like can be used as desired. Selectable markers include, for example, ampicillin resistance gene (hereinafter sometimes abbreviated as ^ampr ) and the like.
In addition, if necessary, a nucleotide sequence (signal codon) encoding a signal sequence suitable for the host may be added to the 5' end of the YjbI-encoding DNA (or replaced with a native signal codon). For example, when the host is Escherichia, the PhoA signal sequence, OmpA signal sequence, etc. are used; when the host is Bacillus, the α-amylase signal sequence, subtilisin signal sequence, etc. are used, respectively.

Cells of the genus Escherichia, Bacillus and the like are used, for example.
As Escherichia bacteria, for example, Escherichia coli (Escherichia coli) K12 DH1 [Proceedings of the National Academy of Sciences of the USA (Proc. Natl. Acad. Sci. USA) 60, 160 (1968)], Escherichia coli JM103 [Nucleic Acids Research, 9, 309 (1981)], Escherichia coli JA221 [Journal of Molecular Biology Biology), Vol. 120, 517 (1978)], Escherichia coli HB101 [Journal of Molecular Biology, Vol. 41, 459 (1969)], Escherichia coli C600 [Genetics, Vol. 39, 440 (1954)]. )] and the like are used.
Examples of Bacillus spp. include Bacillus subtilis MI114 [Gene, Vol. 24, 255 (1983)], Bacillus subtilis 207-21 [Journal of Biochemistry, Vol. , 87 (1984)].

Transformation can be performed according to known methods depending on the type of host.
Escherichia spp., for example, Proc. Natl. Acad. Sci. USA, Vol. 69, 2110 (1972) and Gene ( Gene), vol. 17, 107 (1982).
Bacillus can be transformed according to the method described in, for example, Molecular & General Genetics, Vol. 168, 111 (1979).

As described above, cells containing an expression vector containing the YjbI-encoding DNA of the present invention can be obtained. The YjbI of the present invention can also be produced by culturing the above cells.

YjbI of the invention or cells of the invention can catalyze the oxidation of oxidative chromogenic chromogens and the reduction of hydroperoxides, and thus can be used as reagents for the detection of hydroperoxides. Accordingly, the present invention provides a hydroperoxide detection reagent (hereinafter, the detection reagent of the present invention) comprising a cell containing YjbI or a nucleic acid encoding YjbI. Conventionally, horseradish peroxidase has been a typical enzyme that catalyzes the reduction of hydrogen peroxide by an oxidative chromogenic chromogen and can detect hydrogen peroxide by coloration. No enzyme is known that can catalyze the reduction of lipid peroxides or fatty acid peroxides. Therefore, by using the detection reagent of the present invention, not only hydrogen peroxide but also lipid peroxide and fatty acid peroxide can be detected simply and easily using the color reaction of the oxidative chromogenic chromogen. In addition, unlike horseradish peroxidase, which is a plant-derived protein, YjbI of the present invention is a microorganism-derived protein, so it is excellent in that it can be prepared inexpensively and in large quantities. Furthermore, the detection reagent of the present invention can detect only lipid peroxides and fatty acid peroxides under basic conditions, and it is also advantageous in that the detection target can be easily selected. Since lipid peroxide and fatty acid peroxide are indicators of oxidative stress in living organisms, the reagent for detection of the present invention can also be used as a reagent for detecting oxidative stress as another aspect. The hydroperoxide that can be detected with the detection reagent of the present invention is not particularly limited as long as it is reduced by the oxidative chromogenic chromogen in the presence of the YjbI of the present invention or the cell of the present invention. may be similar to

The detection reagent of the present invention may further contain heme. As shown in Examples described later, the YjbI of the present invention has high amino acid sequence identity with a protein of unknown function in the genus Bacillus, suggesting that it is a heme protein. Heme may be a complex consisting of a divalent iron atom and porphyrin, or a complex consisting of a divalent iron atom and a porphyrin structural isomer or porphorin analogue. Porphyrin structural isomers include porphycene, colphycene, hemiporphycene, and the like. Porphyrin analogs include corroles, phthalocyanines, chlorins, and the like.

The detection reagent of the present invention may further contain an oxidative color-forming chromogen. YjbI of the present invention can catalyze the oxidation of oxidative chromogenic chromogens by hydroperoxides and induce color reaction. Therefore, oxidative chromogenic chromogens can make the detection and quantification of hydroperoxides with the detection reagents of the present invention more rapid and convenient. The oxidative coloring chromogen contained in the detection reagent of the present invention is not particularly limited as long as it is a chromogen that develops color by oxidation with hydroperoxide in the presence of YjbI of the present invention. It may be the same as the drug substance.

As described above, hydroperoxide can be detected by using the YjbI of the present invention or the cells of the present invention. Therefore, the present invention also provides a hydrochloride method in a test sample, which comprises reacting a test sample with an oxidative color-forming chromogen in the presence of the YjbI of the present invention or the cells of the present invention, and detecting the coloration of the reaction solution. A method for detecting peroxides (hereinafter referred to as the detection method of the present invention) is provided. The detection method of the present invention may further use heme and an oxidative color-forming chromogen in the same manner as the detection reagent of the present invention.

The test sample used in the detection method of the present invention is not particularly limited as long as it enables the detection method of the present invention. Examples include biological samples (blood, plasma, serum, lymph, urine, sweat, saliva, etc.) body fluids or fractions thereof).

In the detection method of the present invention, the concentration of YjbI of the present invention is not particularly limited as long as it is suitable for detection of hydroperoxide, and is usually 1 to 100 kU/L. When the cells of the present invention are used, the number of cells is adjusted so that the concentration of YjbI in the cells is the above concentration.

In the detection method of the present invention, the concentration of the oxidative color-forming chromogen is not particularly limited as long as it is suitable for detecting hydroperoxide, but is usually 0.01 to 10 g/L.

The detection method of the present invention is usually carried out in an aqueous medium of pH 4.0-10.0, preferably pH 6.0-9.0. In particular, when the detection method of the present invention is carried out in an aqueous medium of pH 8.7 to 9.0, hydrogen peroxide cannot be detected, but lipid peroxide or fatty acid peroxide can be detected, which is preferable. Examples of the aqueous medium include deionized water, distilled water, buffer solutions, etc., and buffer solutions are preferred. Examples of the buffer include phosphate buffer, citrate buffer, borate buffer, carbonate buffer, Good's buffer and the like.

Also, YjbI of the present invention or cells of the present invention can catalyze the reduction of hydroperoxides and thus can be used as compositions for reducing hydroperoxides. Accordingly, the present invention provides a composition for hydroperoxide reduction (hereinafter, composition of the invention) comprising a cell comprising YjbI or a nucleic acid encoding YjbI. The composition of the present invention not only reduces the hydroperoxide contained in the composition by the YjbI of the present invention or the cell of the present invention, but also reduces the hydroperoxide in the subject to which the composition is administered, ingested or applied. It can also be redeemed. Hydroperoxides that can be reduced with the compositions of the invention can be similar to hydroperoxides that can be detected with the detection reagents of the invention. In addition, the composition of the present invention may further contain heme as well as the detection reagent of the present invention.

Examples of the composition of the present invention include, but are not limited to, pharmaceutical compositions, food compositions, feed compositions, cosmetic compositions, and the like.

When the composition of the present invention is a pharmaceutical composition, the dosage form of the pharmaceutical composition includes powders, granules, pills, soft capsules, hard capsules, tablets, chewable tablets, rapidly disintegrating tablets, syrups, liquids, Suspensions, suppositories, ointments, creams, gels, stickies, inhalants, injections and the like. These formulations are prepared according to a conventional method.

When administered in the form of an injection, it can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, transdermally, intraarticularly, intrasynovially, intrathecally, intraperiosteally, sublingually, intraorally, etc. Preferred, particularly intravenous or intraperitoneal administration. Intravenous administration may be either drip infusion or bolus administration.

When the composition of the present invention is a food composition (or food additive), the food composition may be in an orally ingestible form such as a solution, suspension, powder, solid molding, or the like. , is not particularly limited. Specific examples include supplements, beverages, confectionery, instant foods, oil and fat foods, flour products, seasonings, processed livestock products, and fermented foods. In particular, when the food composition of the present invention is a fermented food, the bacteria used for fermentation may be the cells of the present invention. For example, when the food composition of the present invention is natto, Bacillus subtilis var. Hydroperoxides contained in natto can be reduced by fermenting soybeans with natto.

When the composition of the present invention is a feed composition (or feed additive), examples of the feed composition include pet food, livestock or aquaculture feed additives, and the like.

When the composition of the present invention is a cosmetic composition, it is not particularly limited, but basic cosmetics (e.g., lotion, milky lotion, makeup base, serum, night cream, pack, makeup remover (cleansing gel, etc.), lip cream, nail cream, etc.), makeup cosmetics (e.g., foundation (solid, cream, liquid, etc.), sunscreen, BB cream, CC cream, concealer, lipstick, lip gloss, eyeshadow, eyeliner, blush, mascara, bronzer, etc.), hair care cosmetics (hair treatment agents (e.g., hair treatment, out bath treatment, hair serum, split end coat agent, etc.), hair styling agents (e.g., brushing lotion, curler lotion, pomade, tick , set hair spray, hair mist, hair liquid, styling foam, hair gel, water grease, etc.), shaving products (eg, shaving cream, aftershave lotion, etc.).

Subjects to which the composition of the present invention is administered, ingested or applied include humans and animals other than humans (e.g., dogs, cats, mice, rats, hamsters, guinea pigs, rabbits, pigs, cows, chickens, parakeets, mynahs, goats, horses, sheep, monkeys, etc.).

The amount of administration, ingestion, or application of the composition of the present invention varies depending on the subject to be administered, ingested, or applied, the target disease, symptoms, administration route, etc. For example, the YjbI of the present invention contained in the composition of the present invention The daily administration, intake or application amount is usually 0.02 to 100 mg/kg body weight, preferably 0.2 to 50 mg/kg body weight, more preferably 0.5 to 20 mg/kg body weight, orally or parenterally. can be administered or ingested to Administration, ingestion or application may be divided into multiple times per day. In addition, the dose may be increased or decreased depending on symptoms. When the cells of the present invention are used, the number of cells is adjusted so that the concentration of YjbI in the cells is the above concentration.

The composition of the invention may further comprise a reducing agent. YjbI of the present invention is capable of catalyzing the reduction of hydroperoxides by said reducing agents. Thus, the reducing agent can have greater activity in catalyzing the reduction of hydroperoxides by the compositions of the present invention. The reducing agent contained in the composition of the present invention is a substance capable of reducing hydroperoxide in the presence of YjbI of the present invention, and is not particularly limited as long as it is not toxic to the body. Examples include genistein, genistein-7- glucoside, genistein-acetyl glycoside, genistein-malonyl glycoside, daidzein, daidzein-7-glucoside, daidzein-acetyl glycoside, daidzein-malonyl glycoside, glycitein, glycitein-7-glucoside, glycitein-malonyl glycoside, hesperetin, equol, quercetin, catechin , epicatechin, epigallocatechin, epicatechin gallate, epigallocatechin gallate, tannic acid, theaflavin, thearubigin, azaleatin, fisetin, galangin, gossipetin, kaemperide, kaempferol, morin, myricetin, natsudaidain, pachypodol, ramnazin, ramnetin, tamarixetine, astragalin, azalein, hyperoside, isoquercitrin, quercitrin, robinin, rutin, spireoside, resveratrol, anthocyanin, chlorogenic acid, ellagic acid, lignan, curcumin, guaiacol, syringol, vanillin, ethylvanillin, eugenol, canolol , caffeic acid, sinapinic acid, coumaric acid, ferulic acid, protocatechuic acid, 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde, 4-hydroxyphenylacetic acid, vanillic acid, syringic acid, naringenin, eriodictyol-7-rutinoside, hesperetin -7-rutinoside, kaempferol-3-rutinoside, kaempferol-3-glucoside, eriodictyol glycoside, naringenin-7-neohesperiodoside, resveratrol glucoside, tocopherol and the like.

In addition, since the YjbI of the present invention or the cell of the present invention can induce a color reaction by catalyzing the oxidation of the oxidative chromogenic chromogen by hydroperoxide, can be used as Accordingly, the present invention provides a color reaction inducer (hereinafter referred to as the inducer of the present invention) that contains cells containing YjbI or a nucleic acid encoding YjbI and is induced by an oxidative chromogenic chromogen. As described above, representative compounds that can catalyze the oxidation-reduction reaction between the oxidative color-forming chromogen as a reducing agent and hydrogen peroxide as an oxidizing agent and can induce a color reaction by oxidation of the oxidative color-forming chromogen are oxidized. The enzyme is currently horseradish peroxidase. The color reaction is widely used as a medically or biologically effective tool. For example, in clinical tests, the above-described color reaction is used when detecting a marker antigen present in a biological sample of a subject by ELISA. Alternatively, in basic biological research, the above color reaction is used for nucleic acid analysis means (Southern blot, Northern blot, etc.) and protein analysis means (Western blot, etc.). The YjbI of the present invention or the cells of the present invention are superior in that they can substitute for horseradish peroxidase, are easy to prepare in large quantities, and can use as substrates fatty acid peroxides and lipid peroxides that cannot be used with HRP. . The oxidative color-forming chromogen capable of inducing a color reaction with the inducing agent of the present invention may be the same as the oxidative color-forming chromogen described above. The inducing agent of the present invention may further contain heme, similar to the detection reagent of the present invention.

The inducer of the invention may further comprise a hydroperoxide. YjbI of the present invention can catalyze the oxidation of oxidative chromogenic chromogens by hydroperoxides, thereby inducing a color reaction. Thus, hydroperoxides are capable of enhancing the degree of coloration induced by the inducing agents of the present invention. The hydroperoxide contained in the inducer of the present invention is not particularly limited as long as it is reduced by the oxidative chromogenic chromogen in the presence of the YjbI of the present invention or the cell of the present invention. may be similar.

As described above, the use of the YjbI of the present invention or the cells of the present invention can induce the color reaction of the oxidative chromogenic chromogen. Therefore, the present invention also provides a method for inducing a color reaction of an oxidative chromogenic chromogen, which comprises reacting a hydroperoxide with an oxidative chromogenic chromogen in the presence of the YjbI of the present invention or the cell of the present invention ( Hereinafter, the induction method of the present invention) is provided. The derivation method of the invention, like the detection method of the invention, may additionally employ heme.

In the induction method of the present invention, the concentrations of YjbI and the oxidative chromogenic chromogen of the present invention may be the same as in the detection method of the present invention. The hydroperoxide concentration in the induction method of the present invention is not particularly limited as long as it is suitable for inducing the color reaction of the oxidative color-forming chromogen, but is usually 0.01 to 10 g/L. In addition, the induction method of the present invention is usually carried out in an aqueous medium of pH 4.0-10.0.

In addition, since the YjbI of the present invention or the cells of the present invention can catalyze the oxidation of lignin model compounds guaiacol and syringol by hydroperoxides, they can be used as lignin degrading agents. Accordingly, the present invention provides a lignin-degrading agent (hereinafter, the degrading agent of the present invention) comprising a cell containing YjbI or a nucleic acid encoding YjbI. At present, the decomposition, modification, and removal of lignin using biotechnology have not been put to practical use, but by using the decomposing agent of the present invention, lignin can be decomposed, and cellulose can be easily purified. become. In particular, since lipoxidase released from plant cells by crushing plants produces fatty acid peroxides by oxidizing fatty acids, it is not necessary to newly add fatty acid peroxides from the outside for the decomposition of lignin. It's easy. The decomposing agent of the present invention may further contain heme, like the detection reagent of the present invention. Further, the decomposing agent of the present invention may further contain a hydroperoxide, similar to the detection reagent of the present invention, for the purpose of increasing the rate of lignin decomposition.

As described above, lignin can be degraded by using the YjbI of the present invention or the cells of the present invention. Accordingly, the present invention also provides a method for degrading lignin in a plant sample (hereinafter referred to as the degrading method of the present invention), comprising contacting the YjbI of the present invention or the cell of the present invention with the plant sample.

The plant sample used in the decomposition method of the present invention is not particularly limited as long as it contains lignin. Examples of plants include pasture grasses, cereals, rice, and trees that produce wood. Such plant samples include Italian ryegrass, orchardgrass, guinea grass, turfgrass, smoothbromgrass, timothy, tall fescue, hybrid ryegrass, bahiagrass, festolium, perennial ryegrass, meadow fescue, rosegrass, red clover, alfalfa, white clover, Oat, sorghum, corn, sugar cane, rice, wheat, barley, rye, Japanese red pine, Japanese black pine, yew, ginkgo biloba, dogwood, spruce, Japanese maple, Japanese larch, Japanese cedar, Japanese hemlock, Japanese sawara, Sakhalin fir, nezuko, cypress, cypress, Japanese white pine, fir, aodamo, aohada, red oak, asada, isunoki, Japanese maple, barberry, styrax, Japanese walnut, persimmon, katsura, yellowfin, paulownia, camphor, chestnut, zelkova, kojii, cherry, sawa walnut, linden, white oak, white birch, tabunoki, Examples include plant samples derived from boxwood, horse chestnut, porcupine, black locust, sen harigiri, elm, alder, beech, magnolia, makanba, dogwood, oak, mizume, yachidamo, willow, and yamaguwa. A plant sample may be a sample derived from a whole plant or a sample derived from a portion thereof. Parts include above-ground parts (eg, trunks, branches, bark, leaves, flowers, fruits and seeds) or below-ground parts (eg, roots). In addition, the form of the sample is not particularly limited as long as it does not interfere with the contact between the YjbI of the present invention or the cells of the present invention and the plant sample. mentioned.

In the decomposition method of the present invention, the concentration of YjbI of the present invention is not particularly limited as long as it is suitable for lignin decomposition, but is usually 1 to 100 kU/L. When the cells of the present invention are used, the number of cells is adjusted so that the concentration of YjbI in the cells is the above concentration.

The decomposition method of the present invention is usually carried out in an aqueous medium of pH 4.0-10.0, preferably pH 6.0-9.0.

EXAMPLES The present invention will be described in more detail below with reference to examples, but these are merely examples and do not limit the scope of the present invention.

Materials and Methods (1) Acquisition, Identification, and Genome Analysis of Bacteria 1 g of the collected soil was suspended in 5 mL of sterilized physiological saline, and 10 μl of the supernatant was separated. Lignin medium ₍ pH 7.5) containing 0.5% Lignin ₍ Sigma _- Aldrich), 0.2% _K2HPO4 , 0.2% _KH2PO4 , 0.2% NaCl, 0.01% _MgSO4.7H2O , and 0.01% yeast extract and cultured with shaking at 32°C for one week. 100 μL of the medium in which suspension was observed on a solid medium prepared in advance by adding 1.5% agar to the lignin medium and heating, was separated, applied, and cultured at 30° C. for another week. A bacterium TKS3 capable of metabolizing phenols was obtained by isolating the colonies that appeared on the solid medium. Bacterial species identification was entrusted to Koga Isotope Co., Ltd. and analyzed by MALDI/TOFMS. In addition, the genome was entrusted to the Molecular Genetics Laboratory of Toyohashi University of Technology and analyzed using the MiSeq sequencer.

(2) Genomic DNA extraction
Genomic DNA was extracted and purified from a medium containing about 1×10 ⁸ cells/mL of TKS3 cells using Wizard (registered trademark) Genomic DNA Purification Kit from Promega.

(3) Gene sequence amplification
The sequence of the TKS3 gene corresponding to yjbI of the genus Bacillus (hereinafter referred to as "yjbI" unless otherwise specified) was amplified by PCR under the following conditions. 10 μM of each primer (5'-GGATCCGATGGGACAATCG-3' (SEQ ID NO: 3) and 5'-CTCGAGATGTGAACCATCCTCC-3' (SEQ ID NO: 4)) 1.5 μL, 10×PCR Buffer for KOD -Plus- 5 μL, 2 mM dNTPs After preparing a reaction solution containing 5 µL, 1 µL of the extracted TKS3 genomic DNA, 33 µL of sterilized water, and 1 µM of 1 U/µL KOD-Plus-Polymerase, the reaction solution was vortexed. PCR of the reaction solution was performed by repeating 30 times a reaction cycle of 94°C for 15 seconds, 65°C for 30 seconds, and 68°C for 2 minutes using a thermal cycler.

(4) Restriction enzyme treatment of gene sequence
The solution containing the amplified DNA sequences was passed through the NucleoSpin® Purify according to the Gel and PCR Clean-up kit and protocol, and add 1 μL of BamHI, 1 μL of XhoI, 2 μL of 10× Universal Buffer K, and 14 μL of sterilized water from Takara Bio Inc. to 2 μL of the collected solution. The restriction enzyme treatment was carried out by reacting at °C for 1 hour. Then NucleoSpin® Purified using Gel and PCR Clean-up.

(5) Restriction enzyme treatment of vector
1 μL of BamHI, 1 μL of XhoI, 2 μL of 10×K Buffer K, and 15 μL of sterile water were added to 1 μL of 1 μg/μL plasmid vector pET21b(+) solution and allowed to react at 37° C. for 1 hour for restriction enzyme treatment. . Then NucleoSpin® Purified using Gel and PCR Clean-up.

(6) Preparation of recombinant E. coli Ligation is performed by adding 7.5 μL of Mighty MIX from Takara Bio Inc. to a solution containing 3.5 μL of restriction enzyme-treated yjbI DNA solution and 3 μL of vector DNA solution, and reacting at 25°C for 5 minutes. gone. Thereafter, the entire amount of the solution was added to ECOS ^™ Competent E. coli BL21 (DE3) manufactured by Nippon Gene Co., Ltd. on ice, stirred with a vortex, and then incubated on ice for 5 minutes. Furthermore, it is incubated at 42°C for 45 seconds, vortexed, immediately spread on LB solid medium (1.5% agar added to LB medium, heated), and allowed to stand at 30°C for 24 hours to culture the recombinant E. coli. colonies were obtained. As a control, E. coli BL21 (DE3) transformed with the pET21b(+) vector was also prepared.

(7) Culture and preservation of bacteria
TKS3 was cultured with shaking in 500 mL of LB medium (1% polypeptone, 1% NaCl, 0.5% yeast extract, pH 7.4) for 24 hours. For the recombinant E. coli, after culturing in 500 mL of LB medium for 3 hours, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to 1 mM, and YjbI was added to the recombinant E. coli by further culturing for 8 hours. highly expressed. These cells were recovered by separating pellets with a centrifuge, suspending them with physiological saline, and then centrifuging them again. The collected cells were stored at -80°C.

(8) Preparation of peroxidized linoleic acid solution In this test, peroxidized linoleic acid, which is a representative peroxidized fatty acid in the living body, was used. 50 μL of linoleic acid, 10 μL of 50,000 U/mL lipoxidase (Sigma-Aldrich), 830 μL of 500 mM phosphate buffer, 10 μL of 0.5 M EDTA, and 4100 μL of distilled water were mixed well and pumped for about 60 minutes. It was produced by bubbling with air.

(9) Preparation of 3,3′,5,5′-tetramethylbenzidine (TMB) solution 20 mg of TMB was added to 1 mL of a solution obtained by mixing ethanol and acetone at 3:1 to prepare a TMB solution.

(10) Activity measurement of each electron donor (reducing agent) and linoleic acid peroxide
The oxidation activity of electron donors (reducing agents) by TKS3 or YjbI was evaluated using 10 μL of the above linoleic acid peroxide solution, 10 μL of 1M MOPS buffer (pH 8.7), 195 μL of distilled water, and TKS3 (or YjbI expression vector). E. coli) 10 mg/mL added physiological saline 5 μL is mixed with 10 μL of each electron donor (reducing agent) substrate solution (20 mg/mL TMB, 100 mM guaiacol, 100 mM syringol, 20 mg /mL 3,3'-diaminobenzidine (DAB)) was added, and the specific activity was calculated from the change in absorbance for 10 seconds (TMB was used to measure the activity of recombinant E. coli with the YjbI expression vector). . The wavelength and extinction coefficient for measuring each molecule after the reaction are as follows; TMB: 653 nm, ε= 3.9×10 ⁴ (M ⁻¹ cm ⁻¹ ), Guaiacol: 456 nm, ε= 1.2×. 10 ⁴ (M ⁻¹ cm ⁻¹ ), syringol: 468 nm, ε=2.8×10 ⁴ (M ⁻¹ cm ⁻¹ ), 3,3′-diaminobenzidine (DAB): 480 nm, ε= 5.5× 10 ³ (M ^-1 cm ^-1 ). In addition, K _m and V _max values were calculated by Lineweaver-Burk plots from the absorbance of TMB when the final concentration of linoleic acid peroxide was changed to 70, 139, 278, 417, 696, 1113, 1391, and 2783 μM. It was calculated by At this time, the protein concentration in each sample was calculated by the Bradford method.

(11) Activity Staining First, a sample buffer was prepared by mixing 1.2 mL of 0.5M Tris-HCl (pH 6.8), 1 mL of glycerol, 0.5 mL of 0.5% BPB, and 4.8 mL of distilled water. Total protein was extracted by dissolving 10 mg of TKS3 in 1 mL of B-PER ^TM , 10 μL aliquot was mixed with an equal volume of sample buffer, and 15 μL aliquot was added to 15% After applying to wells of a polyacrylamide gel, electrophoresis was performed in an SDS buffer. After electrophoresis, the gel was taken out and shaken in 50 mL of distilled water for 5 minutes. Furthermore, this gel was transferred to a mixed solution of 1 mL of 1 M acetate buffer (pH 5.0), 0.5 mL of TMB solution, 200 μL of linoleic acid peroxide solution, and 15 mL of distilled water, and allowed to stand at room temperature for 5 minutes. Activity stained. After photographing the results of activity staining, the gel was shaken in 500 mL of distilled water for 30 minutes, and all proteins were stained with a CBB staining solution (CBB Stain One).

(12) Measurement of activity change due to pH 10 μL of various buffers (1 M acetate buffer of pH 4.0, 5.0, 6.0, 1 M MOPS buffer of pH 6.0, 7.0, 8.0, 8.7, pH 8.7, 9.6) Tris-HCl buffer), 10 μL of linoleic acid peroxide solution or 3% hydrogen peroxide, 195 μL of distilled water, and 5 μL of TKS3 10 mg/mL added physiological saline, and 10 μL of TMB was added to the solution. , the specific activity was calculated from the amount of change in absorbance for 10 seconds.

(13) Evaluation of heat resistance
TKS3 10 mg/mL added physiological saline was heated to 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, and 90°C for 10 minutes and cooled to room temperature. μl was aliquoted. To this, 10 μL of linoleic acid peroxide solution, 10 μL of 1M MOPS buffer (pH 8.7), 195 μL of distilled water and 10 μL of TMB were added, and the residual activity was calculated from the amount of change in absorbance after 60 seconds.

(14) Activity comparison between hemin and TKS3 A hemin solution was prepared by adding 200 µL of a 10% SDS solution to 1 mg of hemin. Heat-treated samples were prepared by heating TKS3 10 mg/mL added physiological saline or hemin solution at 96°C for 10 minutes in a heat block. 10 μL of 20 mg/mL TMB, linoleic acid peroxide solution or 10 μL of 3% hydrogen peroxide, 1 M MOPS buffer (for heat resistance evaluation: pH 8.7, for pH effect evaluation: pH 8.0 and pH 8.7) 5 μL of each was added to a mixed solution of 10 μL and 195 μL of distilled water, and the activity was compared by measuring the amount of change in absorbance after 60 seconds.
Alternatively, add 5 μL of hemin solution or TKS3 solution to 10 μL of 3% hydrogen peroxide or distilled water (the final concentration of hydrogen peroxide at this point is 2%) and incubate at room temperature for 10 minutes to remove hydrogen peroxide. compared the resistance to After that, add 185 μL of distilled water, 10 μL of 1M MOPS buffer (pH 8.7), and 10 μL of TMB to the solution. Then, the residual activity was calculated from the amount of change in absorbance after 60 seconds.

Results (1) Identification of TKS3 species
MALDI/TOFMS and genome analysis results confirmed that TKS3 was Bacillus licheniformis.

(2) Peroxylinoleic acid reduction activity of TKS3
TKS3 expresses a protein with reducing activity for fatty acid peroxides, with linoleic acid peroxide as an electron acceptor (oxidant) and tetramethylbenzidine, diaminobenzidine, guaiacol, or syringol as electron donors (reducing agents). (Table 1). In addition, the protein was presumed to have heme from the property of using phenols as electron donors.

(3) Identification of a gene and amino acid sequence encoding a protein having fatty acid peroxide reduction activity
Based on the molecular weight estimated from the results of activity staining of the TKS3-derived extracted protein (Fig. 1) and the results of genome analysis, it is speculated that a protein with high sequence identity to YjbI, a protein of unknown function in the genus Bacillus, has the activity. rice field. Therefore, E. coli transformed with an expression vector containing the gene sequence corresponding to yjbI from the TKS3 genome was cloned, and the protein was expressed, and its reducing activity against linoleic acid peroxide was confirmed (Table 2). FIG. 2 shows the nucleotide sequence of yjbI of TKS3 and the amino acid sequence encoded by the nucleotide sequence.

(4) Determination of K _m and V _max values in the reduction reaction of linoleic acid peroxide by TKS3
A Lineweaver-Burk plot was generated by measuring the activity in the presence of TMB and varying concentrations of peroxylinoleic acid (Fig. 3). These results indicated that the K _m and V _max values of the fatty acid peroxide reduction activity using TKS3 as the enzyme and linoleic acid peroxide as the substrate were 389 μM and 51 μmol/min/mg protein, respectively.

(5) Effect of pH on the reduction activity of linoleic acid peroxide by TKS3
TKS3 was used to evaluate the effect of pH on the reduction activity of linoleic acid peroxide (Fig. 4). We also evaluated the effect of using hydrogen peroxide as a substrate (Fig. 5a, b). These results indicated that the use of TKS3 or YjbI enables specific detection of peroxidized fatty acids at a pH of 8.7 or higher.

(6) Evaluation of heat resistance of linoleic acid peroxide reduction activity of TKS3
The resistance to heat of the peroxylinoleic acid reduction activity of TKS3 was evaluated (Fig. 6).

(7) Comparison of linoleic acid peroxide reduction activity of hemin and TKS3 The peroxidase-like activity of hemin is not inactivated by heat, but the linoleic acid reduction activity of TKS3 is inactivated by heating (96°C, 10 minutes). (Fig. 7a). In addition, the peroxidase-like activity of hemin maintained its hydrogen peroxide reducing activity under any pH condition, indicating that hydrogen peroxide and fatty acid peroxide could not be differentiated (Fig. 7b). Furthermore, even under the conditions of hydrogen peroxide treatment (2%, 10 minutes) at which the peroxidase-like activity of normal heme or heme protein is inactivated, the peroxylinoleic acid reduction activity of TKS3 is hardly affected. (Fig. 7c).

Unlike HRP, which is a plant-derived protein, cells containing YjbI or YjbI-encoding nucleic acids are expected to be prepared in large quantities at low cost because YjbI is a microorganism-derived protein. can be used as a substrate, and it is highly useful in that only fatty acid peroxides and lipid peroxides can be detected under basic conditions. In addition, cells containing YjbI or a nucleic acid encoding YjbI can induce a color reaction by catalyzing the oxidation of an oxidative chromogenic chromogen by hydroperoxide. Peroxidase (HRP) can be substituted. Furthermore, in a subject to which YjbI or a cell containing YjbI-encoding nucleic acid is administered, ingested, applied, etc., active oxygen involved in DNA damage can be reduced. In recent years, it has been reported that hydroxylated fatty acids obtained by reducing fatty acid peroxides, which are a type of hydroperoxide, have physiological effects such as anti-inflammatory effects and metabolic regulation. expected to be effective. Furthermore, when cells containing YjbI or YjbI-encoding nucleic acids are used as lignin-degrading agents, lipoxidase released from the cells by plant crushing oxidizes fatty acids to produce peroxidized fatty acids. It is convenient in that there is no need to add fatty acid peroxide to the

Claims (9)

A reagent for specific detection of lipid peroxide or fatty acid peroxide under pH 8.7 to 9.0 conditions, including any of the following (i) to (iii);
(i) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2;
(ii) A protein consisting of an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 2, and having an activity to catalyze the reduction of lipid peroxide or fatty acid peroxide and/or an oxidative coloring type a protein consisting of an amino acid sequence having activity to catalyze chromogen oxidation;
(iii) a cell containing a nucleic acid encoding (i) or (ii); 2. The reagent of claim 1, wherein the cell containing the nucleic acid encoding (i) is Bacillus licheniformis deposited under Accession No. NITE P-02670. A composition for specific reduction of lipid peroxide or fatty acid peroxide under pH 8.7 to 9.0 conditions, comprising any of the following (i) to (iii);
(i) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2;
(ii) A protein consisting of an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 2, and having an activity to catalyze the reduction of lipid peroxide or fatty acid peroxide and/or an oxidative coloring type a protein consisting of an amino acid sequence having activity to catalyze chromogen oxidation;
(iii) a cell containing a nucleic acid encoding (i) or (ii); 4. The composition of claim 3, wherein the cell containing the nucleic acid encoding (i) is Bacillus licheniformis deposited under Accession No. NITE P-02670. 5. The composition according to claim 3 or 4, wherein the composition is a pharmaceutical composition, a food composition, a feed composition or a cosmetic composition. A lipid peroxide- or fatty acid peroxide-specific color reaction inducer by an oxidative chromogenic chromogen under pH 8.7 to 9.0 conditions, including any of the following (i) to (iii);
(i) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2;
(ii) A protein consisting of an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 2, and having an activity to catalyze the reduction of lipid peroxide or fatty acid peroxide and/or an oxidative coloring type a protein consisting of an amino acid sequence having activity to catalyze chromogen oxidation;
(iii) a cell containing a nucleic acid encoding (i) or (ii); The inducer according to claim 5 or 6, wherein the cell containing the nucleic acid encoding (i) is Bacillus licheniformis deposited under Accession No. NITE P-02670. In the presence of any of the following (i) to (iii), in an aqueous medium of pH 8.7 to 9.0, a test sample and an oxidative color-forming chromogen are reacted, and the coloration of the reaction solution is detected. A method for specific detection of lipid peroxides or fatty acid peroxides in a test sample, comprising:
(i) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2;
(ii) A protein consisting of an amino acid sequence having 95% or more identity with the amino acid sequence represented by SEQ ID NO: 2, and having an activity to catalyze the reduction of lipid peroxide or fatty acid peroxide and/or an oxidative coloring type a protein consisting of an amino acid sequence having activity to catalyze chromogen oxidation;
(iii) a cell containing a nucleic acid encoding (i) or (ii); 9. The detection method according to claim 8, wherein the cell containing the nucleic acid encoding (i) is Bacillus licheniformis deposited under accession number NITE P-02670.

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