The present disclosure, pyridoxamine or producible recombinant microorganism salt thereof, and a pyridoxamine or a salt thereof using the recombinant microorganism.
BACKGROUND
[0002]
　Pyridoxamine and salts thereof Vitamin B 6 is a kind of, are known to have a saccharification reaction inhibitory effect. Pyridoxamine and salts thereof, for example, advanced glycation end products involved in various aging reaction; inhibiting accumulation in the body of the (Advanced Glycation End Products AGE). AGE is the generic term for substances produced by glycation of proteins, accumulation of AGE are diabetes, atherosclerosis, chronic renal failure, there is a worsening of the diseases such as Alzheimer's disease. Therefore, pyridoxamine and salts thereof are promising substances which are expected to allow the prevention and treatment of these diseases by preventing the accumulation of AGE.
[0003]
　Further, pyridoxamine and salts thereof are known to have activity as schizophrenia drugs, research towards its practical application have made various. Other, it has been advanced development of pyridoxamine and various health foods and cosmetics utilizing the physiological activities of the salts thereof.
　Pyridoxamine and salts thereof can be chemically synthesized. For example WO 2006/066806 discloses a method of chemically synthesizing the pyridoxamine dihydrochloride alanine and formic acid as the starting material. Furthermore, WO 2005/077902 discloses a method for chemically synthesizing pyridoxamine from pyridoxine.
[0004]
　On the other hand, it has also been studied to synthesize the pyridoxamine biologically. WO 2007/142222 discloses a method for obtaining a pyridoxamine from pyridoxal using a specific microorganism Achromobacter sp like. Furthermore, WO 2007/142222 by culturing the Acremonium Fushijioidesu in the presence pyridoxine, also discloses experiments a certain amount is converted into pyridoxal.
　JP-9-107985 discloses vitamin B belonging to the genus Rhizobium 6 a microorganism having an ability to produce, under aerobic conditions, cultured in medium, vitamins B produced 6 Vitamin B obtained from culture 6 production method of discloses.
[0005]
　Vitamin B 6 as the research on the synthesis, vitamin B at least one of activity by culturing organisms with enhanced than the parent organism of yaaD and yaaE of WO 2004/035010 Patent Bacillus subtilis (Bacillus subtilis) 6 a It discloses a method of manufacturing. .. Journal of Molecular Catalysis B: Enzymatic, 2010, vol 67, p 104-110 is pyridoxamine modified so as to enable use of L- glutamic acid by sequence alterations - discloses pyruvate aminotransferase (PPAT) . The E. coli expressing the PPAT with this modified sequence, by incubating pyridoxal presence of saturating amounts, it has been described that could produce pyridoxamine.
Summary of the Invention
Problems that the Invention is to Solve
[0006]
　However, not obtain a high reaction yield by pyridoxamine synthesis by chemical synthesis. For example, in the method described in WO 2006/066806, since the pyridoxamine dihydrochloride is synthesized through many chemical reactions, the reaction yield becomes low. The method described in WO 2005/077902 produces dimers and trimers of pyridoxamine, not much higher reaction yield.
　Meanwhile, in the method disclosed in WO 2007/142222 is a method using a microorganism of the particular species, the conversion efficiency from pyridoxal to pyridoxamine are large variations by microbial species, also intended much higher overall There was no. Further, in the culture experiments Acremonium Fushijioidesu in pyridoxine presence of described in WO 2007/142222, most of the product was pyridoxal instead of pyridoxamine. Further, in the description of JP-A-9-107985, JP-B vitamins resulting 6 is mostly a pyridoxol (pyridoxine), generation of pyridoxamine was only.
[0007]
　In WO 2004/035010, by culturing in a medium a microorganism obtained by highly expressed genes yaaD and yaaE, vitamin B 6 but have described that is obtained, the product pyridoxine, pyridoxal, pyridoxamine, a mixture of such pyridoxamine phosphate, it was not possible to obtain selectively the pyridoxamine.
　As described above, pyridoxamine or chemical synthesis (e.g., WO 2006/066806 and WO 2005/077902) a salt thereof, there is going through a number of reaction and purification steps, higher yield The resulting non. Although in the microorganisms are those having the pyridoxamine synthesizing ability (e.g. WO 2007/142222 Patent and Hei 9-107985 Patent Publication), takes time be newly discovered such a microorganism, also, vitamin B 6 not possible to selectively synthesize pyridoxamine or a salt thereof in the group, not even obtained high pyridoxamine production efficiency. Also, from the approach thus screening specific microbial species from microbial species that naturally occurring vitamin B 6 molecular biological information as to which enzymes or genes for highly producing a are important not be obtained.
[0008]
　Furthermore vitamin B 6 Although some examples using a recombinant microorganism prepared by using genetic recombination techniques with respect to the production of (WO 2004/035010 Patent and Journal of Molecular Catalysis B:. Enzymatic , 2010, vol 67, p. 104-110), vitamin B using a conventional medium during material production 6 or (WO 2004/035010 was obtained in non-selective), or a saturated amount of pyridoxal is an expensive raw material and pyridoxamine are obtained using in (Journal of Molecular Catalysis B:. . Enzymatic, 2010, vol 67, p 104-110), to produce better inexpensively and selectivity of pyridoxamine or a salt thereof has not been achieved .
[0009]
　In view of the above situation, the present disclosure, pyridoxamine or from pyridoxine or pyridoxine or a salt thereof with a recombinant microorganism, and a recombinant microorganism which can be inexpensively produced in pyridoxamine or high production efficiency salt thereof salt thereof to provide a method for inexpensively producing a salt thereof with high production efficiency.
Means for Solving the Problems
[0010]
　The present disclosure includes the following aspects.
<1>
　has the gene encoding the pyridoxamine synthase having pyridoxine enzyme activity oxidase synthesizing pyridoxamine from encoding gene and pyridoxal a
　respective genes encoding gene and the pyridoxamine synthase encoding said pyridoxine oxidase Although inherent in or bacterial cells a gene has been introduced from the outside of cells is a gene whose expression is enhanced, the recombinant microorganism.
<2>
　the pyridoxamine synthase, pyridoxamine - pyruvic transaminase, pyridoxamine - oxaloacetic transaminase, aspartate transaminase, or pyridoxamine phosphate transaminase, recombinant microorganisms described <1>.
<3>
　the pyridoxine oxidase is represented by enzyme number EC1.1.3.12, recombinant microorganism according to <1> or <2>.
<4>
　the gene encoding pyridoxine oxidase is derived from Microbacterium luteolum, recombinant microorganism according to any one of <1> to <3>.
<5>
　gene encoding the pyridoxine oxidase,
　or having the nucleotide sequence of (a) SEQ ID NO: 5,
　(B) a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 5, and pyridoxine or has a nucleotide sequence encoding a protein having a thin oxidase activity,
　(c) SEQ ID NO: protein or having a nucleotide sequence encoding, or having the amino acid sequence
　has (d) is an amino acid sequence having 80% or more sequence identity to the amino acid sequence of SEQ ID NO: 1, and having a pyridoxine oxidase activity having a nucleotide sequence encoding a protein, a
　recombinant microorganism according to any one of <1> to <4>.
<6>
　the pyridoxamine synthase partial amino acid sequence of the following (c), the partial amino acid sequence (d), the partial amino acid sequence (e), the partial amino acid sequence (f), the partial amino acid sequence (g) and the partial amino acid sequence ( wherein at least one of h), and having an enzyme activity to synthesize pyridoxamine from pyridoxal recombinant microorganism according to any one of <1> to <5>. (C)
X 8 X 9 X 10 X 11 X 12 X 13 (SEQ ID NO: 　　39) (X 8
Represents L, M, I or
　　V, X 9 represents H or
　　Q, X 10 is, G, represents C or
　　A, X 11 represents E or
　　D, X 12 is, P or represents
　　a, X 13 is V, I, represents L or
a) (d) X 14 X 15 TPSGTX 16 X 17 (SEQ ID NO: 　　40) (X 14 represents H or 　　S, X 15 is D or an 　　E, X 16 is, I, represents V or 　　L, X 17 represents N or T) (e) X

18 DX 19 VSX 20 X 21 (SEQ ID NO:
　　41) (X 18 is, V, represents I or
　　A, X 19 is, A, represents T or
　　S, X 20 represents S, A or
　　G, X 21 is F, W, or an
V) (f) X 22 X 23 X 24 KCX 25 GX 26 X 27 P (SEQ ID NO: 　　42) (X 22 represents G or 　　S, X 23 is, P, S or 　　a, X 24

Is N, G, S, represents A or
　　Q, X 25 represents L or
　　M, X 26 is A, S represents C or
　　G, X 27 is P, T, S or A expressed)
(g) X 28 X 29 X 30 X 31 SX 32 GX 33 X 34 (SEQ ID NO:
43) (X 28 represents G or
　　D, X 29 represents V or
　　I, X 30 is V represents T, a, S, M, I or
　　L, X 31 represents F, M, L, I or
　　V, X 32Represents S, G, A, T, I, L or
　　H, X 33 is R, represents M or
　　Q, X 34 represents G, R, A, D, H or
K) (h ) X 35 X 36 RX 37 X 38 HMGX 39 X 40 a (SEQ ID NO:
　　44) (X 35 represents L or
　　V, X 36 represents T, I, V or
　　L, X 37 is, I, represents V or
　　L, X 38 represents G or
　　S, X 39 is, P, represents a or
　　R, X 40 represents T, V or S)
<7>
　the pyridoxamine synthase represented by enzyme number EC2.6.1.30, recombinant microorganism according to any one of <1> to <6>.
<8>
　the gene encoding pyridoxamine synthase is derived from Mesorhizobium loti, recombinant microorganism according to any one of <1> to <7>.
<9>
　gene encoding the pyridoxamine synthase,
　(a) or having any of the nucleotide sequences of SEQ ID NO: 6 and SEQ ID NO: 25 to SEQ ID NO: 31,
　18 nt ~ in the nucleotide sequence of SEQ ID NO: 10 3 '18 nt ~ 3 in any of the nucleotide sequences of the region to end or SEQ ID NO: 32 to SEQ ID NO: 38' or with a region of up to end,
　(b) SEQ ID NO: 6 and SEQ ID NO: 25 to SEQ ID NO: DNA having a nucleotide sequence complementary to any of the nucleotide sequences of 31, or 18 th in the nucleotide sequence of SEQ ID NO: 10 nucleotides to the 3 'to the end region or SEQ ID NO: 32 of any of to SEQ ID NO: 38 a region up to 18 nt ~ 3 'terminus in the nucleotide sequence A DNA which hybridizes with the DNA under stringent conditions with the complement nucleotide sequence, and either has a nucleotide sequence encoding a protein with the enzymatic activity to synthesize pyridoxamine from pyridoxal,
　(c) SEQ ID NO: 2 and SEQ ID NO: 18 or has a nucleotide sequence encoding a protein having any of the amino acid sequence of the ~ SEQ ID NO: 24, or
　(D) is SEQ ID NO: 2 and SEQ ID NO: 18 to SEQ has an amino acid sequence having 80% or more sequence identity to at least one of the amino acid sequence of ID NO: 24, and the enzyme activity to synthesize pyridoxamine from pyridoxal having a nucleotide sequence encoding a protein, a having,
　recombinant microorganism according to any one of <1> to <8>.
<10>
　gene encoding the pyridoxamine synthase,
　(a) or having the nucleotide sequence of SEQ ID NO: 6,
　(b) DNA under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 6 in hybridized, and either has a nucleotide sequence encoding a protein with the enzymatic activity to synthesize pyridoxamine from pyridoxal,
　or has a nucleotide sequence encoding a protein having the amino acid sequence of (c) SEQ ID NO: 2, or
　(d ) SEQ ID NO: has an amino acid sequence having 80% or more sequence identity to the amino acid sequence, and having a nucleotide sequence encoding a protein, a having an enzyme activity to synthesize pyridoxamine from pyridoxal,
　<1> - according to any one of <9> The recombinant microorganism.
<11>
　the pyridoxamine synthase represented by enzyme number EC2.6.1.31 or EC2.6.1.1, recombinant microorganism according to any one of <1> to <5>.
<12>
　The pyridoxamine gene encoding synthase is derived from Escherichia coli, recombinant microorganism according to any one of <1> to <5> and <11>.
<13>
　gene encoding the pyridoxamine synthase,
　(a) or having the nucleotide sequence of SEQ ID NO: 8,
　(b) DNA under stringent conditions with a nucleotide sequence complementary to a nucleotide sequence of SEQ ID NO: 8 in hybridized, and either has a nucleotide sequence encoding a protein with the enzymatic activity to synthesize pyridoxamine from pyridoxal,
　or has a nucleotide sequence encoding a protein having the amino acid sequence of (c) SEQ ID NO: 4, or
　(d ) has an amino acid sequence having 80% or more sequence identity to the amino acid sequence of SEQ ID NO: 4, and having a nucleotide sequence encoding a protein, a having an enzyme activity to synthesize pyridoxamine from pyridoxal,
　<1> - <5> and <11> - <12> Of recombinant microorganism according to any one.
<14>
　further comprising a gene encoding the hydrogen peroxide degrading enzyme having the enzymatic activity of producing oxygen from hydrogen peroxide, recombinant microorganism according to any one of <1> to <13>.
<15>
　gene encoding the hydrogen peroxide degrading enzyme, but inherent in or bacterial cells a gene has been introduced from the outside of cells is a gene whose expression is enhanced, according to <14> pairs modified microorganisms.
<16>
　the hydrogen peroxide decomposing enzyme is expressed in enzyme number EC 1.11.1.6, recombinant microorganism according to <14> or <15>.
<17>
　gene encoding the hydrogen peroxide degrading enzyme,
　(a) or having the nucleotide sequence of SEQ ID NO: 7,
　a DNA stringent having a nucleotide sequence complementary to a nucleotide sequence of (b) SEQ ID NO: 7 hybridized under the conditions, and either has the enzymatic activity to produce oxygen from hydrogen peroxide,
　(c) or has a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO: 3, or
　(d) of SEQ ID NO: 3 has an amino acid sequence having 80% or more sequence identity to the amino acid sequence, and having a nucleotide sequence encoding a protein, a having the enzymatic activity of producing oxygen from hydrogen peroxide,
　<14> - <16> the recombinant microorganism according to any one of the.
<18>
　is a recombinant E. coli, the recombinant microorganism according to any one of <1> to <17>.
<19> <1>
　and treated to <18> culture or the recombinant microorganism or the culture of a recombinant microorganism or the recombinant microorganism according to any one of, a pyridoxine or a salt thereof contact is to produce a pyridoxamine or a salt thereof in the presence of oxygen, pyridoxamine or a salt thereof.
<20>
　Processed product of said recombinant microorganism or culture of the recombinant microorganism or the recombinant microorganism or the culture, including the pyridoxine oxidase and the pyridoxamine synthase method according to <19>.
<21>
　treatment of the recombinant microorganisms or culture of the recombinant microorganism or the recombinant microorganism or the culture, further comprising a hydrogen peroxide degrading enzyme production method according to <20>.
<22>
　treatment of the recombinant microorganism or the culture, heat treatment, cooling treatment, mechanical disruption of the cells, sonication, freeze-thaw treatment, drying treatment, the pressure or vacuum treatment, osmotic pressure treatment, cell autolysis, detergent treatment, an enzyme treatment, the cell separation process, treated by the process comprising one or more selected from the group consisting of purification treatment and extraction process, which of the <19> - <21> the method according to one or.
<23>
　include either or both of the following (A) and (B), The process according to any one of <19> ~
<22>: (A) the recombinant microorganism or the recombinant the solution containing the processed product of the culture or the recombinant microorganism or the culture of a microorganism, can be added separately to pyridoxine or continuously added or several times a salt thereof,
(B) the recombinant microorganism or said set in a solution containing the processed product of the culture or the recombinant microorganism or the culture of recombinant microorganisms, the pyridoxamine molar concentration of the amino acid that is consumed by the synthetase 1 times or more with respect to pyridoxine or molar concentration of the salt and It is controlled to be.
<24>
　The pyridoxamine acid consumed by synthesizing enzyme is L- alanine, D- alanine, L- glutamic acid or D- glutamate The method according to <23>.
The invention's effect
[0011]
　According to the present disclosure, pyridoxine or recombinant microorganism capable and inexpensively produced pyridoxamine or high production efficiency a salt thereof from a salt, and high production efficiency pyridoxine or pyridoxamine, or a salt thereof from its salt using recombinant microorganisms how to low cost production in it is provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012]
[1] pyridoxine hydrochloride concentration in the test 7 shows the measurement results of pyridoxal hydrochloride salt concentration and pyridoxamine dihydrochloride salt concentration.
[Figure 2] pyridoxine hydrochloride concentration in the test 8 shows the results of measurement of pyridoxal hydrochloride salt concentration and pyridoxamine dihydrochloride salt concentration.
[Figure 3] pyridoxine hydrochloride concentration in the test 9 shows the results of measurement of pyridoxal hydrochloride salt concentration and pyridoxamine dihydrochloride salt concentration.
Shows the alignment of sequences [Figure 4-1] SEQ ID NO: 2 and SEQ ID NO: 18 to SEQ ID NO: 24.
Shows the alignment of sequences [Figure 4-2] SEQ ID NO: 2 and SEQ ID NO: 18 to SEQ ID NO: 24.
DESCRIPTION OF THE INVENTION
[0013]
　The present disclosure, the gene and pyridoxal encoding pyridoxine oxidase has a gene encoding the pyridoxamine synthase having an enzyme activity to synthesize pyridoxamine,
　each of the genes encoding the gene and the pyridoxamine synthase encoding said pyridoxine oxidase There is inherent in or bacterial cells a gene has been introduced from the outside of cells is a gene whose expression is enhanced, a recombinant microorganism (hereinafter, referred to recombinant microorganism according to the present disclosure) provide. Note that the introduction in this disclosure, refers to the introduction so as to be expressed in bacterial cells.
[0014]
　Previously, by biological methods by a chemical method, a method of and inexpensively produce pyridoxine or pyridoxamine, or a salt thereof from a salt thereof with high productivity has not been known. The structure of pyridoxamine are shown below.
[0015]
[Formula 1]

[0016]
　However, surprisingly, by using a treated product of the culture or the recombinant microorganism or the culture of a recombinant microorganism or the recombinant microorganism having the above configuration, pyridoxamine or a pyridoxine or a salt thereof the ability to inexpensively produced with high production efficiency of the salt present inventor has found. The reason is not necessarily clear, the two types of enzymes, respectively, by inherent or cells are introduced from outside of cells but its expression is enhanced, from the introduction or enhanced gene it is possible to express a high expression level of the enzyme, the amount of pyridoxine or raw materials which are by-products such as and consumption produced in equilibrium and the process of the reaction in the process of generating the pyridoxamine, or a salt thereof from a salt thereof, the the two cooperative action of enzymes, it is presumed that it is a to become advantageous to the generation of pyridoxine or pyridoxamine, or a salt thereof from a salt thereof.
　Furthermore, when a processed product of the culture or the recombinant microorganism or the culture of a recombinant microorganism or the recombinant microorganism according to the present disclosure is presumed to be temporarily generated pyridoxal as an intermediate product that. However, pyridoxal is highly reactive to an aldehyde, react spontaneously with the amino group donor alanine, etc. even in the absence of a catalyst such as an enzyme in the pH conditions near Accordingly neutral pyridoxamine and secondary to produce a product. Thus, in the spontaneous reaction of pyridoxal-products are produced, because not high selectivity of pyridoxamine generation, production efficiency of pyridoxamine is considered not high. In contrast, in the case of using the treated product of the culture or the recombinant microorganism or the culture of a recombinant microorganism or the recombinant microorganism according to the present disclosure, pyridoxal intermediates promptly by pyridoxamine synthase is converted to pyridoxamine, accumulation of pyridoxal is suppressed, the production of by-products is suppressed. As a result, the high pyridoxamine production efficiency can be achieved.
[0017]
　Such above-described two kinds of cooperative action of the enzyme, which has not been found so far, by using a pyridoxine or a salt thereof by which performs each time a reaction of the multi-step as chemical synthesis methods without the need, it has become possible to produce the pyridoxamine, or a salt thereof with high production efficiency. Moreover, the use of the recombinant microorganism according to the present disclosure, vitamin B 6 can be avoided by-produced in large quantities of other substances contained in the group. Also, pyridoxine is industrially cheaply available starting materials as compared to pyridoxal, the ability pyridoxine starting material, it becomes possible to inexpensively manufacture the pyridoxamine, or a salt thereof.
[0018]
　
　pyridoxine oxidase, an enzyme which is also known as pyridoxine 4-oxidase. Pyridoxine oxidase may be an enzyme represented by enzyme number EC1.1.3.12. Pyridoxine oxidase, by oxidizing pyridoxine with oxygen, an enzyme having an enzyme activity that catalyzes the conversion into pyridoxal. Incidentally, pyridoxal and pyridoxine but may also be present as a salt by the environment around, for simplicity of notation, references in this disclosure to salt for a description of the activity of the enzyme is denoted omitted. Pyridoxine oxidase, upon oxidation of pyridoxine it is to consume oxygen to produce hydrogen peroxide. For the generation of harmful hydrogen peroxide in organisms, high yields also be used to produce another substance pyridoxine oxidase from pyridoxine has been considered to not be achieved. However, the use of the recombinant microorganism according to the present disclosure, the effect of the unexpected that pyridoxine or a salt thereof is produced in pyridoxamine or a salt thereof as the raw material can be achieved with high production efficiency is obtained.
[0019]
　Pyridoxine oxidase of the EC1.1.3.12, for example Enterobacter cloacae (Enterobacter cloacae), Mezorizobiumu Roti (Mesorhizobium loti), micro Mycobacterium Ruteoramu (Microbacterium luteolum), tailed Black Mycobacterium anthropi (Ochrobactrum anthropi), Psudomonas (Pseudomonas ) sp. It may be pyridoxine oxidase such as from MA-1. Incidentally, pyridoxine oxidase-mycobacterial Ruteoramu has the amino acid sequence of SEQ ID NO: 1.
[0020]
　
　The pyridoxamine synthase used in the present disclosure, refers to any enzyme having an enzyme activity to synthesize pyridoxamine from pyridoxal. Incidentally, pyridoxal and pyridoxamine are may also be present as a salt by the environment around, for simplicity of notation, references in this disclosure to salt for a description of the activity of the enzyme is denoted omitted. Examples of pyridoxamine synthase, for example, pyridoxamine represented by enzyme number EC2.6.1.30 - pyruvic transaminase, pyridoxamine represented by EC2.6.1.31 - oxaloacetic transaminase, EC2.6. aspartate transaminase represented by 1.1, and pyridoxamine phosphate transaminase and the like represented by EC2.6.1.54.
　Note aspartate transaminase represented by EC2.6.1.1 is a holoenzyme that the pyridoxal phosphate as a coenzyme, to transfer the amino group of the aspartic acid to 2-oxoglutarate, generate glutamate and oxaloacetate It has the enzymatic activity to be. In the aspartate transaminase apoenzyme which is not bound to pyridoxal phosphate state, an amino group of glutamic acid or aspartic acid can be synthesized metastasized pyridoxamine to pyridoxal known (JOURNAL OF BIOLOGICAL CHEMISTRY, 1962 January month, Vol.237, No.1, p.127-132). That apoenzyme of aspartate transaminase represented by EC2.6.1.1 is pyridoxamine of EC2.6.1.31 - a oxaloacetic transaminase. Therefore, aspartate transaminase, when pyridoxine or amount Searle phosphoric acid is not present is small coenzymes are present as apoenzyme synthesize pyridoxamine.
　Pyridoxamine synthase in the synthesis of pyridoxal or pyridoxamine, or a salt thereof from a salt thereof, oxidizing the amino group moiety of a particular amino acid (= O), to produce a pyridoxamine or a salt thereof by transferring the amino group having the enzymatic activity. For example, pyridoxamine - pyruvic transaminase are available either in L- alanine and D- alanine, pyridoxamine - oxaloacetic aspartate transaminase states acid transaminase and apoenzyme D- aspartate, L- aspartic acid, D- glutamic acid, and, any of L- glutamic acid are available, pyridoxamine phosphate transaminase is available D- glutamic acid.
[0021]
　The pyridoxamine - pyruvate transaminase, e.g., Proteobacteria49, Actinobacteria gate may be one derived from a microorganism belonging to the spirochete Gate or Firmicutes. Pyridoxamine - pyruvate transaminase, e.g., Mezorizobiumu Roti (Mesorhizobium loti), tailed Black Mycobacterium anthropi (Ochrobactrum anthropi) and Pseudomonas (Pseudomonas) genus (e.g., Pseudomonas sp.MA-1) pyridoxamine such as from - pyruvate transaminase it may be. Incidentally, for example, pyridoxamine of Mesorhizobium loti - pyruvic transaminase has an amino acid sequence of SEQ ID NO: 2.
[0022]
　Pyridoxamine - pyruvate transaminase, other, for example, Mesorhizobium sp. YR577 (Mesorhizobium sp YR577.) From to pyridoxamine - pyruvate transaminase (SEQ ID NO: 19 - (having the amino acid sequence of SEQ ID NO: 18) pyruvic transaminase, pseudoephedrine amino Arthrobacter Sari sila butoxy Dance (Pseudaminobacter salicylatoxidans) from To pyridoxamine having the amino acid sequence), derived from Bauldia litoralis pyridoxamine - having the amino acid sequence of pyruvate transaminase (SEQ ID NO: 20), pyridoxamine from Skermanella stibiiresistens - having the amino acid sequence of pyruvate transaminase (SEQ ID NO: 21), Rhizobium sp . AC44 / 96 (. Rhizobium sp AC44 / 96) from To pyridoxamine - (having the amino acid sequence of SEQ ID NO: 22) pyruvic transaminase, pyridoxamine from Erwinia Toretana (Erwinia Toletana) - amino pyruvate transaminase (SEQ ID NO: 23 it may be a pyruvate transaminase (having the amino acid sequence of SEQ ID NO: 24) - having the sequence), pyridoxamine derived from Herbiconiux ginsengi.
[0023]
　The pyridoxamine - oxaloacetic transaminase, for example, E. coli (Escherichia coli), Rabbit (Oryctolagus cuniculus) and brown rat (Rattus norvegicus) pyridoxamine from such - may be oxaloacetic transaminase. Incidentally, for example, pyridoxamine of Escherichia coli - oxaloacetic transaminase has an amino acid sequence of SEQ ID NO: 4. The aspartate transaminase, for example, E. coli (Escherichia coli), or may be aspartate transaminase such as from Trichoderma viride (Trichoderma viride). Further, the pyridoxamine phosphate transaminase, e.g. Clostridium butyricum (Clostridium butyricum) may be a pyridoxamine phosphate transaminase derived.
[0024]
　
　recombinant microorganism according to the present disclosure may further comprise a gene encoding the hydrogen peroxide degrading enzyme having an enzymatic activity for decomposing hydrogen peroxide. The hydrogen peroxide decomposition enzyme used in the present disclosure, it refers to any enzyme with hydrogen peroxide decomposing enzyme activity that generated when the pyridoxine oxidase oxidizes pyridoxine. By further comprising a gene encoding the hydrogen peroxide degrading enzyme, organisms and more possible to reduce the accumulation of harmful hydrogen peroxide to enzymatic activity, further improvement of production efficiency of pyridoxamine, or a salt thereof, reaction can continue it is advantageous in terms of extension of time. Further, the hydrogen peroxide degrading enzyme may have an enzymatic activity for reproducing oxygen. The presence of the enzyme activity to play oxygen, especially in the case of performing the production of pyridoxamine, or a salt thereof in a low-oxygen environment, it is possible to increase the concentration of oxygen in the reaction system, further improvement of production efficiency of pyridoxamine, or a salt thereof it is in terms of advantageous.
　Examples of such hydrogen peroxide-degrading enzymes, enzyme number (EC) 1.11.1.1,1.11.1.2,1.11.1.3,1.11.1.5, 1.11.1.6,1.11.1.7,1.11.1.8,1.11.1.9,1.11.1.10,1.11.1.11,1. 11.1.13,1.11.1.14,1.11.1.16,1.11.1.17,1.11.1.18,1.11.1.19,1.11. enzymes expressed are exemplified by 1.21 or 1.11.1.23. Catalase peroxidase represented by catalase and EC1.11.1.21 represented by enzyme number EC1.11.1.6 Among these are preferable from the viewpoint of oxygen regenerative capacity, an enzyme number EC1.11.1.6 more preferably catalase represented.
[0025]
　Wherein the hydrogen peroxide degrading enzymes, for example Listeria Zerigeri (Listeria seeligeri), E. (Escherichia coli), it may be a catalase such as from Saccharomyces cerevisiae (Saccharomyces cerevisiae). Alternatively, the hydrogen peroxide decomposition enzyme may be catalase peroxidase derived in, for example, E. coli (Escherichia coli). Incidentally, for example, catalase Listeria seeligeri has the amino acid sequence of SEQ ID NO: 3.
[0026]
　The pyridoxine oxidase, pyridoxamine synthase, and hydrogen peroxide decomposition enzyme are both known amino acid sequence having the enzymatic activity (e.g., an amino acid sequence encoded by a gene naturally present in the organism, for example the above-exemplified a protein having remained unaltered amino acid sequence) that is encoded by a gene having the natural microorganisms, such as microorganisms may not lose their enzymatic activity for such amino acid sequence (above enzyme activity) range may be a protein having an amino acid sequence plus the sequence modifications. Such modifications, insertion of an amino acid residue, deletion, and addition of additional amino acid residues to substitutions and amino acid sequences N-terminus or C-terminus or both. Insertion of amino acid residues, if there are one or more of the deletions and substitutions, insertions, each of deletions and substitutions, if present, for example, 1 to 30 amino acid residues or 1-20 amino acid residues, or 1-10 amino acid residues, or may be 1-5 amino acid residues, insertion of amino acid residues, deletion and the total number of substituents is, for example 1-50 amino acid residues, or 1-30 amino acid residues, or 1-10 amino acid residues or may be a 1-5 amino acid residues. As the number of amino acid residues added to the ends, first end per example 1-50 amino acid residues, if present, or 1-30 amino acid residues, or 1 to 10 amino acid residues, or 1-5, it may be an amino acid residue. Amino acid residues such additional, may form a signal sequence for secretion or the like into the extracellular. Examples of the signal sequence, and the like OmpA signal sequence of E. coli.
[0027]
　Alternatively, each enzyme, known amino acid sequence having the enzymatic activity (e.g., an amino acid sequence encoded by a gene naturally present in the organism) known amino acid sequence having protein or the enzymatic activity having itself (e.g. has an amino acid sequence having amino acid sequence encoded by a naturally occurring gene) 80% or more with respect to, or more than 85%, or 90%, or 95% sequence identity to an organism, the desired enzyme activity (described above enzyme activity) may be a protein having. Here, sequence identity can be assessed in default parameters using e.g. BLAST (registered trademark, National Library of Medicine) program.
[0028]
　For example, pyridoxine oxidase, for example, may be a protein having the amino acid sequence of SEQ ID NO: 1, substitution of amino acid residues to the amino acid sequence of SEQ ID NO: 1, or N-terminal deletion, insertion and amino acid sequence C-terminal or may be a protein having the amino acid sequence of performing one or more of the addition of additional amino acid residues to both. Amino acid residue substitutions, deletions, for example of the order of addition of additional amino acid residues to the N-terminus or C-terminus or both insertion and amino acid sequences are as described above.
　Alternatively, pyridoxine oxidase, SEQ ID NO: 1 amino acid sequence with a protein or to the amino acid sequence of SEQ ID NO: 1 for example, 80% or more, or 85% or more, or 90%, or 95% or more sequence identity it may be a protein having the amino acid sequence.
　As described above, when using a protein having an amino acid sequence similar to the amino acid sequence of SEQ ID NO: 1, the protein should have activity as pyridoxine oxidase. Pyridoxine oxidase activity, for example, an aqueous solution to the test object of proteins pyridoxine as substrate was added in the presence of oxygen, or the generated pyridoxal quantified by high performance liquid chromatography, or generated as pyridoxal and trishydroxymethylaminomethane in such absorbance measurements at 415nm to form a Schiff base and the like can be measured by quantifying the Schiff base with the amines.
[0029]
　Alternatively, pyridoxamine synthase any amino acid sequence of the protein or SEQ ID NO: 2 having the amino acid sequence, the amino acid sequence of SEQ ID NO: 18, the amino acid sequence of SEQ ID NO: 19 of SEQ ID NO: 2 and SEQ ID NO: 18 to SEQ ID NO: 24, amino acid sequence of SEQ ID NO: 20, SEQ amino acid sequence of ID NO: 21, the amino acid sequence of SEQ ID NO: 22, the amino acid sequence of SEQ ID NO: 23, and SEQ ID NO: 24 amino acid sequence at least one for example 80% or more of the, or 85 % or more, or 90% or more, or may be a protein having an amino acid sequence having 95% or more sequence identity. The protein should also have activity as pyridoxamine synthase. In this case, the enzyme activity to synthesize pyridoxamine from pyridoxal, for example, by pyridoxal and L- alanine aqueous tested proteins, including as a substrate was added, the amount of generated pyridoxamine quantified by high performance liquid chromatography It can be measured.
[0030]
　Alternatively, pyridoxamine synthase partial amino acid sequence of the following (c), the partial amino acid sequence (d), the partial amino acid sequence (e), the partial amino acid sequence (f), the partial amino acid sequence (g) and the partial amino acid sequence (h) at least one comprises, and may be a pyridoxamine synthase having an enzyme activity to synthesize pyridoxamine from pyridoxal among. In this case, the enzyme activity to synthesize pyridoxamine from pyridoxal, for example, by pyridoxal and L- alanine aqueous tested proteins, including as a substrate was added, the amount of generated pyridoxamine quantified by high performance liquid chromatography It can be measured. (C)
X 8 X 9 X 10 X 11 X 12 X 13 (SEQ ID NO: 　　39) (X 8 represents L, M, I or 　　V, X 9 represents H or 　　Q, X 10 is, G It represents C or 　　a, X 11 represents E or 　　D, X 12

Represents P or
　　A, X 13 is V, I, represents L or
A) (d) X 14 X 15 TPSGTX 16 X 17 (SEQ ID NO: 　　40) (X 14 represents H or 　　S, X 15 represents a D or 　　E, X 16 is, I, represents V or 　　L, X 17 represents N or T) (e) X 18 DX 19 VSX 20 X 21 (SEQ ID NO: 　　41) (X 18 is, V, represents I or 　　a, X 19

Is, A, represents T or
　　S, X 20 is, S, represents A or
　　G, X 21 is, F, W, or an
V) (f) X 22 X 23 X 24 KCX 25 GX 26 X 27 P (SEQ ID NO: 　　42) (X 22 represents G or 　　S, X 23 represents P, S or 　　a, X 24 represents N, G, S, a or 　　Q, X 25 is L or an 　　M, X 26 represents a, S, C or 　　G, X 27 represents P, T, S or a) (g) X

28 X 29 X 30 X 31 SX 32 GX 33 X 34 (SEQ ID NO:
43) (X 28 represents G or
　　D, X 29 represents V or
　　I, X 30 is V, T, A, S , M, represents I or
　　L, X 31 represents F, M, L, I or
　　V, X 32 represents S, G, a, T, I, L or
　　H, X 33 is R represents M or
　　Q, X 34 represents G, R, a, D, H or
K) (h) X 35 X 36 RX 37 X 38 HMGX 39 X 40 A (SEQ ID NO:
　　44) (X 35 represents L or
　　V, X 36 represents T, I, V or
　　L, X 37 represents I, V or L,
　　X 38 represents G or
　　S, X 39 is, P, represents a or
　　R, X 40 represents T, V or S)
[0031]
　Figure 4-1 and Figure 4-2 shows the alignment of SEQ ID NO: 2 and SEQ ID NO: 18 to SEQ ID NO: 24. In Figure 4-1 and Figure 4-2, pyridoxamine of MlPPAT is Mesorhizobium loti - pyruvate transaminase, MsPPAT the Mesorhizobium sp. Pyruvate transaminase, PsPPAT is pyridoxamine of Pseudaminobacter salicylatoxidans - - pyridoxamine of YR577 pyruvate transaminase, BlPPAT is pyridoxamine of Bauldia litoralis - pyruvate transaminase, SsPPAT is pyridoxamine of Skermanella stibiiresistens - pyruvate transaminase, RsPPAT is Rhizobium sp. Pyruvate transaminase, EtPPAT the pyridoxamine of Erwinia toletana - - AC44 / 96 pyridoxamine in pyruvate transaminase, HgPPAT the pyridoxamine of Herbiconiux ginsengi - represents a pyruvate transaminase. Partial amino acid sequence (c) is, pyridoxamine of Mesorhizobium loti - corresponds to amino acid residues corresponding on alignment to the N-terminus to the 65th ~ 70th amino acid residue of pyruvic transaminase, partial amino acid sequence (d) are, Mesorhizobium loti of pyridoxamine - corresponds to amino acid residues corresponding to the alignment to 144 th to 152 th amino acid residue from the N-terminus of pyruvic transaminase, partial amino acid sequence (e) is pyridoxamine of Mesorhizobium loti - pyruvate transaminase N corresponds to amino acid residues corresponding on alignment from end to 170 th to 176 th amino acid residue, partial amino acid sequence (f) is the Mesorhizobium loti Pyridoxamine - 19 from the N-terminus of pyruvate transaminase Corresponds to the corresponding amino acid residues on alignments to the fourth to 203 th amino acid residue, partial amino acid sequence (g) is pyridoxamine of Mesorhizobium loti - 329 th from N-terminal of pyruvic transaminase to 337 th amino acid residue the equivalent to the corresponding amino acid residue on the alignments, partial amino acid sequence (h) is the Mesorhizobium loti pyridoxamine - from the N-terminus of pyruvate transaminase in 343 th to 353 th amino acid residue with an amino acid residue corresponding on alignment Equivalent to. Partial amino acid sequence (c) is preferably present in the 55 th to 80 th in the region of amino acid residues from the N-terminus of the protein is present in the region of 56 th to 75 th amino acid residue from the N-terminal it is more preferable. Partial amino acid sequence (d) is preferably present in the region of 134 th to 162 th amino acid residue from the N-terminus of the protein is present in the region of 139 th to 157 th amino acid residue from the N-terminal it is more preferable. Partial amino acid sequence (e) is preferably present in the 160th ~ region of 186 amino acid residue from the N-terminus of the protein is present in the region of 165 th to 181 th amino acid residue from the N-terminal it is more preferable. Partial amino acid sequence (f) is preferably present in the region of 184 th to 213 th amino acid residue from the N-terminus of the protein is present in the region of 189 th to 208 th amino acid residue from the N-terminal it is more preferable. Partial amino acid sequence (g) is preferably present in the region of 319 th to 347 th amino acid residue from the N-terminus of the protein is present in the region of 324 th to 342 th amino acid residue from the N-terminal it is more preferable. Partial amino acid sequence (h) is preferably present in the region of 333 th to 363 th amino acid residue from the N-terminus of the protein is present in the region of 338 th to 358 th amino acid residue from the N-terminal it is more preferable. A sequence between the present disclosure
[0032]
　In this disclosure, the term "corresponding amino acid residues in the X amino acid residues from the N-terminus of the enzyme A" in the amino acid sequence of the enzyme B, and the amino acid sequence of the enzyme A were aligned to the amino acid sequence of the enzyme B Occasionally, it refers to the amino acid residues on the amino acid sequence of the enzyme B corresponding the N-terminal amino acid sequence of the enzyme a and X amino acid residue.
[0033]
　As can be seen from Figure 4-1 and Figure 4-2, the partial amino acid sequence (c), the partial amino acid sequence (d), the partial amino acid sequence (e), the partial amino acid sequence (f), the partial amino acid sequence (g) and partial amino acid sequence (h), the group of pyridoxamine - a highly conserved region between the pyruvate transaminase. Therefore, pyridoxamine contains at least one of the partial amino acid sequence (c) ~ partial amino acid sequence (h) - Variation of pyruvate transaminase probability that functions as pyridoxamine synthase in the present disclosure is considered to be high. Further, pyridoxamine of Mesorhizobium loti - amino acid residue corresponding on alignment from the N-terminus to 197 lysine residues pyruvate transaminase is important for binding to pyridoxal, on alignments 68th glutamic acid residue from the N-terminus corresponding amino acid residues are important for catalytic activity, the amino acid residue corresponding on the alignment of amino acid residues and N-terminal corresponding on alignment to 171 th aspartic acid residue from the N-terminus to 146th threonine residue to assist the binding of pyridoxal, amino acid residue corresponding on alignment from the corresponding amino acid residues and N-terminus on alignments 336 arginine residue from the N-terminus to the 345th arginine residues are important to the amino acid recognition and considered Gills are and (Journal of Biological Chemistry, 2008, vol. 283, No. 2 pp1120-1127), on the function of pyridoxine synthase is an important residue. If it is these residues stored, probability that functions as pyridoxine synthase in the present disclosure is considered to be high. However, pyridoxamine of Mesorhizobium loti - amino acid residue corresponding on alignment from the N-terminus of pyruvate transaminase 68th glutamic acid residue may be aspartic acid other than glutamic acid. Further, pyridoxamine of Mesorhizobium loti - amino acid residue corresponding on alignment from the N-terminus of pyruvate transaminase 336 arginine residues may be methionine or glutamine in addition to arginine.
[0034]
　Mesorhizobium loti of pyridoxamine - from the N-terminus of pyruvate transaminase 68th glutamic acid residue as a separate representation of the region containing the corresponding amino acid residues on alignments include the following partial amino acid sequence (c-1) it is. Pyridoxamine synthase may contain the partial amino acid sequence (c-1) in place of the partial amino acid sequence (c). (C-1)
X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19(SEQ ID NO:
　　45) X 1 is represents V, L, I or
　　M, X 2 is I, represents L or
　　V, X 3 represents L, M, I or
　　V, X 4 is H or represents
　　Q, X 5 is, G, represents C or
　　a, X 6 represents E or
　　D, X 7 represents P or
　　a, X 8 represents V, I, a or L ,
　　X 9 represents L, M, P or
　　V, X 10 represents G or
　　a, X 11 represents L or
　　I, X 12 represents E or Q,
　　X 13 represents A or
　　G, X 14 represents A or
　　V, X 15 represents A or
　　L, X 16 represents A, L, H or
　　Y, X 17 is, S, It represents G or
　　a, X 18 represents L, F, V or
　　a, X 19 represents I, F, V or L.
　Partial amino acid sequence (c-1) is pyridoxamine of Mesorhizobium loti - corresponding to amino acid residues corresponding on alignment from the N-terminus of pyruvate transaminase in 63 th to 81 th amino acid residue. Partial amino acid sequence (c-1) is preferably present in the region of 53 th to 91 th amino acid residue from the N-terminus of the protein, in the region of 58-th to 86 th amino acid residue from the N-terminal it is more preferably present.
[0035]
　Mesorhizobium loti of pyridoxamine - from the N-terminus of pyruvate transaminase 146th threonine residue as another representation of the region containing the corresponding amino acid residues on alignments include the following partial amino acid sequence (d-1). Pyridoxamine synthase may contain the partial amino acid sequence (d-1) in place of the partial amino acid sequence (d). (D-1)
X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 TPSGTX 9 X 10 X 11 X 12 X　13 X 14 X 15 X 16 (SEQ ID NO: 46) X 1 is, V, I, It represents an L or M,

X 2 represents V or
I, X 3 is S, A, V, represents C or
F, X 4 is V, I, A represents L or
　　T, X 5 is a C or V
　　represents, X 6 is H, represents N or
　　a, X 7 represents H or
　　S, X 8 represents D or
　　E, X 9 represents I, V or
　　L, X 10 is It represents N or
　　T, X 11 represents P or
　　D, X 12 represents I, V, L or
　　a, X 13 is, D, N, E, a , G, Q, V, R or It represents a
　　P, X 14 represents A, E, and Q or
　　D, X 15 represents I or
　　L, X 16 represents G or A.
　Partial amino acid sequence (d-1), pyridoxamine of Mesorhizobium loti - corresponding to amino acid residues corresponding on alignment from the N-terminus of pyruvate transaminase in 138 th to 158 th amino acid residue. Partial amino acid sequence (d-1) is preferably present in the region of 128 th to 168 th amino acid residue from the N-terminus of the protein, in the region of 133 th to 163 th amino acid residue from the N-terminal it is more preferably present.
[0036]
　Mesorhizobium loti of pyridoxamine - from the N-terminus of pyruvate transaminase in 171 th aspartic acid residue as a separate representation of the region containing the corresponding amino acid residues on alignments include the following partial amino acid sequence (e-1). Pyridoxamine synthase may contain the partial amino acid sequence (e-1) in place of the partial amino acid sequence (e). (E-1)
X 1 X 2 X 3 X 4 X 5 X 6 DX 7 VSX 8 X 9 X 10 X 11 X 12 (SEQ ID NO: 　　47) X 1 is, G, D, or 　　A X 2 is, A, G represents K, T, Q, R or 　　E, X 3 represents Y, N, L or F,

　　X 4 is, L, F, represent M or
　　V, X 5 is, I, represents L or
　　Y, X 6 is, V, represents A or
　　I, X 7 is, A, represents S or T,
　　X 8 is, S, represents a or
　　G, X 9 is, F, represents W or
　　V, X 10 represents G, a or
　　L, X 11 represents G or
　　S, X 12 is M, representing a V or L.
　Partial amino acid sequence (e-1) is, pyridoxamine of Mesorhizobium loti - corresponding to amino acid residues corresponding on alignment from the N-terminus of pyruvate transaminase 165 th to 179 th amino acid residue. Partial amino acid sequence (e-1) is preferably present in the region of amino acid residues from the N-terminus of the 155-th to 189-th protein, in the region of 160 th to 184 th amino acid residue from the N-terminal it is more preferably present.
[0037]
　Mesorhizobium loti of pyridoxamine - from the N-terminus of pyruvate transaminase 197th lysine residue as another representation of the region containing the corresponding amino acid residues on alignments include the following partial amino acid sequence (f-1) it is. Pyridoxamine synthase may include a partial amino acid sequence (f-1) in place of the partial amino acid sequence (f). (F-1)
X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 KCX 10 GX 11 X 12 PX 13 X 14 X 15 X 16 X 17 X 18 X 19S (SEQ ID NO:
48) X 1 represents A, S, V or
I, X 2 is, D, represents G or
A, X 3 represents I, L, F, V or
　　M, X 4 represents Y, F, L or
　　C, X 5 represents V or
　　I, X 6 represents T or
　　a, X 7 represents G or
　　S, X 8 is, P, S or represents
　　a, X 9 represents N, G, S, and Q or
　　a, X 10 represents L or
　　M, X 11 represents a, S, C or
　　G, X 12 is, P, T, S or a,
　　X 13 is, G, represents A or
　　S, X 14 represents L or
　　V, X 15 is, T, S or
　　A, X 16 represents M, I, L, V or F,
　　X 17 represents M, L, V, a or
　　I, X 18 represents G, a, H or
　　S, X 19 represents V, I or a.
　Partial amino acid sequence (f-1) is, pyridoxamine of Mesorhizobium loti - corresponding to amino acid residues corresponding on alignment from the N-terminus of pyruvate transaminase in 188 th to 211 th amino acid residue. Partial amino acid sequence (f-1) is preferably present in the region of 178 th to 221 th amino acid residue from the N-terminus of the protein, in the region of 183 th to 216 th amino acid residue from the N-terminal it is more preferably present.
[0038]
　Mesorhizobium loti of pyridoxamine - from the N-terminus of pyruvate transaminase 336 arginine residue as another representation of the region containing the corresponding amino acid residues on alignments include the following partial amino acid sequence (g-1) is. Pyridoxamine synthase may contain the partial amino acid sequence (g-1) in place of the partial amino acid sequence (g). (G-1)
X 1 X 2 X 3 X 4 X 5 SX 6 GX 7 X 8 (SEQ ID NO: 　　49) X 1 represents Y, F, H or 　　S, X 2 represents G or D, 　　X 3 represents V or 　　I, X 4 represents V, T, a, S, M, I or 　　L, X 5 represents F, M, L, I or V,

　　X 6 represents S, G, A, T, I, L or
　　H, X 7 is R, represents M or
　　Q, X 8 represents G, R, A, D, H or K.
　Partial amino acid sequence (g-1) is, pyridoxamine of Mesorhizobium loti - corresponding to amino acid residues corresponding on alignment from the N-terminus of pyruvate transaminase in 328 th to 337 th amino acid residue. Partial amino acid sequence (g-1) is preferably present in the region of 318 th to 347 th amino acid residue from the N-terminus of the protein, in the region of 323 th to 342 th amino acid residue from the N-terminal it is more preferably present.
[0039]
　Mesorhizobium loti of pyridoxamine - from the N-terminus of pyruvate transaminase 345th arginine residue as another representation of the region containing the corresponding amino acid residues on alignments include the following partial amino acid sequence (h-1) it is. Pyridoxamine synthase may contain the partial amino acid sequence (h-1) instead of the partial amino acid sequence (h). (H-1)
X 1 X 2 X 3 X 4 X 5 RX 6 X 7 HMGX 8 X 9 AX 10 X 11 (SEQ ID NO: 50) X 1 is, L, Q, K, A , F, Y or W represents, X 2 represents G, N, H or D, X 3 is, K, represents the R or 　　N, X 4

Represents L or
　　V, X 5 represents T, I, V or
　　L, X 6 is, I, represents V or
　　L, X 7 represents G or
　　S, X 8 is, P, represents a or
　　R, X 9 is, T, represents the V or
　　S, X 10 represents Q, R, E, K, H, Y or
　　G, X 11 represents P or G.
　Partial amino acid sequence (h-1) is, pyridoxamine of Mesorhizobium loti - corresponding to amino acid residues corresponding on alignment from the N-terminus of pyruvate transaminase 340 th to 355 th amino acid residue. Partial amino acid sequence (h-1) is preferably present in the region of amino acid residues from the N-terminal 330-th to 365 th of the protein, the 335 th to the region of 360 amino acid residue from the N-terminal it is more preferably present.
[0040]
　Further, pyridoxamine synthase, for example, may be a protein having the amino acid sequence of SEQ ID NO: 2, N-terminal substitutions, deletions, insertions and amino acid sequence of amino acid residues to the amino acid sequence of SEQ ID NO: 2 or it may be a protein having one or more amino acid sequences were performed of adding the C-terminus or additional amino acid residues to both. Amino acid residue substitutions, deletions, for example of the order of addition of additional amino acid residues to the N-terminus or C-terminus or both insertion and amino acid sequences are as described above.
　Alternatively, pyridoxamine synthase, SEQ ID NO: 2 amino acid sequence with a protein or to the amino acid sequence of SEQ ID NO: 2, for example, 80% or more, or 85% or more, or 90%, or 95% or more sequence identity to it may be a protein having an amino acid sequence having.
　As described above, when using a protein having an amino acid sequence similar to the amino acid sequence of SEQ ID NO: 2, the enzyme activity the protein to synthesize pyridoxamine from activity (pyridoxal as pyridoxamine synthase, in the present disclosure pyridoxamine synthase activity also referred) should have a. Enzyme activity to synthesize pyridoxamine from pyridoxal (pyridoxamine synthase activity), for example, pyridoxal and necessary amino acids as a substrate (in the case of a protein having an amino acid sequence similar to SEQ ID NO: 2 amino acid sequences, for example L- alanine) a the aqueous solution tested proteins, including adding, the amount of generated pyridoxamine can be measured by quantifying by high performance liquid chromatography.
　Alternatively, pyridoxamine synthase, for example, may be a protein having the amino acid sequence of SEQ ID NO: 4, N-terminal substitutions, deletions, insertions and amino acid sequence of amino acid residues to the amino acid sequence of SEQ ID NO: 4 or it may be a protein having one or more amino acid sequences were performed of adding the C-terminus or additional amino acid residues to both. Amino acid residue substitutions, deletions, for example of the order of addition of additional amino acid residues to the N-terminus or C-terminus or both insertion and amino acid sequences are as described above.
　Alternatively, pyridoxamine synthase, SEQ ID NO: protein having the amino acid sequence of 4 or SEQ ID NO: 4 amino acid sequence to, for example, 80% or more, or 85% or more, or 90%, or 95% or more sequence identity to it may be a protein having an amino acid sequence having.
　As described above, when using a protein having an amino acid sequence similar to the amino acid sequence of SEQ ID NO: 4, the enzyme activity the protein to synthesize pyridoxamine from activity (pyridoxal as pyridoxamine synthase, in the present disclosure pyridoxamine synthase activity also referred) should have a. Enzyme that synthesizes pyridoxamine from pyridoxal (pyridoxamine synthase activity), for example, in the case of a protein having an amino acid sequence similar to pyridoxal and the required amino acids (amino acid sequence of SEQ ID NO: 4 as a substrate e.g. L- glutamic acid or L- the aqueous solution protein in the test subject, including an aspartic acid or a salt thereof) added, the amount of generated pyridoxamine can be measured by quantifying by high performance liquid chromatography.
[0041]
　Further, the hydrogen peroxide degrading enzyme, for example, may be a protein having the amino acid sequence of SEQ ID NO: 3, substitution of amino acid residues to the amino acid sequence of SEQ ID NO: 3, deletion, insertion and amino acid sequence it may be a protein having the amino acid sequence of performing one or more of the additional N-terminal or C-terminus or additional amino acid residues to both. Amino acid residue substitutions, deletions, for example of the order of addition of additional amino acid residues to the N-terminus or C-terminus or both insertion and amino acid sequences are as described above.
　Alternatively, the hydrogen peroxide degrading enzyme, SEQ ID NO: 3 protein or having the amino acid sequence of SEQ ID NO: 3 amino acid sequence with respect to, for example, 80% or more, or 85% or more, or 90%, or 95% or more sequence identity it may be a protein having an amino acid sequence having sex.
　As described above, when using a protein having an amino acid sequence similar to the amino acid sequence of SEQ ID NO: 3, the protein should have activity as hydrogen peroxide decomposing enzyme. Hydrogen peroxide decomposition activity, for example, an aqueous solution to the test object of the protein of hydrogen peroxide as a substrate was added, can be measured by quantifying the reduction in amount of hydrogen peroxide as an index and decrease in absorbance at 240 nm.
[0042]
　
　gene encoding pyridoxine oxidase may be any gene encoding pyridoxine oxidase described above. Genes encoding pyridoxamine synthase may be any gene encoding pyridoxamine synthase described above. Gene encoding hydrogen peroxide-degrading enzyme may be any gene encoding the hydrogen peroxide degrading enzyme described above. Enzymes encoded by these genes are known amino acid sequence having the enzymatic activity (e.g., an amino acid sequence encoded by a gene naturally present in the organism) not limited to, a different modification to the known amino acid sequence it may be an enzyme having the amino acid sequence.
[0043]
　Such genes, such as genes exemplified microorganisms (microbes from which it is derived) has naturally on a microorganism having the enzyme, may be a known gene, a range of desired enzymatic activity is obtained the inner, it may be a gene obtained by modifying the nucleotide sequence to encode a modified modified amino acid sequence from the known amino acid sequence of the enzyme as described above. Examples of such modified amino acid sequence, as described above, amino acid sequence similarity and the like in the amino acid sequence of either of SEQ ID NOs: 1-4. Further, the nucleotide sequence of the gene that codes for a specific amino acid sequence can be varied within the scope of codon degeneracy. In this case, better to use a frequently used codon in a microorganism as a host of the recombinant microorganism is preferred in terms of expression efficiency of the gene.
[0044]
　Incidentally, the nucleotide sequence of the gene can also be designed from the amino acid sequence to be encoded based on the codon table. Nucleotide sequences designed, to a known nucleotide sequence may be obtained by modified using genetic recombination techniques, it may be obtained by chemically synthesizing a nucleotide sequence.
　As a method for modifying nucleotide sequences, such as site-specific mutagenesis (Kramer, W. And frita, H.J., Methods in Enzymology, vol.154, P.350 (1987)), recombinant PCR method (PCR Technology, Stockton Press (1989), a method of chemically synthesizing the DNA of a particular part, the gene ultraviolet irradiation processing method, a strain carrying a gene hydroxylamine treatment, or a chemical agent in the process, such as nitrosoguanidine or nitrous acid how to, and a method of using a commercially available mutagenesis kit.
[0045]
　For example, the gene encoding the pyridoxine oxidase, genes encoding the pyridoxamine synthase and a gene encoding the hydrogen peroxide degrading enzyme, are both known nucleotide sequence encoding a polynucleotide having the enzyme activity ( for example, it may be a DNA having remained unaltered nucleotide sequence having a naturally occurring gene is) in an organism such as a microorganism exemplified above, for such nucleotide sequences, enzyme encoded its enzyme activity may be a DNA having the nucleotide sequence plus the sequence modifications in a range not losing the (enzyme activity described above). Such modifications, insertion of a nucleotide, deletion, and addition of substitutions and nucleotide sequence 5 'terminus or 3' terminus or additional nucleotides to both. Insertion of nucleotides, if there are one or more of the deletions and substitutions, insertions, deletions and each substitution, when present, for example, 1 to 90 nucleotides or 1 to 60 nucleotides, or 1 to 30 amino acids, residues or 1-20 amino acid residues, or 1-15 nucleotides or 1-10 nucleotides, or may be 1-5 nucleotides, insertion of nucleotides, deletion and the total number of substituents is, for example, 1 to 100 nucleotides,,, or 1-50 nucleotides or 1-30 nucleotides, or one to 10 nucleotides, or may be 1-5 nucleotides. Insertion or deletion of nucleotides may be present in the local, but is preferably not generated a large frame shift as a whole nucleotide sequence. Further, the number of nucleotides added to the ends, first end per example 1 to 150 nucleotides, if present, or 1-100 nucleotides, or 1-50 nucleotides, or 1 to 30 nucleotides, or 1 to 10 nucleotides, or it may be from 1 to 5 nucleotides. Such additional nucleotides may encode a signal sequence for secretion or the like into the extracellular.
[0046]
　Alternatively, the gene encoding each enzyme is known nucleotide sequence encoding a polynucleotide having the enzymatic activity (e.g., the nucleotide sequence of the gene naturally present in the organism) DNA having itself or the known nucleotide sequence having a nucleotide sequence having 80% or more, or 85%, or 90%, or 95% sequence identity with respect to, encoding an enzyme having the desired enzymatic activity (above enzyme activity) DNA it may be. Here, sequence identity can be assessed in default parameters using e.g. BLAST (registered trademark, National Library of Medicine) program.
[0047]
　Alternatively, the gene encoding each enzyme is known nucleotide sequence encoding a polynucleotide having the enzymatic activity (e.g., the nucleotide sequence of the gene naturally present in the organism) DNA having itself or the known nucleotide sequence having a nucleotide sequence that hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to, or may be a DNA encoding an enzyme having the desired enzymatic activity (above enzyme activity). Further, the hybridization under stringent conditions can be carried out as follows.
　In hybridization, a DNA composed from a reference nucleotide sequence complementary to the nucleotide sequence or subsequence thereof as a probe, hybridization is performed with respect to the DNA of interest, probe after washing under stringent conditions and the target to check that you are significantly hybridize to a nucleic acid. The length of the probe, for example a continuous 20 or more nucleotides, preferably 50 nucleotides or more, more preferably 100 nucleotides or more, more preferably it is possible to use more than 200 nucleotides. Have the same nucleotide length as the reference become nucleotide sequence, it is also preferable to use as a probe a DNA complementary over the entire length. And conditions for hybridization can be exemplified by conditions under which the skilled worker is generally used to detect specific hybridization signals. Preferably, it means stringent hybridization conditions and stringent washing conditions. For example, 6 × SSC (saline sodium citrate) (1 × SSC composition: 0.15 M NaCl, 0.015M sodium citrate, pH7.0), 0.5% SDS, 5 × Denhardt and 100 mg / ml herring sperm DNA conditions that incubation overnight at 55 ° C. with the probe and the like in a solution containing. Then the like can be exemplified by washing the filters in 0.2 × SSC in 42 ° C.. Stringent conditions are conditions of 0.1 × SSC, 50 ° C. in a filter cleaning step, as further stringent conditions, and the like conditions of 0.1 × SSC, 65 ° C. in the same process it can.
[0048]
　For example, the gene may be, for example, a DNA having the nucleotide sequence of SEQ ID NO: 5 (nucleotide sequence of pyridoxine oxidase gene-mycobacterial Ruteoramu), complementary to the nucleotide sequence of SEQ ID NO: 5 encoding pyridoxine oxidase a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence may range and DNA having a nucleotide sequence encoding a protein having a pyridoxine oxidase activity.
　Alternatively, the gene encoding pyridoxine oxidase, for example, may be a DNA having the nucleotide sequence of SEQ ID NO: 5 nucleotide substitutions to the nucleotide sequence of SEQ ID NO: 5, deletion, insertion and nucleotide sequences 5 'terminus or 3' terminus or a nucleotide sequence of performing one or more of the addition of additional nucleotides to both, or may be a DNA having a nucleotide sequence encoding a protein having a pyridoxine oxidase activity. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
　Alternatively, pyridoxine oxidase, SEQ ID NO: 5 nucleotides DNA having the sequence or sequence number for the nucleotide sequence of the 5, for example, 80% or more, or 85%, or 90%, or 95% or more sequence identity a nucleotide sequence may be a DNA having a nucleotide sequence encoding a protein having a pyridoxine oxidase activity.
[0049]
　Genes encoding pyridoxamine synthase, for example, the nucleotide sequence of SEQ ID NO: 6 - or the nucleotide sequence of SEQ ID NO: 8 (pyridoxamine of Mezorizobiumu Roti nucleotide sequence of pyruvate transaminase gene) (E. coli pyridoxamine - oxaloacetic transaminase gene nucleotide sequence ) may be a DNA having, a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to a nucleotide sequence of SEQ ID NO: 6 or 8, encoding a protein and having a pyridoxamine synthase activity it may be a DNA having the nucleotide sequence.
　Alternatively, genes encoding the pyridoxamine synthase, for example, may be a DNA having the nucleotide sequence of SEQ ID NO: 6 or 8, nucleotide substitutions to the nucleotide sequence of SEQ ID NO: 6 or 8, deletion, insertion and a 5 'terminus or 3' terminus or the nucleotide sequence was carried out one or more of the addition of additional nucleotides to both the nucleotide sequence, DNA having a nucleotide sequence encoding a protein with pyridoxamine synthase activity it may be. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
[0050]
　Alternatively, pyridoxamine synthase, SEQ ID NO: 6 or 8 nucleotides DNA having the sequence or SEQ ID NO: 6 or 8 nucleotides sequence to example 80%, or 85%, or 90%, or 95% or more a nucleotide sequence having sequence identity may be a DNA having a nucleotide sequence encoding a protein having pyridoxamine synthase activity.
[0051]
　Alternatively, genes encoding the pyridoxamine synthase, for example, may be a DNA having any nucleotide sequence of SEQ ID NO: 6 and SEQ ID NO: 25 to SEQ ID NO: 31, SEQ ID NO: 6 and SEQ ID NO: 25 to SEQ a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to any of the nucleotide sequences of No. 31, a protein may be a DNA having a nucleotide sequence encoding a and having a pyridoxamine synthase activity .
　Alternatively, genes encoding the pyridoxamine synthase, for example, may be a DNA having any nucleotide sequence of SEQ ID NO: 6 and SEQ ID NO: 25 to SEQ ID NO: 31, SEQ ID NO: 6 and SEQ ID NO: 25 to SEQ nucleotide substitutions to any of the nucleotide sequences of No. 31, deletion, insertion and nucleotide sequences of the 5 'terminus or 3' terminus or one or more nucleotides was carried out the addition of additional nucleotides to both a sequence may be a DNA having a nucleotide sequence encoding a protein having pyridoxamine synthase activity. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
　Alternatively, pyridoxamine synthase, the nucleotide sequence of the DNA or SEQ ID NO: 6, having any of the nucleotide sequences of SEQ ID NO: 6 and SEQ ID NO: 25 to SEQ ID NO: 31, SEQ ID NO: 25 (Mesorhizobium sp YR577 of pyridoxamine -. Pyruvate the nucleotide sequence of the nucleotide sequence of the gene) encoding the transaminase, SEQ ID NO: 26 (Pseudaminobacter salicylatoxidans of pyridoxamine - nucleotide sequence of the nucleotide sequence of the gene coding for pyruvate transaminase), pyridoxamine of SEQ ID NO: 27 (Bauldia litoralis - pyruvate transaminase the nucleotide sequence of the coding nucleotide sequence of the gene), SEQ ID NO: 28 ( Skermanella stibiiresistens of pyridoxamine - nucleotide sequence of pyruvate nucleotide sequence of the gene encoding acid transaminase), SEQ ID NO: 29 (Rhizobium sp of AC44 / 96 pyridoxamine -. The nucleotide sequence of the nucleotide sequence of the gene coding for pyruvate transaminase), SEQ ID NO: 30 - the nucleotide sequence of (Erwinia toletana of pyridoxamine nucleotide sequence of the gene encoding pyruvate transaminase), and SEQ ID NO: 31 - at least one of the nucleotide sequences of (Herbiconiux ginsengi of pyridoxamine nucleotide sequence of the gene encoding pyruvate transaminase) for example, 80% or more One Or 85% or more, or 90% or more, or a nucleotide sequence having 95% or more sequence identity, or may be a DNA having a nucleotide sequence encoding a protein having pyridoxamine synthase activity.
[0052]
　When expressing in a recombinant microorganism prokaryotic as a host such as E. coli, it may be codon optimized to facilitate expression. For example, what is the highest frequency of use among the codons encoding each amino acid in a prokaryotic as the host may be modified nucleotide sequence to be used frequently as a codon for the amino acids. From this point of view, any as DNA containing a gene encoding the pyridoxamine synthase, for example, to 18 th nucleotide in the nucleotide sequence of SEQ ID NO: 10 3 'region up end or SEQ ID NO: 32 to one of SEQ ID NO: 38 'may be used a DNA having a region to the end, the 18 th in the nucleotide sequence of SEQ ID NO: 10 nucleotides to the 3' 18 nt ~ 3 of Kano nucleotide sequence region to the end or SEQ ID NO: 32 through SEQ a protein having an 18 nt ~ 3 'hybridizes with DNA under stringent conditions with a region complementary to the nucleotide sequence to end, and pyridoxamine synthase activity of any nucleotide sequence of ID NO: 38 the DNA having a nucleotide sequence encoding It may be used. The nucleotide sequence of SEQ ID NO: 10, 17 nucleotides from the 5 'end has a upstream region, there is a start codon 18 nt to 20 nt. Thus, may be used range up to 18 nt ~ 3 'end of this nucleotide sequence as a gene region encoding the pyridoxamine synthase. Similarly, the nucleotide sequence of SEQ ID NO: 32 to SEQ ID NO: 38, either, 17 nucleotides from the 5 'end has a upstream region, there is a start codon 18 nt to 20 nt. Thus, may be used range up to 18 nt ~ 3 'end of these nucleotide sequences as a gene region encoding the pyridoxamine synthase.
　Alternatively, genes encoding the pyridoxamine synthase, for example, of any of the nucleotide sequences of the 18 th nucleotide in the nucleotide sequence of SEQ ID NO: 10 ~ 3 'region to end or SEQ ID NO: 32 to SEQ ID NO: 38 18 th 'may be a DNA having the region of to the end, nucleotides 1-3 of 18 th in the nucleotide sequence of SEQ ID NO: 10' nucleotides 1-3 or nucleotide in the region of up to end or SEQ ID NO: 32 to SEQ ID NO: 38 18 th nucleotides to the sequence 3 'region nucleotide substitutions relative to the terminal, deletion, insertion and 5 nucleotide sequence' terminus or 3 'terminus or one or more of the addition of additional nucleotides to both a nucleotide sequence was carried out, having a pyridoxamine synthase activity Protein or may be a DNA having a nucleotide sequence encoding. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
[0053]
　Alternatively, pyridoxamine synthase to '18 nt ~ 3 of any nucleotide sequence of the region or SEQ ID NO: 32 to SEQ ID NO: 38 to the end' terminal 18 nt ~ 3 in the nucleotide sequence of SEQ ID NO: 10 18 nt ~ 3 'region up end of the nucleotide sequence of the DNA, or SEQ ID NO: 10 having a region (Mesorhizobium loti of pyridoxamine - codon optimized nucleotide sequence of the gene coding for pyruvate transaminase), of SEQ ID NO: 32 18 nt ~ 3 'region up end of the nucleotide sequence (Mesorhizobium sp YR577 of pyridoxamine -. codon optimized nucleotide sequence of the gene coding for pyruvate transaminase), Region of up to 18 nt ~ 3 'terminus of the nucleotide sequence of SEQ ID NO: 33 (pyridoxamine of Pseudaminobacter salicylatoxidans - codon optimized nucleotide sequence of the gene coding for pyruvate transaminase), 18 th nucleotide of the nucleotide sequence of SEQ ID NO: 34 ~ 3 'to the end region (of Bauldia litoralis pyridoxamine - codon optimized nucleotide sequence of the gene coding for pyruvate transaminase), 18 th nucleotides 1-3 of the nucleotide sequence of SEQ ID NO: 35' to terminal region (the Skermanella stibiiresistens pyridoxamine - codon optimized nucleotide sequence of the gene coding for pyruvate transaminase) Region of up to 18 nt ~ 3 'terminus of the nucleotide sequence of SEQ ID NO: 36 (Rhizobium sp of AC44 / 96 pyridoxamine -. Codon optimized nucleotide sequence of the gene coding for pyruvate transaminase), the nucleotide sequence of SEQ ID NO: 37 18 nt ~ 3 'region up end (Erwinia toletana of pyridoxamine - pyruvate transaminase
[0054]
　Gene encoding the hydrogen peroxide degrading enzyme, for example, may be a DNA having the nucleotide sequence of SEQ ID NO: 7 (nucleotide sequence of catalase gene of Listeria Zerigeri), complementary nucleotide to the nucleotide sequence of SEQ ID NO: 7 a DNA which hybridizes with the DNA under stringent conditions with the sequence, and may be a DNA having a nucleotide sequence encoding a protein having a hydrogen peroxide decomposition activity.
　Alternatively, genes encoding the hydrogen peroxide degrading enzyme, for example, may be a DNA having the nucleotide sequence of SEQ ID NO: 7, substitution of a nucleotide to the nucleotide sequence of SEQ ID NO: 7, deletion, insertion and nucleotide a 5 'terminus or 3' terminus or additional nucleotide nucleotide sequence was carried out one or more of the addition of on both sequences, DNA having a nucleotide sequence encoding a protein having a hydrogen peroxide decomposition activity it may be. Nucleotide substitutions, deletions, for example of the order of addition of additional nucleotides to the N-terminus or C-terminus or both insertion and nucleotide sequences are as described above.
　Alternatively, the hydrogen peroxide degrading enzyme, SEQ ID NO: 7 nucleotides DNA having the sequence or SEQ ID NO: 7 nucleotide sequence with respect to, for example, 80% or more, or 85%, or 90%, or 95% or more sequence identity a nucleotide sequence having sex, or may be a DNA having a nucleotide sequence encoding a protein having a hydrogen peroxide decomposition activity.
[0055]
　
　in recombinant the microorganism according to the present disclosure, the gene encoding the gene and pyridoxamine synthase encoding pyridoxine oxidase, respectively, bacteria inherent in the body but may be a gene with enhanced expression may be a gene that has been introduced from the outside of cells to intracellular. Furthermore, the gene is endogenous to cells with enhanced expression, both genes introduced from extracellular to intracellular may be present in the recombinant microorganism. Further, in a recombinant the microorganism according to the present disclosure, but inherent in cells encoding the gene expression encodes enhanced pyridoxine oxidase, and / or pyridoxine oxidase introduced into intracellular from extracellular in addition to the gene, further not expressed enhanced inherent in the cells (e.g., promoter unmodified) genes encoding pyridoxine oxidase may be present. Similarly, inherent in cells in addition to the gene, which encodes the gene expression encodes pyridoxamine synthase is enhanced, and / or pyridoxamine synthase introduced from extracellular to intracellular, further , not expressed enhanced inherent in the cells (e.g. promoter unmodified) genes encoding pyridoxamine synthase may be present. In this disclosure, for each of the genes encoding genes and pyridoxamine synthase encoding pyridoxine oxidase, one or more of the introduced genes from strengthening and extracellular expression of genes endogenous to cells into intracellular because doing, even if they exist originally gene unreinforced expressed intracellularly, significantly higher expression level than the expression amount of the original gene which is not reinforced the expression is obtained, thereby high pyridoxamine production efficiency can be achieved.
　When the recombinant microorganism according to the present disclosure further comprises a gene encoding the hydrogen peroxide degrading enzyme, a gene encoding the hydrogen peroxide degrading enzyme may be a gene inherent in bacteria, fungi inherent in the body but may be a gene with enhanced expression may be a gene that has been introduced from the outside of cells to intracellular. However, since the increase amount of generated hydrogen peroxide the reaction rate increases, to increase the expression level of hydrogen peroxide degrading enzyme, to enhance the expression of a gene encoding the hydrogen peroxide degrading enzyme endogenous to cells it, and introducing a gene encoding the hydrogen peroxide degrading enzyme in the cells from the extracellular, it is preferable to perform at least one of.
[0056]
　In other words, each of the genes encoding genes and pyridoxamine synthase encoding pyridoxine oxidase may but inherent in the genome of the host microorganism be a gene with enhanced expression by operating the substitution or the like of the promoter and it may be a gene that has been introduced from the outside of cells to intracellular using a vector such as a plasmid. In addition, genes that are not expressed enhanced inherent in the genome of the host microorganism before recombination may also be present. In the present disclosure, whether the respective genes encoding the gene and pyridoxamine synthase encoding pyridoxine oxidase introduced from extracellular host microorganism, or the expression of the gene endogenous to the host bacterium by substitution or the like of the promoter by strengthening, increased expression of the gene, have granted pyridoxamine production capacity due to the combination of the two enzymes. Here, each of the genes encoding the gene and pyridoxamine synthase encoding pyridoxine oxidase, in the case of reinforcing introduction or expression, and introduction of the extracellular host microorganism, the gene expression by substitution or the like of the promoter of enhancements it may be performed only one, both may be performed.
[0057]
　The recombinant microorganism according to the present disclosure, may or may not have a gene encoding the hydrogen peroxide degrading enzyme. When the recombinant microorganism according to the present disclosure further comprises a gene encoding the hydrogen peroxide degrading enzyme, the gene encoding the hydrogen peroxide degrading enzyme, a gene inherent in the genome of the host microorganism before recombination may be, but is inherent in the genome of the host microorganism may be a gene with enhanced expression by operating the substitution or the like of the promoter, to the intracellular from extracellular using a vector such as a plasmid it may be an introduced gene. The gene encoding the hydrogen peroxide degrading enzyme, in the case of reinforcing introduction or expression, and introduction of the extracellular host microorganism of the strengthening of the gene expression by substitution or the like of the promoter, even if only one of to the good, both may be performed.
[0058]
　The gene encoding the gene and pyridoxamine synthase encoding pyridoxine oxidase, Without increasing the above-described gene expression, sufficient production capacity for high production of pyridoxamine, or a salt thereof can not be obtained. Therefore, in the recombinant microorganism according to the present disclosure, both of the gene encoding the gene and pyridoxamine synthase encoding pyridoxine oxidase, respectively, of the enhanced expression of genes underlying the introduction and cells from extracellular It has done at least one. Incidentally, introduction of the enzyme gene from extracellular is necessarily not limited to introduction to compensate for the enzyme gene that is not present in the host microorganism, performed for the purpose of increasing the expression also gene endogenous to the host microorganism it may be. The gene that is not present in the host microorganism can be readily ascertained using enzyme database, eg KEGG and BRENDA.
[0059]
　If enhancing expression of the gene by replacing the promoter of the gene endogenous to the host microorganism with a different promoter, enhanced expression of genes in host microorganism as such alternative promoters (promoter newly introduced) it is not particularly as long as it (can be enhanced than before promoter replacement) limit, or a inducible promoter in constitutive promoter. Substitution of promoters, general genetic recombination techniques can be performed using. The arrangement of a promoter endogenous to the host microorganism, unless, may be left partially or completely adversely affect as a practical problem for expression by the newly introduced promoter.
　When the host microorganism is, for example, prokaryotes, Examples of usable promoters Promoters newly introduced, trp promoter, lac promoter derived from E. coli, and GAPDH promoter, PL promoter and PR promoter derived from lambda phage, Bacillus subtilis-derived gluconic acid synthetase promoter (gnt), alkali protease promoter (apr), neutral protease promoter (npr), alpha-amylase promoter (amy) and the like. Furthermore, uniquely modified or designed promoter sequence as tac promoter may also be utilized.
[0060]
　When the host microorganism is, for example, filamentous fungi, examples of usable promoters Promoters newly introduced, cellobiohydrolase (cbh) promoter, endoglucanase (egl) promoter, xylanase III (XYN3) promoter, U6 promoter, alpha-amylase (amy) promoter, and the like.
[0061]
　When the host microorganism is, for example, yeast, examples of usable promoters Promoters newly introduced, alcohol dehydrogenase (ADH1) promoter, phosphoglycerate kinase (PGK1) promoter, peptide chain elongation factor (TEF) promoter , include glycerol 3-phosphate dehydrogenase (GPD) promoter, galactokinase (GAL1) promoter, metallothionein (CUP1) promoter, repressible acid phosphatase (PHO5) promoter and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter It is. Incidentally, from the sequence of the promoter is not limited to the yeast as a host microorganism. Such as cytomegalovirus (CMV) promoter, may be used as the promoter of the foreign.
[0062]
　When introducing the genes from extracellular host microorganism (foreign (heterogenous) gene), examples of the method to introduce genes into bacterial cells (intracellular), to express the enzyme encoded by the gene if it is possible, not particularly limited, transformed with a plasmid carrying the enzyme gene introduction into the genome of the gene, and can be combinations thereof. When introducing genes the expression vector incorporating the gene may be introduced in the cells. The expression vector is not particularly limited as long as it incorporates the nucleotide sequence of the gene, a plasmid vector shown from the viewpoint of enhancing the transformation efficiency and translation efficiency, a structure as shown below and more preferably, which is a phage vector.
[0063]
　Expression vector comprises a nucleotide sequence of the gene is not particularly limited as long as it can transform the host microorganism. If necessary, in addition to said nucleotide sequence, the nucleotide sequence constituting the other areas (hereinafter, simply referred to as "other regions".) May be contained. Other regions, for example, a recombinant microorganism obtained by transformation, and the control area required to produce the desired enzymes, and the like region required for autonomous replication.
　Further, from the viewpoint of facilitating the selection of the recombinant microorganism may further contain a nucleotide sequence encoding a selection gene can be a selectable marker.
　The control area needed to produce the desired enzyme, the promoter sequence (including an operator sequence to control transcription.), Ribosome binding sequence (SD sequence) include transcription termination sequences and the like.
[0064]
　Expression vectors that can be used when a yeast is used as a host microorganism, in addition to the nucleotide sequence of the gene, from the viewpoint of the expression efficiency of the gene, it is preferable to contain a promoter sequence. The promoter sequence may be any one as long as it can express the gene in transformants that yeast as a host microorganism. Examples include host microorganisms include promoters described above as examples of promoters that can be used as a promoter for newly introduced when a yeast.
[0065]
　Further, the expression vector may comprise a secretory signal. Accordingly, if the recombinant microorganism is producing the desired enzyme, it is possible to secrete the enzyme extracellularly.
　The secretion signal, as long as it can secrete the desired enzyme from the yeast as a host microorganism is not particularly limited. From the viewpoint of secretion efficiency, alpha-factor signal sequence, invertase signal sequence, the acid phosphatase signal sequence, it is preferable to use a glucoamylase signal sequence.
[0066]
　The expression vector having a promoter sequence and secretion signal as described above, and specific examples thereof include pRS423, pRS424, YEplac195 like.
[0067]
　Expression vectors which can be used where the filamentous fungus as a host microorganism, in addition to the nucleotide sequence of the gene, from the viewpoint of the expression efficiency of the gene, it is preferable to contain a promoter sequence. The promoter sequence, a filamentous fungus may be used so long as it can express the gene in transformants that the host microorganism. Examples include host microorganisms include promoters described above as examples of usable promoters Promoters newly introduced in the case of filamentous fungi.
[0068]
　Suitable expression vectors against filamentous fungi, van den Hondel, C. A. M. J. J. et al. (1991) In: Bennett, J . W. and Lasure, L. L. (Eds.) More gene Manipulations in Fungi. Academic Press, pp. It is described in the 396-428.
　Further, pUC18, pBR322, pUC100, pSL1180 (Pharmacia Inc.) may be used generally another expression vector used, such as pFB6 and Aspergillus (Aspergillus) pRAX, Trichoderma (Trichoderma) pTEX.
[0069]
　E. coli, expression vectors which can be used where Bacillus subtilis, prokaryotic organisms, such as actinomycetes and host microorganism, in addition to the nucleotide sequence of the gene, from the viewpoint of the expression efficiency of the gene, that contains a promoter sequence It is preferred. In addition to may contain a ribosome binding sequence and a transcription termination sequence and the like of the promoter sequence.
[0070]
　Examples of the promoter sequence, the host microorganism include promoters described above as examples of promoters that can be used as a promoter for newly introduced in the case of prokaryotes.
[0071]
　The ribosome binding sequence, but from E. coli or sequence from B. subtilis, it is not particularly limited as long as it is a sequence which functions in a desired host microorganism such as Escherichia coli or Bacillus subtilis.
　As the ribosome binding sequence, for example, of the sequence complementary to the 3 'terminal region of 16S ribosomal RNA, and the like array created a continuous consensus sequence 4 or more nucleotides by DNA synthesis.
　The transcription termination sequence is not always necessary, those ρ factor independent, available for example lipoprotein terminator, trp operon terminator or the like.
　These arrangement order on the expression vector of the control region, but are not particularly limited, considering the transfer efficiency 5 'end upstream promoter sequences from ribosome binding sequence, the gene encoding the desired enzyme, a transcription termination sequence it is desirable that the line up of the order.
[0072]
　PBR322 host microorganism Specific examples of usable expression vector when prokaryotic has autonomously replicable region in E. coli, pUC18, Bluescript II SK (+ ), pKK223-3, pSC101 and, pUB110 has autonomously replicable region in B. subtilis, pTZ4, pC194, ρ11, φ1 , and the like Fai105.
　As examples of autonomous replication expression vector in the two or more host microorganism can be used as the expression vector pHV14, TRp7, YEp7 and pBS7 like.
[0073]
　In the case of introducing a gene from extracellular host microorganism, the gene may be a gene having a nucleotide sequence that is not present in the genome of the host microorganism, but present in the genome of the host microorganism nucleotides it may be a gene having a sequence. Even if originally the same gene in the genome of the host microorganism is present, a stronger gene expression by the introduction of genes from extracellular is obtained by enhanced enzymatic activity, pyridoxamine or production efficiency of a salt thereof it can be further improved.
[0074]
　Prepared from extracellular (extracellular) of genomic DNA required to introduce genes into bacterial cells (intracellular), cleavage and ligation of DNA, transformation, PCR (Polymerase Chain Reaction), design of oligonucleotides used as primers design, method of synthesis and the like can be carried out in a conventional manner well known to those skilled in the art. These methods, Sambrook, J. , Et al. , "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, are described, for example, (1989). For example, a method of using competent cells is a method using electroporation.
[0075]
　The host microorganism, if the microorganism capable of expressing them if the gene encoding the gene and pyridoxamine synthase encoding pyridoxine oxidase is present is not particularly limited. Examples of host microorganisms, yeast, fungi, and prokaryotes. Examples of the yeast, Saccharomyces (Saccharomyces) yeasts such as Saccharomyces cerevisiae, Schizosaccharomyces (Schizosaccharomyces) yeasts such as Schizosaccharomyces pombe, Hansenula (Hansenula) yeasts, and Pichia (Pichia) yeasts, but and the like. Examples of filamentous fungi, Trichoderma reesei or Trichoderma (Trichoderma) filamentous fungi such viride, Aspergillus, such as Aspergillus niger or oryzae (Aspergillus) genus fungi, Humicola (Humicola) filamentous fungi, such as Humicola insolens, and Acremonium cellulolyticus or Acremonium (Acremonium) genus filamentous fungi such Fusidioides, and the like. Further, examples of prokaryotes, Escherichia, such as Escherichia coli (Escherichia) bacteria, Schewanella sp. AC10 etc. Shewanella (Schewanella) bacteria, Mesorhizobium loti etc. Mezorizobiumu (Mesorhizobium) bacteria, Rhizobium (Rhizobium) bacteria such as Rhizobium meliloti, Bacillus bacteria such as Bacillus (Bacillus) genus Bacillus bacteria such as Bacillus subtilis, Streptomyces lividans include actinomycetes Streptomyces (Streptomyces) genus actinomycetes, such as the like.
[0076]
　These in a host microorganism, for example by introducing an expression vector containing the desired gene (transformed), it is possible to introduce a desired gene into a host microorganism. Then, the introduced gene, for example, by the activity of the contained in the expression vector promoter, it is possible to highly expressed in the resultant recombinant microorganism.
[0077]
　As a method of transferring the recombinant DNA of the cell into an expression vector such as the host microorganism, for example, when the host microorganism is Escherichia coli can be used include competent cell method or electroporation method by calcium treatment. Thus recombinant microorganism obtained, by being cultured, it can be produced stably with high expression levels of the enzyme genes introduced encodes.
　Further, DNA encoding these enzymes can be removed from a recombinant microorganism, it is possible to transfer to other microorganisms. Further, by using this DNA as a template, a DNA fragment encoding the enzyme was amplified by PCR, after the treatment with restriction enzymes or the like, is combined with other vector DNA fragment, it is also easily to be newly introduced into a host microorganism It can be carried out.
[0078]

　In one embodiment, pyridoxamine or a salt thereof is recombinant microorganism according to the present disclosure, the process of culture or the recombinant microorganism or the culture of the recombinant microorganism It objects and is contacted in the presence of oxygen and a pyridoxine or a salt thereof, causing production of pyridoxamine or a salt thereof.
　Examples of pyridoxine salts, salts with pyridoxine and acid. Examples of acids include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts of pyridoxine, for example, pyridoxine hydrochloride.
　Examples of salts of pyridoxamine, and salts with pyridoxamine and acid. Examples of acids are hydrochloric, sulfuric, nitric, acetic, phosphoric acid, and the like. As for the terms used in medicine, the salts of pyridoxamine is preferably applied to the pharmaceutical use is the most advanced in that pyridoxamine dihydrochloride.
　Such a recombinant microorganism, that of the recombinant using a process of culture or the recombinant microorganism or the culture of a microorganism pyridoxamine or a salt thereof, simply using a "recombinant microorganism pyridoxamine or referred to as a manufacturing method of the salt. "
[0079]
　The culture of the recombinant microorganism obtained by culturing a recombinant microorganism, refers to the product made from the cell and the surrounding medium and the like. When the enzyme is extracellularly secreted, the use of such cultures, and substrate and enzyme is more easily contacted, it is possible to improve the efficiency of producing pyridoxamine or a salt thereof. However, may not use the culture, for example, the cells of the recombinant microorganism were pre prepared dried or freeze may be added directly to the reaction system.
[0080]
　During cultivation of recombinant microorganism, the culture medium, carbon sources, nitrogen sources, if medium suitable amount of inorganic substance and other nutrients, can be used in either a synthetic medium or a natural medium. Culture can be carried out using in a liquid medium containing the culture components, shake culture, aeration spinner culture, the ordinary method for culturing, such as continuous culture, or fed-batch culture.
[0081]
　More specifically, the conditions of culture of the recombinant microorganism is the same as the culture conditions of the original host microorganism can be used known conditions.
　The components used in the medium may be a known. For example, meat extract, yeast extract, malt extract, peptone, NZ amine and organic nutrient sources such as potato, glucose, maltose, sucrose, carbon sources such as starch and organic acids, ammonium sulfate, a nitrogen source such as urea and ammonium chloride, phosphorus salts, magnesium, inorganic nutrient sources such as potassium and iron, a combination of vitamins can be suitably used.
　In the culture of a recombinant microorganism transformed by an expression vector containing the selection marker, for example, if the selectable marker is a drug resistance using a medium containing the drug to the corresponding said selectable marker when auxotrophic uses media without nutrients corresponding thereto.
[0082]
　Culture conditions, the recombinant microorganism, medium may be appropriately selected depending on the type of cultivation method, the recombinant microorganism is grown is not particularly limited so long as those are conditions that can produce pyridoxine oxidase and pyridoxamine synthase.
　The pH of the medium is, for example, may be selected in the range of 4-8, it may be in the range of 5-8.
　The culture temperature is, for example, 20 ℃ ~ 45 ℃, preferably 24 ℃ ~ 37 ℃. Culture, depending on the type of microorganisms, may be performed in aerobic, it may be carried out anaerobically.
　Culture period is, for example, 1 to 7 days. Culture period, production of the enzyme of interest may be set to be maximized.
[0083]
　Further, the recombinant The microorganism of the workpiece, to the extent that the recombinant microorganism does not lose activity pyridoxine oxidase and pyridoxamine synthase produced, refers to the product were any processing to recombinant microorganisms. Such processing, for example, heat treatment, cooling treatment, mechanical disruption, sonication, freeze-thaw treatment, drying treatment, pressurization or depressurization treatment, osmotic pressure treatment, autolysis, surfactant treatment and enzyme treatment (eg, cell lysis treatment) such treatment comprising one or more can be mentioned which is selected from the group consisting of. Even if it recombinant microorganism itself killed by such a process, if the activity of the enzymes the microorganism is produced is long remains, it can be used in the reaction.
[0084]
　Wherein the treated product of the culture, to the extent that the recombinant microorganism does not lose activity pyridoxine oxidase and pyridoxamine synthase produced, refers to the product were any processing for culture of the recombinant microorganism. Such treatment, heat treatment, cooling treatment, mechanical disruption of the cells, sonication, freeze-thaw treatment, drying treatment, pressurization or depressurization treatment, osmotic pressure treatment, cell autolysis, surfactant treatment, enzymatic treatment (e.g. cell destruction), cell separation process, and the like processes comprising one or more selected from the group consisting of purification treatment and extraction process. For example, the cells of the recombinant microorganism is separated from the medium and the like, the isolated cells may be added to the reaction system. Such separation means such as filtration or centrifugation can be used. Alternatively, pyridoxine oxidase and pyridoxamine synthase, and if present was purified process for separating the hydrogen peroxide degrading enzyme from contaminants, a solution containing the enzyme obtained by the purification treatment in the reaction system it may be. Alternatively, cultures, the extract extracted with a mixed solvent of an organic solvent or an organic solvent and water, such as methanol or acetonitrile may be added to the reaction system. Such purification or extracts may be one that does not contain cells of a recombinant microorganism. Without cells of the microorganisms are present, if the residual activity of the enzyme, can be used in the reaction.
[0085]
　Crushing or dissolving treatment of the cells as described above, lysozyme treatment, freezing and thawing, by disrupting the cell membrane of a recombinant microorganism according to known methods, such as sonication, it can be performed.
[0086]
　Recombinant microorganism according to the present disclosure, a treated product of the culture or the recombinant microorganism or the culture of the recombinant microorganism, contact with pyridoxine or a salt thereof is preferably carried out under the following conditions.
　Contacting is preferably performed in a solution containing pyridoxine or a salt thereof as a substrate. The pH of the solution is not particularly limited as long as pyridoxine oxidase and the enzyme activity of pyridoxamine synthase is maintained more be preferably from 6.0 to 9.0, 7.0-8.5 preferable. The temperature of the solution also, pH of the solution, as long as pyridoxine oxidase and the enzyme activity of pyridoxamine synthase is maintained is not particularly limited, and more is possible preferably from 20 ~ 70 ° C., a 25 ° C. ~ 50 ° C. preferable.
　The medium of the solution, water or an aqueous medium, a mixture of organic solvents or water or an aqueous medium and an organic solvent is used. The aqueous medium, such as phosphate buffer, HEPES (N-2-hydroxyethylpiperazine--N- ethanesulfonic acid) buffer, tris [tris (hydroxymethyl) aminomethane] buffer such as hydrochloric acid buffer solution is used . The organic solvent may be any as long as it does not inhibit the reaction, such as acetone, ethyl acetate, dimethyl sulfoxide, xylene, methanol, ethanol, butanol and the like are used.
[0087]
　Contact with treated product of recombinant microorganisms, cultures or the recombinant microorganism or the culture of the recombinant microorganism, and pyridoxine or a salt thereof is carried out in the presence of oxygen. This is because the pyridoxine oxidase consume oxygen when oxidizing pyridoxine or a salt thereof. As the oxygen concentration in the reaction system, for example, the recombinant microorganism, a treated product of the culture or the recombinant microorganism or the culture of the recombinant microorganism, a solution containing pyridoxine or a salt thereof, open to the atmosphere Te, i.e. the reaction is carried out in contact with air, or 0.1 to 20% by volume of 0.5 to 10%, or to carry out the reaction in contact with gas containing 1-5% of oxygen it can. The dissolved oxygen content in such solution may be 0.1 mg ~ 13 mg oxygen /L,0.5mg~10mg oxygen / L or 1 mg ~ 8 mg oxygen / L,.
[0088]
　Incidentally, in the case where a gene the recombinant microorganism encodes a product capable of hydrogen peroxidase oxygen can be reduced oxygen concentration, or even under oxygen reduced or cut off criteria the supply of the reaction It can be performed. For example, it is possible to condition and reaction system was sealed and the reaction system the reaction is carried out at conditions nitrogen purge (nitrogen substituted).
[0089]
　Recombinant microorganism according to the present disclosure, and the set culture recombinant microorganism or the recombinant microorganism or the processed product of the culture, contact with pyridoxine or a salt thereof is preferably carried out in a shaking or stirring. For example, such contact can be carried out in solution. For example, the recombinant microorganism, to a solution containing the processed product of the culture or the recombinant microorganism or the culture of the recombinant microorganism be a pyridoxine or a salt thereof is added in the form or in solid form substrate solution good. Moreover, the recombinant microorganism, to a solution containing the processed product of the recombinant culture of a microorganism or the recombinant microorganism or the culture may be further added an amino acid that is consumed by pyridoxamine synthase. The amino acid, pyridoxine or its salt contained in the substrate solution with pyridoxine or a salt thereof, the recombinant microorganism, to a solution containing the processed product of the culture or the recombinant microorganism or the culture of the recombinant microorganism it may be added. Also, pyridoxine or a salt thereof contained in another of the substrate solution and the substrate solution, or in solid form, the recombinant microorganism, processed product of the culture or the recombinant microorganism or the culture of the recombinant microorganism it may be added to a solution containing.
　In the course the reaction is started or when the reaction to maintain the pH of the reaction solution in an appropriate range, or by adding an acid or alkali. Examples of additives which can be an alkali to the reaction solution, sodium lithium hydroxide, hydroxide, other alkali metal hydroxides such as potassium hydroxide, ammonium hydroxide, calcium hydroxide, dipotassium phosphate, disodium phosphate , potassium pyrophosphate, dissolved in water, such as ammonia, which humoral basic. Examples of possible additives to the reaction solution acid, hydrochloric acid, sulfuric acid, nitric acid, acetic acid, phosphoric acid, and the like.
[0090]
　The contacting, for example, may be performed in an air atmosphere, it may be carried out under partial deoxygenation atmosphere. Deoxygenated atmosphere substituted with an inert gas, reduced pressure can be achieved by combining boil and them. At least, substitution with inert gas, i.e., it is preferable to use an inert gas atmosphere. As the inert gas, e.g., nitrogen gas, can be mentioned helium gas, argon gas, carbon dioxide gas or the like, preferably nitrogen gas. However, when pyridoxine oxidase oxidizes pyridoxine because consumes oxygen, oxygen is preferably towards the atmosphere not removed.
[0091]
　In a preferred embodiment, it is used, the processing of recombinant microorganisms, cultures or the recombinant microorganism or the culture of the recombinant microorganism includes the pyridoxine oxidase and the pyridoxamine synthase. Therefore, by the contact, the are both present in the reaction solution acts on pyridoxine oxidase and the pyridoxamine synthase cooperatively produce with high productivity (manufacturing efficiency) pyridoxamine, or a salt thereof. Although not essential to contain treated or the recombinant microorganism in a state where the processing of the culture living of the recombinant microorganism, be continuously supplied to a substance participating in the reaction by metabolic from the viewpoint that it is, it preferably contains the recombinant microorganisms living state.
[0092]
　Recombinant microorganism, the recombinant microorganism addition timing of the culture or treated product of the recombinant microorganism or the culture is to may be added at once at the start of the reaction, or continuously by dividing into the reaction it may be added. Similarly, pyridoxamine as a raw material is also to may be added at once at the start of the reaction, it may be divided and added during the reaction or sequentially.
[0093]
　The reaction solution may contain an amino acid that is consumed in generating the pyridoxamine, or a salt thereof by pyridoxamine synthase. This amino acid is also to may be added at once at the start of the reaction, it may be added by dividing into the reaction or continuously. For example, pyridoxamine as pyridoxamine synthase - in the case of using pyruvic transaminase, the reaction solution may contain one or more of L- alanine and D- alanine. Further, pyridoxamine as pyridoxamine synthase - when using oxaloacetate transaminase or aspartate transaminase, the reaction solution L- aspartic acid, D- aspartic acid, L- glutamic acid, and of D- glutamate at least one comprise It can have. Incidentally, amino acids are can also be present as a salt by the surrounding environment, in this specification, it is also included described as an amino acid. For example L- glutamic acid and wherein the well L- glutamic acid not forming a salt, also encompasses the form a salt L- glutamic acid (e.g. L- sodium glutamate monohydrate). Counterion when forming the salts include sodium as the cation, such as potassium ion. Examples of the anion chloride ion, acetate ion and nitrate ion.
[0094]
　Pyridoxine or concentration in the reaction solution of its salts, for example, 0.1 mM ~ 500 mM, or 0.4 mM ~ 200 mM or 0.5 mM ~ 100 mM, or it may be a 0.8 mM ~ 50 mM,. When pyridoxine or concentration of a salt becomes high, there is a tendency that the enzymatic activity of pyridoxine oxidase is inhibited. Therefore, it is preferable that the concentration is not too excessively high in pyridoxine or reaction solution of a salt thereof. That is, a pyridoxine or concentration in the reaction solution of a salt, for example, may be controlled to maintain the concentration within the above range. For example, pyridoxamine or a salt thereof according to the present disclosure, the following (A) and may include either or both of (B):
(A) culturing the recombinant microorganism or the recombinant microorganism a solution containing an object or treated product of the recombinant microorganism or the culture can be added separately to pyridoxine or continuously added or several times a salt thereof,
(B) the recombinant microorganism or the recombinant microorganism in a solution containing the processed product of the culture or the recombinant microorganism or the culture, so that the molar concentration of the amino acid that is consumed by the pyridoxamine synthase is 1 times or more with respect to pyridoxine or molar concentration of the salt control to it.
　Examples of the amino acids, L- alanine, D- alanine, L- glutamic acid, D- glutamate, L- aspartic acid, D- aspartic acid.
[0095]
　For such control, for example, pyridoxine or its salt instead of adding the entire amount at once in the reaction solution may be added in several times. For the reaction to a concentration of pyridoxine is reduced by progress, by adding pyridoxine or a salt thereof at a time interval, it is possible to avoid that pyridoxine or concentration of a salt becomes excessively high. Such sequential addition, for example, at a time interval in the range of 0.5 hour to 10 hours, pyridoxine or a salt more than once, and preferably be added more than 3 times. Also, pyridoxine or a salt thereof, continuously or may be added to the reaction solution. In the case of continuous addition also by adjusting the addition rate, it is possible to avoid that pyridoxine or concentration of a salt becomes excessively high. For this adjustment, a pyridoxine or concentration of a salt in the reaction solution may be measured at a specific timing may be continuously monitored.
[0096]
　Further, the concentration of amino acids (amino acids consumed in generating the pyridoxamine, or a salt thereof by pyridoxamine synthase) is, for example, 0.1 mM ~ 2000 mM, or a 0.2 mM ~ 1000 mM, or 0.4 mM ~ 500mM, or 0.5mM ~ 400mM or may be a 1mM ~ 300mM or 2mM ~ 250mM,,. Manner of addition of amino acids is not particularly limited, may be added at once the total amount may be added in several times, it may be added continuously. Such sequential addition, for example, at a time interval in the range of 0.5 hour to 10 hours, amino acids more than once, and preferably be added more than 3 times. The timing of addition, when added in portions pyridoxine or a salt thereof into a plurality of times can be a timing simultaneously adding a pyridoxine or a salt thereof, may be added separately.
[0097]
　The concentration of amino acids (amino acids consumed in generating the pyridoxamine, or a salt thereof by pyridoxamine synthase) (molar concentration) is preferably maintained higher than pyridoxine or concentration of a salt (molar). The concentration of the amino acid to maintain higher than the concentration of pyridoxine or a salt thereof, the equilibrium of the reaction can be shifted to pyridoxamine or preferential direction into a salt thereof, further to enhance the production efficiency of the pyridoxamine, or a salt thereof be able to. Molar concentration of the amino acid is preferably 1.0 times or more than pyridoxine or molar concentration of the salt, more preferably 1.5 times or more, still more preferably 2.0 times or more , further preferably 5.0 times or more, but it may be 10.0 times.
[0098]
　The recombinant microorganism, a treated product of the culture or the recombinant microorganism or the culture of the recombinant microorganism, a method of contacting a pyridoxine or a salt thereof, wherein the recombinant microorganism, culture of said recombinant microorganism or adding processing of the recombinant microorganism or the culture solution containing pyridoxine or a salt thereof, a method that allows the reaction to proceed while stirring, the process of culture or the recombinant microorganism or the culture of the recombinant microorganism object was added to the solution containing pyridoxine or a salt thereof, a method that allows the reaction to proceed with shaking, the recombinant microorganism, processed product of the culture or the recombinant microorganism or the culture of the recombinant microorganism and pyridoxine or its after thoroughly mixing a salt in solution, and a method that allows the reaction to proceed on standing. Preferably, progress from the viewpoint of reaction efficiency, the recombinant microorganism, said set of modified culture of a microorganism or treated product of the recombinant microorganism or the culture was added to a solution containing pyridoxine or a salt thereof, while stirring the reaction and a method for.
[0099]
　There is no particular limitation on the reaction vessel can be used for the reaction. Preferably, the recombinant microorganism is added, it is possible stirred as a solution and is well mixed including processed and the pyridoxine or a salt thereof of the culture or the recombinant microorganism or the culture of the recombinant microorganism, pyridoxine it is preferably a reaction vessel having a temperature control function to keep within the optimum temperature range of oxidase and pyridoxamine synthase.
[0100]
　The recombinant microorganism, a treated product of the culture or the recombinant microorganism or the culture of the recombinant microorganism, pyridoxine or contact time with a salt thereof (reaction time), pyridoxine oxidase and the enzyme activity of pyridoxamine synthase it is not particularly limited as long as it is maintained, for example, 30 minutes to 100 hours, or may be two hours to 50 hours. Further, the reaction may be carried out batchwise, during the reaction, the substrate and the microorganism may be carried out in semi-batch be added separately to one or both of the culture or the treated product into a plurality of times, successively it may be carried out in the formula. For semi-batch or continuous type, new materials and the recombinant microorganism, since the operation such that one or both of the culture or the treated product is supplied is performed, the upper limit of the reaction time is particularly restricted not.
[0101]
　In the above method, by using a process of pyridoxine or recombinant microorganism or the culture or the recombinant microorganism or the culture according to the present disclosure a salt thereof as a raw material, as is the case in chemical synthesis methods without going through the complicated steps, it is possible to inexpensively, pyridoxamine for preparing a salt at a high production efficiency. Pyridoxamine or a salt thereof obtained by the method described above can be used, for example, in the manufacture of products using its physiological activity. For example, diabetes, atherosclerosis, chronic renal failure, prevention and treatment of diseases and schizophrenia caused by accumulation of AGE, such as Alzheimer's disease, are available in health food, applications such cosmetics.
[0102]
　Incidentally, the term "process" in the present disclosure is not only independent step, if even that can not be clearly distinguished from other processes is achieved intended purpose of the process, included in the term It is. The numerical range expressed by using "to" in the present specification indicates a range including numerical values described as the minimum and maximum values respectively before and after "to".
　In this disclosure, when referring to the amount of each component in the composition, if substances corresponding to the component in the composition there are plural, unless otherwise indicated, the plurality of substances present in the composition means of the total amount.
Example
[0103]
　The embodiments further by the following examples illustrating but the present disclosure is not intended to be the following embodiment thereof all. Also shows the component contents in the composition of Example "%" are by weight unless otherwise specified.
[0104]
　
　pyridoxine hydrochloride, pyridoxal hydrochloride, pyridoxamine dihydrochloride was determined by high performance liquid chromatography. These analytical conditions are as follows.

　Column: Shodex (registered trademark) Asahipak ODP-50 6E (Showa Denko KK)
　guard column: Shodex (registered trademark) Asahipak ODP-50G 6A (Showa Denko KK)
　Column temperature: 30 ° C.
　Pump flow rate: 1.0 ml / min
　eluent: 50 mM phosphate buffer (pH 2.0)
　detection: UV254nm
[0105]
　Creating Comparative Example 1 pno expression strain
　of Microbacterium Luteolum from pyridoxine 4-oxidase gene was synthesized by entrusting those codon optimized E. coli GenScript Corporation, to obtain a synthetic DNA having the nucleotide sequence of SEQ ID NO: 9. The synthetic DNA was amplified a DNA fragment containing the objective gene by PCR as a primer, an oligonucleotide having the nucleotide sequence of the oligonucleotide and SEQ ID NO: 13 having the nucleotide sequence of SEQ ID NO: 12 as a template. The amplified DNA fragment was treated with BamHI and SalI, and DNA fragment obtained, and treated with BamHI and SalI of pUC18 (manufactured by Takara), and ligated by using ligation high (Toyobo Co., Ltd.). Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in.
[0106]
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence of SEQ ID NO: 9. The resulting plasmid was named pUC18-pno. Here, pno is an abbreviation of pyridoxine-4-oxidase.
[0107]
　Example 1 PPAT-pno creation of expression strains
　Mesorhizobium loti MAFF303099 from pyridoxamine - the nucleotide sequence of the pyruvate aminotransferase gene was synthesized by entrusting those codon optimized E. coli GenScript Corp., having the nucleotide sequence of SEQ ID NO: 10 obtaining a synthetic DNA. The synthetic DNA was amplified a DNA fragment containing the objective gene by PCR as a primer, an oligonucleotide having the nucleotide sequence of the oligonucleotide and SEQ ID NO: 15 having the nucleotide sequence of SEQ ID NO: 14 as a template. The amplified DNA fragment was treated with EcoRI and BamHI, and a DNA fragment obtained, a treatment product of pUC18-pno prepared in Comparative Example 1 was treated with EcoRI and BamHI, ligated High (manufactured by Toyobo Co., Ltd.) It was ligated using. Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in.
[0108]
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence shown in SEQ ID NO: 10. The resulting plasmid was named pUC18-ppat-pno. Here, PPAT is pyridoxamine - an abbreviation for pyruvate aminotransferase.
[0109]
　Example 2 ppat-pno-kat Creating expression strain
　The nucleotide sequence of the Listeria seeligeri from catalase gene was synthesized by entrusting those codon optimized E. coli GenScript Corp., obtaining a synthetic DNA having the nucleotide sequence of SEQ ID NO: 11 It was. The synthetic DNA was digested with SalI and HindIII, and DNA fragments obtained, a treatment product of pUC18-ppat-pno created treated with SalI and HindIII in Example 1, the ligation high (Toyobo Co., Ltd.) It was ligated using. Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in. Here, kat is an abbreviation of catalase.
[0110]
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence shown in SEQ ID NO: 11. The resulting plasmid was named pUC18-ppat-pno-kat.
[0111]
　Example 3 aspC-pno creation of expression strains
　derived from Escherichia coli W3110 by PCR as a primer, an oligonucleotide having the nucleotide sequence of the oligonucleotide and SEQ ID NO: 17 having the nucleotide sequence of SEQ ID NO: 16 using genomic DNA of Eschrichia coli W3110 as a template pyridoxamine - oxaloacetic to amplify a DNA fragment containing the acid transaminase gene. The amplified DNA fragment was treated with EcoRI and BamHI, and a DNA fragment obtained, a treatment product of pUC18-pno prepared in Example 1 was treated with EcoRI and BamHI, ligated High (manufactured by Toyobo Co., Ltd.) It was ligated using. Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in.
[0112]
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence shown in SEQ ID NO: 8. The resulting plasmid was named pUC18-aspC-pno. Here, aspC the pyridoxamine - an abbreviation for oxaloacetate transaminase.
[0113]
　Example 4 aspC-pno-kat Creating expression strain
　those nucleotide sequences Listeria seeligeri derived catalase gene was codon-optimized E. coli synthesized commissioned GenScript Corp., obtaining a synthetic DNA having the nucleotide sequence of SEQ ID NO: 11 It was. The synthetic DNA was digested with SalI and HindIII, and DNA fragments obtained, a treatment product of pUC18-aspC-pno created treated with SalI and HindIII in Example 3, the ligation high (Toyobo Co., Ltd.) It was ligated using. Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in. Here, kat is an abbreviation of catalase.
[0114]
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence shown in SEQ ID NO: 11. The resulting plasmid was designated aspC-pno-kat.
[0115]
　Production of pyridoxamine for test 1 pyridoxine as a raw material
　pUC18, Comparative Example 1, and DH5α transformed with each plasmid prepared in Example 1, LB medium containing ampicillin 100 [mu] g / ml in baffled Erlenmeyer flasks 500 ml 100 ml to inoculate, for 24 hours with shaking cultured at 30 ℃. The culture was centrifuged 20 min at 8000 rpm, and the obtained cells as precipitate was suspended in water 800μl prepare cell suspension.
　Pyridoxine hydrochloride and L- alanine was dissolved in water to prepare a substrate solution containing pyridoxine hydrochloride (200 mM) and L- alanine (800 mM). The pH of the substrate solution, was adjusted to pH8.0 with sodium hydroxide. The substrate solution 100μl and the cell suspension 300μl were mixed in 2.0ml tubes, the tube 6 hours at 37 ° C. opens the lid was performed by shaking at 1,000 rpm. The reaction solution was partially collected, and analyzed by the analytical conditions shown above. The results obtained reaction yields in Table 1. It should be noted that the yields shown in Table 1, represents the molar amount of the ratio of pyridoxamine dihydrochloride salt obtained to the molar amount of pyridoxine hydrochloride in the substrate solution.
[0116]
[Table 1]

[0117]
　As shown in Table 1, both of the genes encoding genes and pyridoxamine synthase encoding pyridoxine oxidase when introduced from outside the cell, pyridoxamine dihydrochloride was produced at a high production efficiency. In the transformant containing the plasmid produced in Comparative Example 1, spontaneous reaction of the pyridoxal and amino group donor is presumed to have occurred. As mentioned above, this because the case of the spontaneous reaction of the formation of by-products, high pyridoxamine production efficiency is not obtained. In the results of Table 1 also, in the case of using a transformant containing a plasmid prepared in Comparative Example 1, high pyridoxamine productivity has not been obtained.
[0118]
　Test 2 pyridoxine production of pyridoxamine as a raw material
　of DH5α transformed with each plasmid prepared in pUC18 and Example 3, was inoculated into LB medium 100ml containing ampicillin 100 [mu] g / ml in baffled Erlenmeyer flasks 500 ml, 30 ℃ in it was shaking cultured for 24 hours. The culture was centrifuged 20 min at 8000 rpm, and the obtained cells as precipitate was suspended in water 800μl prepare cell suspension.
　Pyridoxine hydrochloride and L- sodium glutamate monohydrate was dissolved in water to prepare a substrate solution containing pyridoxine hydrochloride (200 mM) and L- sodium glutamate monohydrate (800 mM). The pH of the substrate solution, was adjusted to pH8.0 with sodium hydroxide. The substrate solution 300μl and the cell suspension 900μl were mixed in 2.0ml tubes, tubes for 24 hours at 37 ° C. opens the lid was performed by shaking at 1,000 rpm. The reaction solution was partially collected, and analyzed by the analytical conditions shown above. The results obtained reaction yield in Table 2. It should be noted that the yields shown in Table 2, represents the molar amount of the ratio of pyridoxamine dihydrochloride salt obtained to the molar amount of pyridoxine hydrochloride in the substrate solution.
[0119]
Table 2
[Table 2]

[0120]
　As shown in Table 2, when a gene encoding a gene and pyridoxamine synthase encoding pyridoxine oxidase is introduced from outside the cell, pyridoxamine dihydrochloride was produced at a high production efficiency.
[0121]
　Test 3 pyridoxine production of pyridoxamine as a raw material (oxygen Investigation of concentration)
　the DH5α transformed with the plasmid prepared in Example 1 (pUC18-ppat-pno) , ampicillin 100 [mu] g / ml in baffled Erlenmeyer flasks 500ml It was inoculated into LB medium 100ml, including, for 24 hours with shaking cultured at 30 ℃. The culture was centrifuged 20 min at 8000 rpm, and the obtained cells as precipitate was suspended in water 800μl prepare cell suspension.
　Pyridoxine hydrochloride and L- alanine was dissolved in water to prepare a substrate solution containing pyridoxine hydrochloride (200 mM) and L- alanine (800 mM). The pH of the substrate solution, was adjusted to pH8.0 with sodium hydroxide. The substrate solution 100μl and cell suspension 300μl were mixed in 2.0ml tubes. Reaction conditions open the lid of the tube, conditions capped tubes, and in conditions capped after nitrogen purge, for 24 hours at 37 ° C., was performed by shaking at 1,000 rpm. The reaction solution was partially collected, and analyzed by the analytical conditions shown above. The results obtained reaction yields in Table 3. It should be noted that the yields shown in Table 3, represents the molar amount of the ratio of pyridoxamine dihydrochloride salt obtained to the molar amount of pyridoxine hydrochloride in the substrate solution.
[0122]
Table 3
[Table 3]

[0123]
　As shown in Table 3, if the gene encoding the gene and pyridoxamine synthase encoding pyridoxine oxidase to produce pyridoxamine or a salt thereof with a recombinant microorganism which has been introduced from outside the cell, the oxygen supply is limited who conditions the supply of oxygen is not restricted than conditions was higher pyridoxamine or yield of a salt thereof.
[0124]
　Test 4 (Examination of effects of the hydrogen peroxide degrading enzyme) pyridoxine production of pyridoxamine as a raw material
　plasmid prepared in Example 1 (pUC18-ppat-pno) and plasmid prepared in Example 2 (pUC18-ppat-pno- the DH5α transformed with each kat), was inoculated into LB medium 100ml containing ampicillin 100 [mu] g / ml in baffled Erlenmeyer flasks 500 ml, for 24 hours by shaking culture at 30 ° C.. The culture was centrifuged 20 min at 8000 rpm, and the obtained cells as precipitate was suspended in water 800μl prepare cell suspension.
　Pyridoxine hydrochloride and L- alanine was dissolved in water to prepare a substrate solution containing pyridoxine hydrochloride (200 mM) and L- alanine (800 mM). The pH of the substrate solution, was adjusted to pH8.0 with sodium hydroxide. The substrate solution 100μl and cell suspension 300μl were mixed in 2.0ml tubes. The reaction for 24 hours at 37 ° C. under conditions capped tube, was carried out by shaking with 1,000 rpm. The reaction solution was partially collected, and analyzed by the analytical conditions shown above. when the generation amount of pyridoxamine dihydrochloride in the case of using the DH5α transformed with pUC18-ppat-pno was 1.0, pyridoxamine second case of using DH5α transformed with pUC18-ppat-pno-kat the amount of the relative value of the hydrochloride (PM generation speed ratio) shown in Table 4.
[0125]
Table 4
[Table 4]

[0126]
　As shown in Table 4, by having a gene recombinant microorganism according to the present disclosure further encodes a hydrogen peroxide-degrading enzyme, pyridoxamine or more improved production efficiency of a salt thereof under conditions of oxygen supply is limited it can be seen that.
[0127]
　Test 5 (Examination of effects of the hydrogen peroxide degrading enzyme) pyridoxine production of pyridoxamine as a raw material
　plasmid (pUC18-aspC-pno) prepared in Example 3 and plasmid prepared in Example 4 (pUC18-aspC-pno- the DH5α transformed with each kat), was inoculated into LB medium 100ml containing ampicillin 100 [mu] g / ml in baffled Erlenmeyer flasks 500 ml, for 24 hours by shaking culture at 30 ° C.. The culture was centrifuged 20 min at 8000 rpm, and the obtained cells as precipitate was suspended in water 800μl prepare cell suspension.
　Pyridoxine hydrochloride and L- sodium glutamate monohydrate was dissolved in water to prepare a substrate solution containing pyridoxine hydrochloride (200 mM) and L- sodium glutamate monohydrate (800 mM). The pH of the substrate solution, was adjusted to pH8.0 with sodium hydroxide. The substrate solution 100μl and cell suspension 300μl were mixed in 2.0ml tubes. The reaction for 24 hours at 37 ° C. under conditions capped tube, was carried out by shaking with 1,000 rpm. The reaction solution was partially collected, and analyzed by the analytical conditions shown above. when the generation amount of pyridoxamine dihydrochloride in the case of using the DH5α transformed with pUC18-aspC-pno was 1.0, pyridoxamine second case of using DH5α transformed with pUC18-aspC-pno-kat the amount of the relative value of the hydrochloride (PM generation speed ratio) shown in Table 5.
[0128]
Table 5
[Table 5]

[0129]
　As shown in Table 5, by having a gene recombinant microorganism according to the present disclosure further encodes a hydrogen peroxide-degrading enzyme, pyridoxamine or more improved production efficiency of a salt thereof under conditions of oxygen supply is limited it can be seen that.
[0130]
　Thus, according to the pyridoxamine, or a salt thereof according to the present disclosure, it has been shown to be capable of producing low cost pyridoxamine or a salt thereof with pyridoxine or high production efficiency from a salt thereof.
[0131]
　Test 6 (Reference): Examination of pyridoxine concentration pno
　inoculated with plasmid prepared in Comparative Example 1 (pUC18-pno) the DH5α transformed into LB medium 2ml containing ampicillin 100 [mu] g / ml in the test tube of 500 ml, 37 ℃ in it was shaking cultured for 24 hours. The culture was centrifuged 3 minutes at 13,000 rpm, and the obtained cells as precipitate was suspended in water 1,000μl prepare cell suspension.
　Pyridoxine hydrochloride salt was dissolved in water to prepare a substrate solution containing pyridoxine hydrochloride (500 mM). The substrate solution was adjusted to pH8.0 with sodium hydroxide. A predetermined amount of substrate solution, Tris-HCl (1M) 100μl , by mixing the cell suspension 100 [mu] l and water, the reaction solution 1,000μl concentration of pyridoxine hydrochloride are each concentration shown in Table 6 and 2. It was prepared in 0ml tube. The reaction is 24 hours at 37 ° C. Close the lid of the tube, was carried out by shaking with 1,000 rpm. The reaction solution was partially collected, and centrifuged, by measuring the absorbance at 415 nm, was quantitated generated pyridoxal hydrochloride. The PL generation amount in each pyridoxine hydrochloride concentration, indicating relative values pyridoxine hydrochloride concentration for pyridoxal hydrochloride in the case of 0.8mM the (PL generation amount ratio) in Table 6.
[0132]
Table 6
[Table 6]

[0133]
　As shown in Table 6, if the concentration of pyridoxine hydrochloride in the reaction solution was increased to more than a predetermined value, PL generation amount ratio decreased contrarily. That is, inhibition of enzymatic activity of pyridoxine oxidase was observed.
[0134]
　Test 7 pyridoxine manufacture of pyridoxamine as a raw material (all material total volume batch addition)
　of DH5α transformed with the plasmid prepared in Example 1 (pUC18-ppat-pno) , ampicillin in baffled Erlenmeyer flasks 2000 ml 100 [mu] g / ml was inoculated into LB medium 400ml containing, for 24 hours by shaking culture at 30 ° C.. The culture was centrifuged 20 min at 8000 rpm, and the obtained cells as a precipitate to prepare a cell suspension was suspended in water 10 ml.
　Pyridoxine hydrochloride salt was dissolved in water, adjusted to pH8.0 with sodium hydroxide and substrate solution containing pyridoxine hydrochloride (500 mM) of (1) was prepared. The L- alanine was dissolved in water, adjusted to pH8.0 with sodium hydroxide, was prepared L- alanine (1,000 mM) substrate solution containing a (2). Bacterial cell suspension 4.0ml to 100ml baffled Erlenmeyer flask, substrate solution (1) 4.0ml, was added substrate solution (2) 2.0 ml, for 24 hours at 37 ° C., with shaking at 200rpm reaction It was. The reaction solution was partially collected, and the concentration of pyridoxamine dihydrochloride was analyzed by the analytical conditions shown above. The results are shown in Figure 1. In FIGS. 1 to 3, PN is the concentration of pyridoxine hydrochloride, PL is the concentration of pyridoxal hydrochloride, PM represents the concentration of pyridoxamine dihydrochloride. In addition, hr represents the time of the unit of "time".
[0135]
　As shown in FIG. 1, it is added the total amount of high concentration of pyridoxine hydrochloride and L- alanine from the start, the production of pyridoxamine dihydrochloride proceeded.
[0136]
　Test 8 pyridoxine production of pyridoxamine as a raw material (pyridoxine added in several times into the hydrochloride and L- alanine both)
　the DH5α transformed with the plasmid prepared in Example 1 (pUC18-ppat-pno) , 2000ml baffle per inoculated into LB medium 400ml containing ampicillin 100 [mu] g / ml of Erlenmeyer flask for 24 hours by shaking culture at 30 ° C.. The culture was centrifuged 20 min at 8000 rpm, and the obtained cells as a precipitate to prepare a cell suspension was suspended in water 10 ml.
　Pyridoxine hydrochloride salt was dissolved in water, adjusted to pH8.0 with sodium hydroxide and substrate solution containing pyridoxine hydrochloride (500 mM) of (1) was prepared. The L- alanine was dissolved in water, adjusted to pH8.0 with sodium hydroxide, was prepared L- alanine (1,000 mM) substrate solution containing a (2). 100ml baffled Erlenmeyer flask bacterial cell suspension 4.0 ml, substrate solution (1) 0.5 ml and substrate solution (2) 0.25 ml was added, and reacted by shaking at 200rpm at 37 ° C.. Substrate solution to 1 hour after the start of the reaction (1) was further added 0.5ml and substrate solution 0.25 ml. Substrate solution (1) 0.5 ml and substrate solution (2) 0.25 ml was further added 7 hours after the reaction initiation. The reaction was continued until 24 hours after the start of the reaction. The reaction solution was partially collected, and the concentration of pyridoxamine dihydrochloride was analyzed by the analytical conditions shown above. The results are shown in Figure 2. In this test the pyridoxine hydrochloride and L- alanine are added each time both equimolar.
[0137]
　As shown in FIG. 2, even relatively large amounts of pyridoxine hydrochloride, in the case where the total amount from the beginning was added in several times without addition, the production efficiency of pyridoxamine dihydrochloride was further improved . This is because the addition divided into a plurality of times, maintained at a relatively low concentration levels of pyridoxine can be presumed to be because the activity of pyridoxine oxidase is more easily exhibited.
[0138]
　Production of pyridoxamine for the test 9 pyridoxine as a raw material (additive and L- alanine bulk added in portions pyridoxine hydrochloride plurality of times)
　the DH5α transformed with the plasmid prepared in Example 1 (pUC18-ppat-pno) , It was inoculated into LB medium 400ml containing ampicillin 100 [mu] g / ml in baffled Erlenmeyer flasks 2000 ml, for 24 hours by shaking culture at 30 ° C.. The culture was centrifuged 20 min at 8000 rpm, and the obtained cells as a precipitate to prepare a cell suspension was suspended in water 10 ml.
　Pyridoxine hydrochloride salt was dissolved in water, adjusted to pH8.0 with sodium hydroxide and substrate solution containing pyridoxine hydrochloride (500 mM) of (1) was prepared. The L- alanine was dissolved in water, adjusted to pH8.0 with sodium hydroxide, was prepared L- alanine (1,000 mM) substrate solution containing a (2). Bacterial cell suspension 4.0ml to 100ml baffled Erlenmeyer flask, substrate solution (1) 0.5 ml and substrate solution (2) 2.0 ml was added and reacted by shaking at 200rpm at 37 ° C.. The reaction starts after 1 hour after the substrate solution (1) 0.5 ml was further added, was added substrate solution (1) 0.5 ml a further 7 hours after start of the reaction. The reaction was continued until 24 hours after the start of the reaction. The reaction solution was partially collected, and the concentration of pyridoxamine dihydrochloride was analyzed by the analytical conditions shown above. The results are shown in Figure 3. Is always kept high found the following molar concentrations also L- alanine than the molar concentration of pyridoxine hydrochloride in this test.
[0139]
　As seen from comparison between FIGS. 2 and 3, when the concentration of L- alanine is maintained higher than the concentration of pyridoxine hydrochloride, be a relatively large amount of pyridoxine hydrochloride, pyridoxamine dihydrochloride efficiency was further improved productivity. This is due to the presence of high concentrations of L- alanine than pyridoxine hydrochloride, equilibrium of the reaction is speculated that not because became more advantageously pyridoxamine generation.
[0140]
　Reference Example 1 Creating ppat expression strains
　synthetic DNA having the nucleotide sequence of the nucleotide sequence of the pyruvate aminotransferase gene was synthesized by entrusting those codon optimized E. coli GenScript Corporation, SEQ ID NO: 10 - Mesorhizobium loti MAFF303099 from pyridoxamine It was obtained. The synthetic DNA was amplified a DNA fragment containing the objective gene by PCR as a primer, an oligonucleotide having the nucleotide sequence of the oligonucleotide and SEQ ID NO: 52 having the nucleotide sequence of SEQ ID NO: 51 as a template. The amplified DNA fragment was treated with EcoRI and BamHI, a DNA fragment obtained, and treated with EcoRI and BamHI of pUC18 (manufactured by Takara), and ligated by using ligation high (Toyobo Co., Ltd.). Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in.
[0141]
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence of SEQ ID NO: 10. The resulting plasmid was named pUC18-PPAT. Here, PPAT is pyridoxamine - an abbreviation for amino pyruvate transferase gene.
[0142]
　Reference Example 2. Creating PPAT-plr expression strain
　The nucleotide sequence of Saccharomyces cerevisiae derived from pyridoxal reductase gene was synthesized by entrusting those codon optimized E. coli GenScript Corporation, to obtain a synthetic DNA having the nucleotide sequence of SEQ ID NO: 53. The synthetic DNA was amplified a DNA fragment containing the objective gene by PCR as a primer, an oligonucleotide having the nucleotide sequence of the oligonucleotide and SEQ ID NO: 55 having the nucleotide sequence of SEQ ID NO: 54 as a template. The amplified DNA fragment was treated with SalI and HindIII, and DNA fragments obtained, and a process treated with SalI and HindIII pUC18-PPAT prepared in Reference Example 1, the ligation high (Toyobo Co., Ltd.) It was ligated using. Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in.
[0143]
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence of SEQ ID NO: 53. The resulting plasmid was named pUC18-ppat-plr. Here, plr is an abbreviation of pyridoxal reductase gene (corresponding to pyridoxine dehydrogenase).
[0144]
　Reference Example 3 PPAT-adh Creating expression strain
　Shewanella sp. AC10 nucleotide sequence from alanine dehydrogenase gene, it introduced a D198A mutation in the amino acid sequence encoding, and outsourced those codon-optimized E. coli GenScript Corporation synthesized Te to obtain a synthetic DNA having the nucleotide sequence of SEQ ID NO: 56. The synthetic DNA was amplified a DNA fragment containing the objective gene by PCR as a primer, an oligonucleotide having the nucleotide sequence of the oligonucleotide and SEQ ID NO: 58 having the nucleotide sequence of SEQ ID NO: 57 as a template. The amplified DNA fragment was treated with BamHI and SalI, and DNA fragment obtained and the treated product was treated with BamHI and SalI a pUC18-PPAT prepared in Reference Example 1, the ligation high (Toyobo Co., Ltd.) It was ligated using. Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. The transformant was cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in.
[0145]
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence of SEQ ID NO: 56. The resulting plasmid was named pUC18-ppat-adh. Here, adh is an abbreviation of the alanine dehydrogenase gene.
[0146]
　Reference Example 4 ppat-adh-plr Creating expression strain
　Shewanella sp. AC10 nucleotide sequence from alanine dehydrogenase gene, introduced a D198A mutation in the amino acid sequence encoding, and what has been codon-optimized E. coli GenScript Corporation entrusted to synthesize, to obtain a synthetic DNA having the nucleotide sequence of SEQ ID NO: 56. The synthetic DNA was amplified a DNA fragment containing the objective gene by PCR as a primer, an oligonucleotide having the nucleotide sequence of the oligonucleotide and SEQ ID NO: 58 having the nucleotide sequence of SEQ ID NO: 57 as a template. The amplified DNA fragment was treated with BamHI and SalI, and DNA fragment obtained and the treated product was treated with BamHI and SalI a pUC18-ppat-plr prepared in Reference Example 2, ligation high (Toyobo Co., Ltd. It was ligated using Ltd.). Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. The transformant was cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in.
[0147]
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence of SEQ ID NO: 56. The resulting plasmid was named pUC18-ppat-adh-plr.
[0148]
　Creating Reference Example 5 adh-plr expression plasmid
　of pUC18-ppat-plr prepared in Reference Example 2 was treated with SalI and HindIII, and recovered pyridoxal reductase gene (plr) fragment, pUC-ppat- prepared in Reference Example 3 adh was treated with BamHI and SalI, and a treated product with BamHI and HindIII of the collected alanine dehydrogenase gene (adh) fragment and pUC18, were ligated using ligation high (manufactured by Toyobo Co., Ltd.). Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus a plasmid was extracted from the resulting transformants, and the resulting plasmid was named pUC18-adh-plr.
[0149]
　Reference Example 6 Msppat-adh-plr expression plasmid created
　Mesorhizobium sp YR577 from pyridoxamine -. The nucleotide sequence of the pyruvate aminotransferase gene designed to codon-optimized E. coli, 'EcoRI-terminated 3' 5 end BamHI the gene introduced were synthesized by entrusting the eurofins Inc., to obtain a synthetic DNA having the nucleotide sequence of SEQ ID NO: 32. The synthetic DNA was treated with EcoRI / BamHI, and the DNA fragment obtained in Reference Example 5 fabricated pUC18-adh-plr ligation of a DNA fragment was treated with EcoRI and BamHI High (manufactured by Toyobo Co., Ltd.) using It was ligated Te. Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in.
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence of SEQ ID NO: 32. The resulting plasmid was named pUC18-Msppat-adh-plr.
[0150]
　Reference Example 7 Psppat-adh-plr create expression plasmids
　Pseudaminobacter salicylatoxidans from pyridoxamine - the nucleotide sequence of the pyruvate aminotransferase gene designed to codon-optimized E. coli, introducing a BamHI 'end EcoRI, 3' end 5 the gene was synthesized by entrusting the eurofins Inc., to obtain a synthetic DNA having the nucleotide sequence of SEQ ID NO: 33. The synthetic DNA was treated with EcoRI / BamHI, and the DNA fragment obtained in Reference Example 5 fabricated pUC18-adh-plr ligation of a DNA fragment was treated with EcoRI and BamHI High (manufactured by Toyobo Co., Ltd.) using It was ligated Te. Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in.
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence of SEQ ID NO: 33. The resulting plasmid was named pUC18-Psppat-adh-plr.
[0151]
　Reference Example 8 Blppat-adh-plr create expression plasmids
　Bauldia litoralis-derived pyridoxamine - the nucleotide sequence of the pyruvate aminotransferase gene designed to codon-optimized E. coli, introducing a BamHI 'end EcoRI, 3' end 5 gene was synthesized by entrusting the eurofins Inc. to obtain a synthetic DNA having the nucleotide sequence of SEQ ID NO: 34. The synthetic DNA was treated with EcoRI / BamHI, and the DNA fragment obtained in Reference Example 5 fabricated pUC18-adh-plr ligation of a DNA fragment was treated with EcoRI and BamHI High (manufactured by Toyobo Co., Ltd.) using It was ligated Te. Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in.
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence of SEQ ID NO: 34. The resulting plasmid was named pUC18-Blppat-adh-plr.
[0152]
　Reference Example 9 Ssppat-adh-plr create expression plasmids
　Skermanella stibiiresistens from pyridoxamine - the nucleotide sequence of the pyruvate aminotransferase gene designed to codon-optimized E. coli, introducing a BamHI 'end EcoRI, 3' end 5 gene was synthesized by entrusting the eurofins Inc. to obtain a synthetic DNA having the nucleotide sequence of SEQ ID NO: 35. The synthetic DNA was treated with EcoRI / BamHI, and the DNA fragment obtained in Reference Example 5 fabricated pUC18-adh-plr ligation of a DNA fragment was treated with EcoRI and BamHI High (manufactured by Toyobo Co., Ltd.) using It was ligated Te. Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in.
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence of SEQ ID NO: 35. The resulting plasmid was named pUC18-Ssppat-adh-plr.
[0153]
　Reference Example 10 Rsppat-adh-plr create expression plasmids
　Rhizobium sp AC44 / 96 from pyridoxamine -. The nucleotide sequence of the pyruvate aminotransferase gene designed to codon-optimized E. coli, 'EcoRI-terminated 3' 5 end entrusted the gene introduced a BamHI to eurofins Inc. synthesized to obtain a synthesized DNA having the nucleotide sequence of SEQ ID NO: 36. The synthetic DNA was treated with EcoRI / BamHI, and the DNA fragment obtained in Reference Example 5 fabricated pUC18-adh-plr ligation of a DNA fragment was treated with EcoRI and BamHI High (manufactured by Toyobo Co., Ltd.) using It was ligated Te. Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in.
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence of SEQ ID NO: 36. The resulting plasmid was named pUC18-Rsppat-adh-plr.
[0154]
　Reference Example 11 Etppat-adh-plr create expression plasmids
　Erwinia Toletana from pyridoxamine - the nucleotide sequence of the pyruvate aminotransferase gene designed to codon-optimized E. coli, introducing a BamHI 'end EcoRI, 3' end 5 gene was synthesized by entrusting the eurofins Inc. to obtain a synthetic DNA having the nucleotide sequence of SEQ ID NO: 37. The synthetic DNA was treated with EcoRI / BamHI, and the DNA fragment obtained in Reference Example 5 fabricated pUC18-adh-plr ligation of a DNA fragment was treated with EcoRI and BamHI High (manufactured by Toyobo Co., Ltd.) using It was ligated Te. Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in.
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence of SEQ ID NO: 37. The resulting plasmid was named pUC18-Etppat-adh-plr.
[0155]
　Reference Example 12 Hgppat-adh-plr create expression plasmids
　Herbiconiux ginsengi from pyridoxamine - the nucleotide sequence of the pyruvate aminotransferase gene designed to codon-optimized E. coli, introducing a BamHI 'end EcoRI, 3' end 5 gene was synthesized by entrusting the eurofins Inc. to obtain a synthetic DNA having the nucleotide sequence of SEQ ID NO: 38. The synthetic DNA was treated with EcoRI / BamHI, and the DNA fragment obtained in Reference Example 5 fabricated pUC18-adh-plr ligation of a DNA fragment was treated with EcoRI and BamHI High (manufactured by Toyobo Co., Ltd.) using It was ligated Te. Escherichia coli DH5α with this ligation product (manufactured by Toyobo Co., Ltd.) was transformed. Transformants were cultured in LB agar medium to select strains having the target plasmid from ampicillin resistant strains. Thus plasmids were extracted from the transformants obtained in.
　According Determination of normal nucleotide sequence, the nucleotide sequence of the introduced DNA fragment to the plasmid was confirmed to be the nucleotide sequence of SEQ ID NO: 38. The resulting plasmid was named pUC18-Hgppat-adh-plr.
[0156]
　Test 10 pyridoxine manufacture of pyridoxamine as a raw material
　of DH5α transformed with each plasmid prepared in Reference Example 6 to Example 12, was inoculated into LB medium 100ml containing ampicillin 100 [mu] g / ml in baffled Erlenmeyer flasks 500ml , for 24 hours with shaking cultured at 30 ℃. Take 40ml of culture was centrifuged 5 minutes at 5000 rpm, and the obtained cells as a precipitate by adjusting the cell suspension was suspended in water 0.9 ml.
　Pyridoxine hydrochloride and L- alanine was dissolved in water to prepare a substrate solution containing pyridoxine hydrochloride (500 mM) and L- alanine (500 mM). The pH of the substrate solution, was adjusted to pH8.0 with 25% aqueous ammonia. Substrate solution 400 [mu] l, water 500μl and cell suspension 100μl were mixed and reacted at 37 ° C.. The reaction solution was partially collected, and analyzed by analytical conditions described above. The results are shown in Table 7. Note that the pyridoxamine production shown in Table 7, expressed in relative amounts as 100 on the molar amount of pyridoxamine dihydrochloride ppat-adh-plr expression strain prepared in Reference Example 4 is produced.
[0157]
[Table 7]

[0158]
　As shown in Table 7, pyridoxamine - as a gene encoding pyruvate aminotransferase, Mesorhizobium loti MAFF303099 from pyridoxamine as that used in Reference Example 4 -. Instead of the aminotransferase gene pyruvate, Mesorhizobium sp YR577 from pyridoxamine - aminotransferase gene pyruvate, Pseudaminobacter salicylatoxidans from pyridoxamine - pyruvate aminotransferase gene, Bauldia litoralis-derived pyridoxamine - aminotransferase gene pyruvate, Skermanella stibiiresistens from pyridoxamine - pyruvate aminotransferase gene, Rhizobium sp AC44 / 96 from pyridoxamine -. pyruvate aminotransferase gene, Erwinia toletana from pyridoxamine - heritage pyruvate aminotransferase Child, or Herbiconiux ginsengi from pyridoxamine - by introducing the pyruvate aminotransferase gene, it was possible to produce pyridoxamine dihydrochloride from pyridoxine hydrochloride. Examples of these references are examples of using pyridoxine dehydrogenase, but are not used pyridoxine oxidase, these pyridoxamine - is an indication that pyruvate aminotransferase can be function as pyridoxamine synthase in the present disclosure .
[0159]
　Disclosure of 2017 May 12 Japanese Patent Application No. 2017-095572, filed on, the entirety of which is incorporated herein by reference.
　All documents described herein, patent applications, and technical standards, each individual publication, patent applications, and to the same extent as if it is marked specifically and individually incorporated by techniques standard reference, It incorporated by reference herein.

WE claims

Pyridoxine oxidase has a gene encoding the pyridoxamine synthase having an enzyme activity to synthesize pyridoxamine from encoding gene and pyridoxal and
　the pyridoxine gene encoding the thin oxidase and respectively extracellular gene encoding the pyridoxamine synthase inherent in or bacterial cells a gene has been introduced from is a gene whose expression is enhanced, the recombinant microorganism.
[Requested item 2]
　The pyridoxamine synthase, pyridoxamine - pyruvic transaminase, pyridoxamine - oxaloacetic transaminase, aspartate transaminase, or pyridoxamine phosphate transaminase recombinant microorganism of claim 1.
[Requested item 3]
　The pyridoxine oxidase is represented by enzyme number EC1.1.3.12, recombinant microorganism according to claim 1 or claim 2.
[Requested item 4]
　The pyridoxine gene encoding Shin oxidase is derived from Microbacterium luteolum, recombinant microorganism as claimed in any one of claims 1 to 3.
[Requested item 5]
　Gene encoding the pyridoxine oxidase,
　or having the nucleotide sequence of (a) SEQ ID NO: 5,
　a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to a nucleotide sequence of (b) SEQ ID NO: 5 and pyridoxine or has a nucleotide sequence encoding a protein having a thin oxidase activity,
　the protein or having a nucleotide sequence encoding, or having the amino acid sequence of (c) SEQ ID NO: 1
　to (d) the amino acid sequence of SEQ ID NO: 1 has an amino acid sequence having 80% or more sequence identity to Te, and pyridoxine having a nucleotide sequence encoding a protein, a having a thin oxidase activity,
　recombinant as claimed in any one of claims 1 to 4 microorganisms.
[Requested item 6]
　The pyridoxamine synthase partial amino acid sequence of the following (c), the partial amino acid sequence of (d), the partial amino acid sequence (e), the partial amino acid sequence (f), the partial amino acid sequence (g) and the partial amino acid sequence (h) out includes at least one, and having an enzymatic activity to synthesize pyridoxamine from pyridoxal recombinant microorganism as claimed in any one of claims 1 to 5. (C)
X 8 X 9 X 10 X 11 X 12 X 13 (SEQ ID NO: 　　39) (X 8 represents L, M, I or 　　V, X 9 represents H or 　　Q, X 10 is, G represents C or 　　a, X 11 represents E or 　　D, X 12 represents P or 　　a, X 13 represents V, I, L or a)

(D) X 14 X 15 TPSGTX 16 X 17 (SEQ ID NO:
　　40) (X 14 represents H or
　　S, X 15 represents D or
　　E, X 16 represents I, V or
　　L, X 17 represents N or
T) (e) X 18 DX 19 VSX 20 X 21 (SEQ ID NO: 　　41) (X 18 is, V, represents I or 　　a, X 19 is, a, represents T or S, 　　X 20 represents S, a or 　　G, X

21 is F, W, or an
V) (f) X 22 X 23 X 24 KCX 25 GX 26 X 27 P (SEQ ID NO: 　　42) (X 22 represents G or 　　S, X 23 is, P, S or 　　a, X 24 represents N, G, S, a or 　　Q, X 25 represents L or 　　M, X 26 represents a, S, C or 　　G, X 27 is P, T, represents an S or a) (g) X 28 X 29 X 30 X 31

SX 32 GX 33 X 34 (SEQ ID NO:
43) (X 28 represents G or
　　D, X 29 represents V or
　　I, X 30 is V, T, A, S, M, I or L
　　represents, X 31 represents F, M, L, I or
　　V, X 32 represents S, G, a, T, I, L or
　　H, X 33 represents R, M or Q,
　　X 34 is, G, R, a, D , H or
K) (h) X 35 X 36 RX 37 X 38 HMGX 39 X 40A (SEQ ID NO:
　　44) (X 35 represents L or
　　V, X 36 represents T, I, V or
　　L, X 37 represents I, V or
　　L, X 38 is, G or S the
　　stands, X 39 is, P, represents a or
　　R, X 40 represents T, V or S)
[Requested item 7]
　The pyridoxamine synthase is expressed in enzyme number EC2.6.1.30, recombinant microorganism as claimed in any one of claims 1 to 6.
[Requested item 8]
　The pyridoxamine gene encoding synthase is derived from Mesorhizobium loti, recombinant microorganism as claimed in any one of claims 1 to 7.
[Requested item 9]
　Gene encoding the pyridoxamine synthase,
　(a) or having any of the nucleotide sequences of SEQ ID NO: 6 and SEQ ID NO: 25 to SEQ ID NO: 31,
　18 nt to 3 'terminus in the nucleotide sequence of SEQ ID NO: 10 or with a region or regions of up to 18 nt ~ 3 'terminus of any of the nucleotide sequence of SEQ ID NO: 32 to SEQ ID NO: 38 up,
　(b) SEQ ID NO: 6 and of SEQ ID NO: 25 to SEQ ID NO: 31 at 18 nt ~ 3 'any of the nucleotide sequences of the region to end or SEQ ID NO: 32 to SEQ ID NO: 38 in the nucleotide sequence of the DNA, or SEQ ID NO: 10 having a nucleotide sequence complementary to any of the nucleotide sequences 18 nt ~ 3 'complementary to the region to end A DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence, and either has a nucleotide sequence encoding a protein with the enzymatic activity to synthesize pyridoxamine from pyridoxal,
　(c) SEQ ID NO: 2 and SEQ ID NO: 18 to SEQ ID NO: or has a nucleotide sequence encoding a protein having any of the amino acid sequence of the 24, or
　(d) SEQ ID NO: 2 and SEQ ID NO: 18 to SEQ ID NO: 24 amino acid sequence 80% or more with respect to at least one of has an amino acid sequence having sequence identity, and has a nucleotide sequence encoding a protein, a having an enzyme activity to synthesize pyridoxamine from pyridoxal,
　recombinant as claimed in any one of claims 1 to 8 microorganisms.
[Requested item 10]
　The pyridoxamine synthase is expressed in enzyme number EC2.6.1.31 or EC2.6.1.1, recombinant microorganism as claimed in any one of claims 1 to 5.
[Requested item 11]
　The pyridoxamine gene encoding synthase is derived from Escherichia coli, recombinant microorganism as claimed in any one of claims 1 to 5 and claim 10.
[Requested item 12]
　The gene encoding the pyridoxamine synthase,
　(a) or having the nucleotide sequence of SEQ ID NO: 8,
　a DNA which hybridizes with the DNA under stringent conditions with a nucleotide sequence complementary to a nucleotide sequence of (b) SEQ ID NO: 8 and, and or has a nucleotide sequence encoding a protein with the enzymatic activity to synthesize pyridoxamine from pyridoxal,
　or has a nucleotide sequence encoding a protein having the amino acid sequence of (c) SEQ ID NO: 4, or
　(d) SEQ ID NO: has an amino acid sequence having 80% or more sequence identity to the amino acid sequence of 4, and having a nucleotide sequence encoding a protein, a having an enzyme activity to synthesize pyridoxamine from pyridoxal,
　claims 1 to 5 and claims 10 to claim 11 Chiizure or recombinant microorganism according to one paragraph.
[Requested item 13]
　Further comprising a gene encoding the hydrogen peroxide degrading enzyme having the enzymatic activity of producing oxygen from hydrogen peroxide, recombinant microorganism as claimed in any one of claims 1 to 11.
[Requested item 14]
　The gene encoding the hydrogen peroxide degrading enzyme, but inherent in or bacterial cells a gene has been introduced from the outside of cells is a gene whose expression is enhanced recombinant microorganism of claim 13.
[Requested item 15]
　Wherein the hydrogen peroxide decomposing enzyme is expressed in enzyme number EC 1.11.1.6, recombinant microorganism of claim 13 or 14.
[Requested item 16]
　Gene encoding the hydrogen peroxide degrading enzyme,
　(a) or having the nucleotide sequence of SEQ ID NO: 7,
　hybridized with DNA under stringent conditions with a nucleotide sequence complementary to a nucleotide sequence of (b) SEQ ID NO: 7 soybean, and either has the enzymatic activity to produce oxygen from hydrogen peroxide,
　a protein or a nucleotide sequence encoding or comprising the amino acid sequence of (c) SEQ ID NO: 3
　in (d) of the amino acid sequence of SEQ ID NO: 3 having a nucleotide sequence encoding a protein, the which has the enzymatic activity having an amino acid sequence having 80% or more sequence identity, and producing oxygen from hydrogen peroxide for,
　any one of claims 13 to claim 15 the recombinant microorganism according to one paragraph or.
[Requested item 17]
　A recombinant E. coli, the recombinant microorganism as claimed in any one of claims 1 to 16.
[Requested item 18]
　And treated product of the culture or the recombinant microorganism or the culture of a recombinant microorganism or the recombinant microorganism according to any one of claims 1 to 17 is brought into contact with pyridoxine or a salt thereof, to produce pyridoxamine or a salt thereof in the presence of oxygen, pyridoxamine or a salt thereof.
[Requested item 19]
　Processed product of said recombinant microorganism or culture of the recombinant microorganism or the recombinant microorganism or the culture, including the pyridoxine oxidase and the pyridoxamine synthase process of claim 18.
[Requested item 20]
　Processed product of said recombinant microorganism or culture of the recombinant microorganism or the recombinant microorganism or the culture, further comprising a hydrogen peroxide-degrading enzyme, the production method according to claim 19.
[Requested item 21]
　Processed product of said recombinant microorganism or the culture, heat treatment, cooling treatment, mechanical disruption of the cells, sonication, freeze-thaw treatment, drying treatment, pressurization or depressurization treatment, osmotic pressure treatment, cell autolysis , surfactant treatment, enzyme treatment, a cell separation process, treated by the process comprising one or more selected from the group consisting of purification treatment and extraction process, any one of claims 18 to claim 20 the method according to.
[Requested item 22]
　Including either or both of the following (A) and (B), claims 18 to process according to any one of claims 21:
(A) culturing the recombinant microorganism or the recombinant microorganism a solution containing an object or treated product of the recombinant microorganism or the culture can be added separately to pyridoxine or continuously added or several times a salt thereof,
(B) the recombinant microorganism or the recombinant microorganism in a solution containing the processed product of the culture or the recombinant microorganism or the culture, so that the molar concentration of the amino acid that is consumed by the pyridoxamine synthase is 1 times or more with respect to pyridoxine or molar concentration of the salt control to it.
[Requested item 23]
　The pyridoxamine acid consumed by synthase L- alanine, D- alanine, an L- glutamic acid or D- glutamate The process of claim 22.