JP2003125784A - New tyrosine decarboxylase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect a bacterium having tyrosine decarboxylase activity by a PCR method using a PCR primer designed on the basis of a base sequence of the enzyme gene of this invention. SOLUTION: This new tyrosine decarboxylase TDCTH acts on tyrosine to form tyramine, is most stable at pH 5.5 and has about 70.2 kDa (SDS-PAGE). The enzyme can be separated and collected from a culture of Tetragenococcus halophilus being a halophilic lactic acid bacterium, etc. The amino acid sequence of the enzyme and the base sequence of a gene encoding the amino acid sequence are identified.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the halophilic lactic acid bacterium Tetragenococcus halophilus ( Tetrageno).
coccus halophilus ) -derived novel tyrosine decarboxylase and tyrosine decarboxylase gene, and
The present invention relates to a method for detecting a microorganism having tyrosine decarboxylation activity by the PCR method using a PCR primer designed based on the nucleotide sequence of the tyrosine decarboxylase gene.

[0002]

2. Description of the Related Art Tyramine is a substance contained in various foods, and as a food with a particularly high content of tyramine, fermented and brewed foods such as cheese, yogurt, beer and wine, as well as citrus fruits, broad beans and chocolate are known. Has been. Tyramine has a stimulatory effect on the sympathetic nerve in the human body, but normally, the amount of tyramine contained in food is very small, and it is rapidly metabolized to glycolic acid by the action of monoamine oxidase in the intestine, liver and kidney. Since it is excreted in urine, there is no concern that tyramine will have a harmful effect on the human body. However, if a large amount of food with a high content of tyramine is ingested, or if the activity of monoamine oxidase is low due to differences in genetic traits, or if a person taking a monoamine oxidase inhibitor to treat depression etc. When a food with a high content is ingested, the metabolism of tyramine is not carried out normally, so that symptoms such as increased blood pressure, sweating, and headache may occur due to the sympathetic nerve stimulating action of tyramine. Therefore, the control of the tyramine content in foods is one of the important issues regarding the quality control of foods.

On the other hand, tyramine is widely produced not only in animals and plants but also in the organisms of microorganisms such as bacteria. Especially in the group of lactic acid bacteria which are bacteria involved in fermentation and brewing, it is known that there are strains capable of producing tyramine. Has been. Therefore, in order to control the content of tyramine in food, it is necessary to detect and detect a strain having a tyramine-producing ability from the group of lactic acid bacteria that survive in the food or the environment. Further, tyramine is known to be produced from tyrosine in cells by a decarboxylation reaction by the action of tyrosine decarboxylase. Therefore, the detection of a strain having a tyramine-producing ability is carried out by examining the tyrosine decarboxylation activity of bacteria and detecting the strain positive for the activity.

[0004] Conventionally, the detection of a strain having tyrosine decarboxylation activity in foods or environments in which tyramine non-producing bacteria are mixed has been performed by culturing a sample bacterium in a tyrosine-containing agar plate medium (Japanese Patent No. 1611644), It has been carried out by a method of detecting a strain in which a halo-forming colony appeared by decomposing tyrosine in the medium. However, in this method, there are strains that do not form a clear halo even if they have tyrosine decarboxylation activity, and thus there is a possibility that even if a strain having a tyramine-producing ability exists, it will not be detected. Alternatively, if there is a halo-forming strain in the surroundings even if it is not a halo-forming strain, there is a problem regarding reliability such that it may be mistaken for a halo-forming strain. Furthermore, since it takes about one week for culturing, it takes a long time for the inspection, which is also a problem.

[0005]

Therefore, the inventor of the present invention
We decided to try a method to detect a strain having tyramine-producing ability by testing for the presence of the tyrosine decarboxylase gene. Gene detection tests are widely performed by those skilled in the art, and the most widely used method is PCR.
It is by law. The method is to design an oligonucleotide PCR primer having a specificity to the gene designed based on the nucleotide sequence of the gene to be detected,
This is a method of detecting the gene in a sample by performing a PCR reaction using the PCR primer using the chromosomal DNA extracted from the sample as a template to obtain an amplified DNA fragment as a reaction product. However, up to now, no example has been known using this method for detecting the tyrosine decarboxylase gene from microorganisms such as lactic acid bacteria that survive in food and the environment.

In order to carry out a test for detecting a tyrosine decarboxylase gene by the PCR method, it is first necessary to know the base sequence of the tyrosine decarboxylase gene. The nucleotide sequence of the tyrosine decarboxylase gene reported so far, and the amino acid sequence of tyrosine decarboxylase, those derived from plants,
For example, the base sequence of the tyrosine decarboxylase gene of parsley and the amino acid sequence of tyrosine decarboxylase (Kawallec
k, P et al, J. Biol Chem., 268 (3): 2189-2194 (199
3))) etc. are known. However, there have been no reports up to now on the nucleotide sequence of the tyrosine decarboxylase gene of bacteria, particularly the lactic acid bacterium group, and the amino acid sequence of tyrosine decarboxylase.

[0007]

Therefore, as a result of earnest research, the present inventor has purified tyrosine decarboxylase from halophilic lactic acid bacterium Tetragenococcus halophilus, which is one of the bacteria belonging to the lactic acid bacterium group, and characterized it. Revealed.
To date, tyrosine decarboxylase of lactic acid bacteria has been used in Streptococcus faecalis ( Streptococcu).
s faecalis ) (Borresen, T. et
al., Biochimica et Biophysica Acta, 993: 108-115 (1
989)) and Lactococcus lactis ( Lactococcus lactis)
subsp. lactis ) (Nishida, JR
akuno Gakuen Univ., 23 (1); 77-78 (1998)), but the enzyme of the present invention is a novel one different from these. Further, the present inventor has clarified for the first time the nucleotide sequence encoding the amino acid sequence of the tyrosine decarboxylase of the present invention and the amino acid sequence of the tyrosine decarboxylase. These base sequences and amino acid sequences were also novel so far unknown. The property and amino acid sequence of the tyrosine decarboxylase of the present invention and the nucleotide sequence of the gene are not only for detecting a strain having tyrosine decarboxylative activity but also for suppressing the production of tyramine in foods by various methods and means. Is also important and is extremely useful for controlling the tyramine content in foods.

Further, the present inventor designed and produced P based on the nucleotide sequence of the tyrosine decarboxylase gene of the present invention.
By the PCR method using a CR primer, it has succeeded in detecting a microorganism including a lactic acid bacterium group having a DNA encoding a tyrosine decarboxylase gene and having a tyrosine decarboxylation activity. In particular, in Tetragenococcus halophilus, the strain in which the tyrosine decarboxylase gene of the present invention was detected was completed by confirming that the strain has tyrosine decarboxylation activity, that is, a tyramine-producing bacterium. Let Hereinafter, the present invention will be described in detail.

The tyrosine decarboxylase according to the present invention has the following properties: (1) Action: It acts on tyrosine to produce tyramine. (2) Optimum pH: When using Mr. Mcllvaine's buffer containing 0.4 mM pyridoxal phosphate 5.5 (17)
% NaCl present). (3) pH stability: most stable at pH 5.5 (17% Na
In the presence of Cl). (4) Stabilization: Relatively stable in the presence of 1.0 M ammonium sulfate. Very stable in the presence of pyridoxal phosphate and 2-mercaptoethanol. (5) Molecular weight: The molecular weight measured by SDS polyacrylamide gel electrophoresis is about 70.2 KDa. (6) Produced by a microorganism belonging to the genus Tetragenococcus, for example, Tetragenococcus halophilus.

The tyrosine decarboxylase according to the present invention has the above-mentioned properties, but no such enzyme has been known so far, and Streptococcus faecalis and Lactococcus lactis subs reported in the past have been reported. It is clearly different from the tyrosine decarboxylase derived from Species lactis, and the present inventors identified this enzyme as a novel enzyme and named it tyrosine decarboxylase TDCTH.

All the enzymes having the above-mentioned properties are included in the tyrosine decarboxylase TDCTH of the present invention. The present inventor further found that the amino acid sequence and the nucleotide sequence of the gene DNA encoding the same. We also proceeded with research from the perspective of, and finally succeeded in determining each sequence. As an example, the amino acid sequence of the tyrosine decarboxylase of the present invention is shown in SEQ ID NO: 1 of the sequence listing, and the base sequence of the tyrosine decarboxylase gene of the present invention is shown in SEQ ID NO: 2. Those having a homology of 40% or more with the sequence have not been reported and are novel.

Further, even if a protein consisting of an amino acid sequence in which a part of amino acids in the above-mentioned amino acid sequence is substituted, deleted, inserted or added, as long as it has tyrosine decarboxylizing activity, all of the proteins are bound by the tyrosine decarboxylase of the present invention. included.

Furthermore, even in the case of a DNA consisting of a nucleotide sequence encoding a protein consisting of a partial substitution, deletion, insertion or addition of the amino acid sequence, the protein encoded by the nucleotide sequence is tyrosine. As long as it has decarboxylation activity, all of the DNA is included in the tyrosine decarboxylase gene of the present invention.

Here, the term "part" varies depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but is 60% or more, preferably 80% or more, with respect to the entire amino acid sequence of the tyrosine decarboxylase. More preferably 9
It means a case where the homology is 0% or more, and more preferably 95% or more.

Furthermore, a DNA that hybridizes with the above-mentioned tyrosine decarboxylase gene under stringent conditions and encodes a protein having tyrosine decarboxylative activity.
Is also included in the tyrosine decarboxylase gene of the present invention. The stringent condition means that the gene is hybridized with any DNA having a homology of 60% or more, preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, at the base level of DNA. Means possible conditions. Or sodium concentration is 15 ~
It is 150 mM, preferably 15 to 30 mM, and the conditions include hybridization at a temperature of 37 to 80 ° C, preferably 50 to 65 ° C.

As described above, all the enzymes having the above-mentioned properties are included in the tyrosine decarboxylase TDCTH of the present invention, and if the amino acid sequence has the properties of the TDCTH, a part thereof is replaced, Even if it is deleted, inserted or added (including a gene having a corresponding nucleotide sequence), it is included in the present invention, and as one example,
Sequence number 1 (FIG. 10) (for the base sequence, sequence number 2: FIG. 11, 12, 13) can be mentioned.

In the above array, Xaa is, for example, A
la, Val, Cys, Trp, Phe, Leu, Il
e, y is c or t, k is g or t, r is a or g, or a deletion. More specifically, SEQ ID NO: 3
Examples of the amino acid sequence (Figs. 14 and 15) and the corresponding nucleotide sequence (SEQ ID NO: 4: Figs. 16, 17, and 18). In addition, this sequence is Tetragenococcus halophilus HSCC 809 strain (FERM P-1850
Although it is derived from 3), strains other than the above also produce this enzyme. However, in that case, although the sequences may differ by several (eg, SEQ ID NO: 1), all have the present enzyme activity, and both are included in the present invention.

The tyrosine decarboxylase of the present invention is Tetragenococcus halophilus.
ccus halophilus ) HSCC 80
9, HSCC 921, HSCC 1010 and other strains. HSCC 809 among these strains
The mycological properties and the results of phylogenetic analysis are shown below. (1) Morphological properties (a) Cell morphology: 4 co-located cocci. (B) Gram stain: positive. (C) Presence or absence of motility: None. (D) Presence or absence of spores: None.

(2) Physiological properties (a) Attitude toward oxygen: facultative anaerobic. (B) Lactic acid production rate: 50% or more based on consumed sugar. (C) Catalase: negative. (D) Fermentation format: homozygous. (E) Growth temperature: Does not grow at optimum temperatures of 25 to 30 ° C, 10 ° C and 45 ° C. (F) Sugar fermentability: Fermentation of melibiose, sorbitol, galactose, mannose, maltose, trehalose, inulin, fructose and glucose. (G) Stereoisomerism of produced lactic acid: L + DL. (H) Salt tolerance: Grows with NaCl 18%. (I) Growth pH: It grows at pH 9.0 and does not grow at pH 5.0. It grows at pH during this period. (J) Litmus milk: acidification; negative, decolorization; negative, coagulation; negative, liquefaction; negative. (K) Production of dextran from sucrose: negative. (L) Arginine degradation: negative.

(3) Systematic classification (a) GC content of DNA: 36.6%. (B) Phylogenetic analysis based on the nucleotide sequence of 16S rRNA:
Tetragenococcus halophilu
s (Tetragenococcus halophilus)

Based on the above-mentioned mycological properties and phylogenetic analysis results, the HSCC 809 strain was identified as Tetragenococcus h.
was identified as a strain belonging to Alofilius ). Furthermore, by the same analysis, HSCC 921, HSC
C 1010 is also Tetragenococcus halophilus
us ). Therefore, each of these strains was treated with Tetragenococcus halophi.
lus ) HSCC809, Tetragenococcus halo
philus ) HSCC 921, Tetragenococcus tetragenococcus
It was named halophilus) HSCC1010.
Among these strains, Tetragenococcus halophi
lus) HSCC 809 of the National Institute of Advanced Industrial Science and Technology Patent Organism to the depositary center FERM P-18
Deposited as 503. Some of these strains may differ in amino acid and / or base sequence, but both strains may also be referred to as tyrosine decarboxylase TDCTH (hereinafter simply referred to as tyrosine decarboxylase, which has the same enzymatic activity). Are also included).

Next, the method for producing the tyrosine decarboxylase of the present invention will be described. The tyrosine decarboxylase of the present invention is a tetragenococcus halophilus capable of producing the tyrosine decarboxylase of the present invention, for example, Tetragenococcus halophilus HSCC809, HSCC 92.
It can be produced by culturing a strain such as 1, HSCC 1010 and collecting the tyrosine decarboxylase from the culture. In addition, examples of the culture method and culture conditions of Tetragenococcus halophilus having the enzyme-producing ability of the present invention will be illustrated below, but are not particularly limited as long as they provide an environment in which the growth and the production of the enzyme of the present invention are possible. Not done.

The medium used for culturing the strain is a carbon source,
Any synthetic medium, natural medium, or semi-synthetic medium may be used as long as it appropriately contains a nitrogen source, an inorganic substance, and other nutrients. Here, the carbon source may be any assimilable carbon compound, such as glucose, fructose,
Carbohydrates such as sucrose, dextrin and starch, organic acids such as acetic acid and propionic acid, alcohols such as glycerol, ethanol and propanol, animal oil and molasses are used. As the nitrogen source of the medium, any nitrogen source can be used as long as it expresses the tyrosine decarboxylase of the present invention. For example, yeast extract, polypeptone, meat extract, corn steep liquor, soybean powder, amino acid, ammonium sulfate, ammonium nitrate and the like can be used. In particular, since the tyrosine decarboxylase of the present invention is derived from tyrosine, it is preferable to add tyrosine or a tyrosine salt in addition to other nitrogen sources. Further, as inorganic salts, salts of cations such as potassium, sodium, calcium, magnesium, manganese, cobalt, zinc, iron and molybdenum, and anions such as sulfuric acid, phosphoric acid, hydrochloric acid and nitric acid are used. In addition, especially Tetragenococcus halophilus is halophilic, so 2-17% Na is added to the medium.
It is preferable to add Cl. Furthermore, if necessary, an inorganic or organic trace component, an antifoaming agent, etc. can be appropriately added to the medium. These nutrient sources can be used alone or in combination.

For the culture of Tetragenococcus halophilus, a liquid culture method is usually preferable, and a culture under static or anaerobic conditions is preferable. Culture temperature is 25 to 37
C., preferably around 30.degree. The pH during culture is preferably pH 5.5 to 9. The culture of Tetragenococcus halophilus is usually carried out in three stages of seed culture, pre-culture and main culture, and the respective culture times vary depending on conditions such as the concentration of NaCl in the medium, but usually 1 It is about 10 days. By the above culture, the present enzyme is produced and accumulated in the culture.

In order to collect the present enzyme from this culture, ordinary enzyme collecting means can be used. Since the present enzyme is an enzyme mainly present in the microbial cells, it is preferable to separate the microbial cells from the culture by an operation such as filtration or centrifugation and collect the present enzyme from the microbial cells. In this case, the bacterial cells can be used as they are, but for example, a method of destroying the bacterial cells using various mechanical disruption means such as a microfluidizer, an ultrasonic crusher, a French press, and a dynamil, cell wall lysis such as lysozyme. A method of lysing a cell wall of an enzyme using an enzyme, a method of extracting an enzyme from a cell using a surfactant such as Triton X-100, etc. can be used alone or in combination. Then, the insoluble matter is removed by filtration or centrifugation to obtain a crude enzyme solution of the present enzyme.

Isolation and purification of the present enzyme from the thus obtained crude enzyme solution can be carried out by a conventional method. For example, ammonium sulfate salting-out method, organic solvent precipitation method, adsorption treatment method using ion exchanger, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, adsorption chromatography, affinity chromatography, electrophoresis method, etc., alone or as appropriate. It is possible to use in combination.

The method of detecting the DNA encoding the tyrosine decarboxylase gene and the microorganism having the tyrosine decarboxylation activity by the PCR method of the present invention will be described below.

In the gene detection method by the PCR method of the present invention, the sample is not particularly limited as long as it can extract DNA, but it is usually a microorganism that survives in food or environment. Furthermore, when a microorganism that survives in food or the environment is used as a sample, the DNA used as a template for the PCR method should be extracted from the microorganism after once culturing the microorganism from the food or the environment. DNA obtained by the above method or DNA obtained by directly extracting from microorganisms collected from food or environment without culturing. Furthermore, in the case of isolating and culturing the microorganisms and extracting the DNA, bacteria are preferable as the microorganisms to be a sample, more preferable are bacteria belonging to the lactic acid bacteria group, and particularly preferable are tetragenococcus.
It is Halophilus.

The PCR primer used in this detection method is designed based on a continuous partial sequence of the base sequence of the tyrosine decarboxylase gene. Examples of the nucleotide sequence of the tyrosine decarboxylase gene include at least one of the following nucleotide sequences. (A) A base sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1. (B) A base sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1 in which some amino acid residues are substituted, deleted, inserted or added. (C) The base sequence shown in SEQ ID NO: 2. (D) A base sequence of a gene DNA that hybridizes with the DNA consisting of the base sequence shown in SEQ ID NO: 2 under stringent conditions and encodes a protein having tyrosine decarboxylase activity.

It is preferably designed, for example, based on a continuous partial sequence of the base sequence shown in SEQ ID NO: 2 in the sequence listing.
The length of the continuous partial sequence is 5 bases or more, usually 5 to 60 bases.
It is a base, preferably 15 to 30 bases. The PCR primer thus designed is artificially synthesized as an oligonucleotide by a DNA synthesizer.

In this detection method, the tyrosine decarboxylase gene is detected by the following method, for example. First, chromosomal DNA is extracted from the sample, and this is used as template D
NA. Examples of the method for extracting the DNA include the standard Marmur method, a modified enzyme method thereof, and a simple method such as benzyl chloride method. In addition, as a method particularly suitable for extraction from bacteria, Sai is
To and Miura method (Biochimica et Biophysica A
cta, 72; 619 to 629 (1963)).

Furthermore, DNA extracted from the above-mentioned sample
And the above PCR primers are combined, and a DNA fragment is amplified by a PCR reaction. This PCR reaction can be performed under normal conditions, for example, double-stranded D
A denaturing step for denaturing NA (eg 94 ° C., 15-30
Second), an annealing step for annealing the single-stranded template DNA and the primer (eg, 54 ° C., 30 seconds to 1 second).
Min), DNA polymerase and 4 kinds of substrates (dNTP)
Primer extension step in the coexistence of
It is performed by repeating the operation for 20 cycles or more, with 0 second to 1 minute) as one cycle. However, the conditions of PCR reaction are
It can be changed according to the Tm value of the primer, the concentration of the template DNA, the size of the amplified fragment, and the like.

As a result of this PCR reaction, a DNA fragment specific to the primer is amplified and obtained as a PCR product, and thus the amplified DNA fragment is subjected to electrophoresis on an agarose gel or the like. By obtaining a band derived from this DNA fragment, the target tyrosine decarboxylase gene of the present invention can be detected.

Further, in the case of the isolated and cultured microorganism, its tyrosine decarboxylation activity can be measured by the method shown in Example 4. It has been confirmed that all the strains in which the tyrosine decarboxylase gene was detected by the detection method of the present invention in the group of lactic acid bacteria containing Tetragenococcus halophilus using this method have tyrosine decarboxylation activity. Particularly, in Tetragenococcus halophilus, it was confirmed that only the strain in which the tyrosine decarboxylase gene of the present invention was detected by the detection method of the present invention was a strain having tyrosine decarboxylation activity, that is, a tyramine-producing bacterium ( Example 4). Therefore, in Tetragenococcus halophilus, the strain in which the tyrosine decarboxylase gene of the present invention is detected matches the tyramine-producing bacterium. As described above, according to the detection method of the present invention, a strain having a tyrosine decarboxylation activity (tyramine-producing bacterium), which cannot be detected by conventional methods, can be detected simply and reliably.

The present invention will be described in more detail based on the following examples, but they are merely illustrative and the present invention is not limited thereto.

Example 1 (Production of tyrosine decarboxylase derived from Tetragenococcus halophilus of the present invention)

(1) Culture of Tetragenococcus halophilus capable of producing tyrosine decarboxylase

(Seed culture) Tetragenococcus halophilus ( Tetragenococcus) , which is a strain capable of producing tyrosine decarboxylase on MRS (Difco) plate agar medium supplemented with 5% sodium chloride and 2% tyrosine
s halophilus ) HSCC809 (FERM
P-18503) was grown.

(Preculture) The cells grown on this plate agar medium were added with 5% sodium chloride and 0.25% tyrosine to give M.
250 ml of RS medium was placed in a medium bottle and sterilized at 120 ° C for 15 minutes, and 1 platinum loop was inoculated into the prepared medium.
The preculture liquid was prepared by statically culturing at 0 ° C. for 120 hours.

(Main culture) Next, 25 L of the above medium was prepared, dispensed into 5 5 L Erlenmeyer flasks, and sterilized in the same manner.
50 ml of the above preculture liquid was aseptically inoculated into each Erlenmeyer flask, and static culture was carried out at 30 ° C. for 120 hours.

(Separation of bacterial cells) After completion of culture, 25 L of culture medium
Cells were collected by centrifugation (6500 g, 15 minutes, 4 ° C) to collect the bacterial cells (wet weight 114 g).
The cells were washed with 0 mM phosphate buffer (pH 6.0). After washing, the cells were suspended in 640 ml of the same buffer solution.

(2) Purification of tyrosine decarboxylase of the present invention Next, the present enzyme was purified by the following method.

Step 1 (Crushing of bacterial cells) The bacterial cell suspension was crushed by passing the bacterial cell suspension through a microfluidizer 2-3 times. After disruption, centrifugation (5000 g, 15 minutes, 4 ° C.) was performed, and the supernatant was collected to obtain a disrupted cell suspension.

Step 2 (Ammonium sulphate fraction) Ammonium sulphate was slowly added to the disrupted cell suspension so that the concentration was 3.0 M, and the mixture was thoroughly stirred. Then, centrifugation (5000 g, 15 minutes, 4 ° C.) was performed, and the supernatant was collected.

Step 3 (Butyl Toyopearl 650, chromatography) The above supernatant solution was added to a Butyl Toyopearl 650 column (5.5 ×).
(15.5 cm), and then 3.0 M ammonium sulfate containing 5
It was washed with 0 mM phosphate buffer (pH 6.0) and then with 50 mM phosphate buffer (pH 6.0) containing 2.0 M ammonium sulfate. Then, it was eluted with 50 mM phosphate buffer (pH 6.0) containing 1.0 M ammonium sulfate, and the active fractions were collected. The collected active fraction was 50 mM containing 2.5 M ammonium sulfate using a dialysis membrane.
It was dialyzed against a phosphate buffer (pH 6.0).

Step 4 (CM-Sepharose Fast Flow Chromatography) After adsorbing the dialysate on the CM-Sepharose Fast Flow column (2.8 × 10 cm), 2.5 M
It was washed with 50 mM phosphate buffer (pH 6.0) containing ammonium sulfate and then eluted with 50 mM phosphate buffer (pH 6.0) containing 2.5 M to 1.0 M ammonium sulfate by the linear concentration gradient method. Then, active fractions eluted with 50 mM phosphate buffer (pH 6.0) containing about 2.0 M ammonium sulfate were collected.
The collected active fraction was dialyzed against a 50 mM phosphate buffer solution (pH 6.0) containing 1.0 M ammonium sulfate using a dialysis membrane, and then concentrated with polyethylene glycol # 20000.

Step 5 (TSK-gel Toyopearl HW55F / Chromatography) The above concentrated liquid is TSK-gel Toyopearl HW
It was applied to a 55F column (2.7 × 90 cm) and eluted with 50 mM phosphate buffer (pH 6.0) containing 1.0 M ammonium sulfate to collect the active fraction.

Step 6 (Enzyme Purified Sample) The active fraction obtained by the above purification procedure was judged to be a substantially homogeneous protein having a molecular weight of about 70 kDa by SDS-polyacrylamide electrophoresis, and thus the purified sample was prepared. It was confirmed that there is. The tyrosine decarboxylation activity of this preparation is 19152 U in total activity and 1 in specific activity.
It was 757 U / mg. The method for measuring the activity of tyrosine decarboxylase was according to the method described in Example 3.

[0048]

[Example 2] (Determination of nucleotide sequence encoding tyrosine decarboxylase and amino acid sequence of tyrosine decarboxylase)

(1) Determination of N-terminal of tyrosine decarboxylase and amino acid sequence of internal peptide The purified preparation obtained in Example 1 was subjected to SDS-polyacrylamide electrophoresis. Then, a band having a molecular weight of about 70 kDa was cut out and used for determining the amino acid sequences of the N-terminal and the internal peptide. The N-terminal amino acid sequences obtained and the amino acid sequences of the two internal peptides are as follows.

○ N-terminal amino acid sequence (SEQ ID NO: 10: FIG. 24) TDNISKNDDLNLNALFI ○ Internal peptide 1 (SEQ ID NO: 11: FIG. 25) TPILGVVAVAGSTEE ○ Internal peptide 2 (SEQ ID NO: 12: FIG. 26) MNDLNLAFYQEASFV

(2) Isolation of tyrosine decarboxylase gene Tetragenococcus halophilus ( Tetrage)
nococcus halophilus ) HSCC
MR with 809 inoculum added with 5% salt and 2% tyrosine
It was grown on S (Difco) plate agar medium. Further, the grown bacterial cells were treated with MRS (manufactured by Difco) liquid medium 100 containing 5% sodium chloride and 0.25% tyrosine.
One platinum loop was inoculated into a medium prepared by sterilizing a medium bottle containing ml at 120 ° C. for 15 minutes, and statically cultured at 30 ° C. for 72 hours. Saito and Miu from the obtained bacterial cells
Ra's method (Biochimica et Bioph
ysica Acta, 72: 619-629 (196).
Chromosomal DNA was extracted according to 3)).

N of tyrosine decarboxylase determined in (1)
Oligonucleotide PCR primer TDF (5'-ACWGA) based on the amino acid sequences of the terminal and internal peptides.
YAYAATTTCWAARAAYGAYGAYYTA
AA-3 '), TDR1 (5'-TCTTCWGTWG
AWCCWGCAACWGCAACAAACWCC-
3 '), TDR2 (5'-ACAAAWGAWGCTT
CTTGATAAAAWGCTARATT-3 ') was prepared, and the chromosomal DNA obtained above was used as a template to prepare the above-mentioned PC.
R primers TDF and TDR1 and TDF and TDR2
The DNA fragment was amplified by the PCR method using. A DNA fragment of about 900 bp was obtained from the former, and a DNA fragment of about 1600 bp was obtained from the latter.

In the above-mentioned oligonucleotide PCR primer, TDF is SEQ ID NO: 7 (FIG. 21), TDR1 is SEQ ID NO: 8 (FIG. 22), and TDR2 is SEQ ID NO: 9 (FIG. 2).
3), respectively. In the above sequence, w is a or t, y is t or c, and r is g or a.

The chromosomal DNA obtained above was cleaved with a restriction enzyme, subjected to agarose electrophoresis, and then subjected to Southern hybridization using a DNA fragment of about 1600 bp amplified by the PCR method as a probe. as a result,
An approximately 3 kb fragment of chromosomal DNA cleaved with HindIII and ScaI reacted with the probe. Next, subcloning of the fragment was tried. That is, again on chromosome D
After cutting NA with HindIII and ScaI and subjecting it to agarose electrophoresis, the band around 3 kb was separated by Ul.
traClean (MO BIO LABORATOR
IES, INC. Product) and pBluesc
Hi of riptIIKS + (manufactured by Stratagene)
It was subcloned into the ndIII and HincII sites. Then used in Southern hybridization 1
Colony hybridization was performed using a 600 bp DNA fragment as a probe, and a transformant carrying a plasmid in which a chromosomal DNA fragment containing the gene for tyrosine decarboxylase was cloned was selected. Next, the plasmid was transformed from the transformant with GFX (AmershamPha).
rmcia Biotech Inc. (Manufactured by K.K.) and the nucleotide sequence of this chromosomal DNA fragment was determined.
The open reading frame of tyrosine decarboxylase was 1878 bp (including stop codon) (SEQ ID NO: 4: FIG. 16, FIG. 17, FIG. 18).

(3) Determination of amino acid sequence of tyrosine decarboxylase The amino acid sequence deduced from the above base sequence consisted of 625 amino acids, and the deduced molecular weight was 70.2 KDa. The amino acid sequences of the N-terminal and internal peptide determined from the purified sample of this enzyme are 2 to 18 amino acids (N-terminal amino acid) of the amino acid sequence deduced from the nucleotide sequence, 286
~ 300 amino acids (internal peptide 1) and 521-535
It matched the amino acid (internal peptide 2) (SEQ ID NO: 3:
14 and 15).

[0056]

Example 3 Properties of the tyrosine decarboxylase of the present invention

(Stability of tyrosine decarboxylase) The stability of this enzyme is determined by adjusting the respective component values to a double concentration of Mc.
It was confirmed by adding 20 μl of tyrosine decarboxylase obtained in Example 1 to 980 μl of llvaine buffer and periodically measuring the remaining activity. The activity of tyrosine decarboxylase is 0.2M acetate buffer (pH 5.0) containing 4 mM tyrosine and 0.4 mM pyridoxal phosphate.
100 μl of the enzyme solution was added to 700 μl, and the mixture was reacted at 37 ° C. for 1 hour, 200 μl of 1M perchloric acid was added to stop the reaction, and the amount of tyramine produced was measured by HPLC.

Salt Concentration Stability The buffer solutions that have been varied so that the final salt concentrations are 0, 5, 10, 15, 17, 20% are adjusted to pH 5.0, and the purified preparations are added to these buffer solutions. In addition, it was stored at 30 ° C.
When the initial activity is 100%, regardless of the salt concentration,
Residual activity after 2 days is 30-40%, after 7 days it is 10%
% Was very unstable. However, the residual activity after 7 days was 60 in the above buffer containing 1M ammonium sulfate instead of sodium chloride.
% Was relatively stable (Fig. 1).

PH stability At a final salt concentration of 17.0%, pH 4.0, 4.5,
Adjust the buffer solution that was changed to 5.0, 5.5 and 6.0,
The above purified preparations were added to these buffer solutions and stored at 30 ° C. The enzyme showed no great difference in stability from pH 4.5 to 6.0, and the residual activity after 2 days was 50% and that after 7 days was 20-25%, which was very unstable when the initial activity was 100%. there were. Among them, pH 5.5 was the most stable. Also, at pH 4.0, when the initial activity is 100%,
The residual activity after 6 hours was 30%, and the residual activity was not observed after 24 hours (FIG. 2).

Temperature stability A buffer solution having a final salt concentration of 17.0% and a pH of 5.0 was prepared, and the above purified preparation was added to this buffer solution at 15 ° C and 23 ° C.
It was stored at ℃, 30 ℃, 37 ℃. This enzyme is 15 ℃ to 3
There is no big difference in stability at 7 ℃, the initial activity is 100%, the residual activity after 2 days is 60%, that after 7 days is 20%.
And was very unstable. Among them, 15 ° C was the most stable (Fig. 3).

Effect of pyridoxal phosphate on stability At final salt concentration of 17.0% and final pyridoxal phosphate concentration of 200 μM, pH 4.0, 4.5, 5.0, 5.
The buffer solutions varied to 5 and 6.0 were prepared, and the above purified preparations were added to these buffer solutions and stored at 30 ° C. pH 4.
No effect was observed on the stability of pyridoxal phosphate at 0, but at pH 4.5 to 6.0, when the initial activity was taken as 100%, the residual activity after 2 days was 70% and that after 7 days was 60%. %, An improvement in stability was observed (Fig. 4).

Effect on stability of 2-mercaptoethanol Final salt concentration was 17.0%, final pyridoxal phosphate concentration was 200 μM, and final 2-mercaptoethanol concentration was 1
At 00 μM, pH 4.0, 4.5, 5.0, 5.5,
The buffer solutions adjusted to 6.0 were adjusted, and the above purified preparations were added to these buffer solutions and stored at 30 ° C. At pH 4.5 to 6.0, when the initial activity was taken as 100%, the residual activity after 7 days was 80% to 90%, indicating an improvement in stability (FIG. 5).

(Effect of each element on the activity of tyrosine decarboxylase) The effect of each element on the activity of tyrosine decarboxylase is
Double-concentration Mcllv containing 4 mM tyrosine and 0.4 mM pyridoxal phosphate adjusted to the respective component values
An enzyme solution 100 prepared by diluting the tyrosine decarboxylase obtained in Example 1 above 50 times with 700 μl of aine buffer solution.
After adding 1 μl and reacting at an appropriate temperature for 1 hour, 1M
The reaction was stopped by adding 200 μl of perchloric acid, and the amount of tyramine produced was measured by HPLC.

Effect of Salt Concentration A buffer solution in which the final salt concentration was changed to 0, 5, 10, 15, 17% was adjusted to pH 5.0 and the enzyme reaction was carried out at 37 ° C. The relative activity decreased with increasing salt concentration, and when the salt concentration was 17% and the activity was 100%, the activity was 150% when the salt concentration was 0% (Fig. 6).

Effect of pH When the final salt concentration is 17%, the pH is 4.0, 4.5, 5.
Adjust the buffer, which was changed to 0, 5.5, and 6.0,
The enzymatic reaction was performed at ° C. 10 activity at pH 5.0
0%, 0% at pH 4.0, 50 at pH 4.5
%, 130% at pH 5.5 and 120% at pH 6.0 (FIG. 7).

Effect of temperature A buffer solution having a final salt concentration of 17% and a pH of 5.0 was prepared, and the enzyme reaction was carried out at 15 ° C, 23 ° C, 30 ° C and 37 ° C. When the activity at 37 ° C was defined as 100%, it was 70% at 30 ° C, 45% at 23 ° C, and 0% at 15 ° C (Fig. 8).

[0067]

Example 4 Detection of Tetragenococcus halophilus (tyramine-producing bacterium) having tyrosine decarboxylation activity by PCR Tetragenococcus halophilus ( Tetrage)
A strain having tyrosine decarboxylation activity (tyramine) by detecting a tyrosine decarboxylation gene by a PCR method using a PCR primer prepared based on the nucleotide sequence of the tyrosine decarboxylase gene of the present invention from a strain of Nococcus halophilus. It was examined whether or not it is possible to specifically detect the production bacteria.

First, based on the nucleotide sequence determined in Example 2 (SEQ ID NO: 4: FIGS. 16, 17, and 18), a tyrosine decarboxylase gene detection primer TDCF (SEQ ID NO: 5: FIG. 19) and TDCR (sequence: No. 6: FIG. 20) was prepared.
For TDCF, a sequence of 32 bases from the 4th to 35th bases of the tyrosine decarboxylase gene (SEQ ID NO: 4), and for TDCR, also from 857th to 88th of SEQ ID NO: 2
The 8th 32 base sequence was used.

Chromosomal DNA was extracted from cells of 24 strains of Tetragenococcus halophilus cultured on MRS plate agar medium containing 5% NaCl and 2% tyrosine. The extraction of DNA from the cells is performed by Saito
And Miura's method.

Furthermore, the chromosomal DNA extracted from this bacterial cell
Was used as a template and a PCR reaction was performed using the PCR primers prepared above. When the product of the PCR reaction was subjected to agarose gel electrophoresis, 7 out of 24 strains of Tetragenococcus halophilus strains (1, 3,
4, 14, 16, 18, 21), a DNA fragment of 885 bp was detected (FIG. 9: photograph of electrophoresis pattern). Therefore, these strains were presumed to be tyramine-producing bacteria.

The tyrosine decarboxylation activity of the strain was determined by adding 700 μl of 0.2 M acetate buffer (pH 5.0) containing 4 mM tyrosine and 0.4 mM pyridoxal phosphate to the bacterial suspension 100.
After adding μl and reacting for 1 hour at 37 ° C., 200 μl of 1M perchloric acid was added to stop the reaction, and the amount of tyramine produced was confirmed by HPLC. As a result, tyrosine decarboxylation activity was observed in the strains (1, 3, 4, 14, 16, 18, and 21) in which the 885 bp DNA fragment was detected by PCR, and the tyrosine decarboxylation activity in the other strains. I was not able to admit.

As described above, in the method for detecting a tyrosine decarboxylase gene by the PCR method of the present invention, the strain of Tetragenococcus halophilus in which the tyrosine decarboxylase gene of the present invention is detected has a tyrosine decarboxylase activity. , I.e., it was confirmed to match the strain that is a tyramine-producing bacterium.

[0073]

INDUSTRIAL APPLICABILITY The present invention is directed to the halophilic lactic acid bacterium Tetragenococcus halophilus.
s halophilus ) -derived novel tyrosine decarboxylase and tyrosine decarboxylase gene. Furthermore,
Rapid by a PCR method using a PCR primer designed based on the nucleotide sequence of the tyrosine decarboxylase gene,
Simple and reliable Tetragenococcus halophilus (tyramine-producing bacterium) with tyrosine decarboxylation activity
And a method for detecting a microorganism.

[0074]

[Sequence list] <110> Higeta Shoyu Co., Ltd. <120> Novel Tyrosine Decarboxylase Derived from Microbes Genus            Tetragenococcus <130> 6517 <141> 2001-10-29 <160> 12 <210> 1 <211> 625 <212> PRT <213> Tetragenococcus halophilus <400> 1 Met Thr Asp Asn Ile Ser Lys Asn Asp Asp Leu Asn Leu Asn Ala Leu                   5 10 15 Phe Ile Gly Asp Lys Ala Glu Asn Ala Asp Leu Phe Lys Ser Thr Leu              20 25 30 Val Lys Leu Val Asn Glu His Met Gly Trp Arg Lys Asn Tyr Met Pro          35 40 45 Gln Asp Lys Pro Leu Ile Ser Glu Asp Glu Lys Thr Ser Lys Ser Phe      50 55 60 Thr Asn Thr Val Asn Lys Met Asp Glu Val Leu Asp Glu Leu Ser Val  65 70 75 80 Arg Leu Arg Thr Asn Ser Val Pro Trp His Ser Pro Arg Tyr Phe Gly                  85 90 95 His Met Asn Ser Glu Thr Leu Met Pro Ser Ile Leu Ala Tyr Thr Tyr             100 105 110 Ala Met Leu Trp Asn Gly Asn Asn Val Ala Tyr Glu Ser Ser Pro Gly         115 120 125 Thr Ser Gln Met Glu Glu Glu Val Gly Lys Asp Phe Ala Lys Leu Phe     130 135 140 Asn Phe Asp His Gly Trp Gly His Ile Val Ala Asp Gly Ser Leu Ala 145 150 155 160 Cys Met Glu Gly Leu Trp Tyr Ala Arg Asn Val Lys Ser Leu Pro Leu                 165 170 175 Ala Met Lys Glu Val Lys Pro Glu Leu Val Ala Gly Lys Ser Asp Trp             180 185 190 Glu Leu Leu Asn Met Pro Xaa Asp Glu Ile Met Asp Ile Leu Asn Ser         195 200 205 Gln Asp Asp Asp Thr Ile Asp Ala Ile Lys Ala His Ser Ala Arg Gly     210 215 220 Gly Met Asp Leu Ser Lys Leu Gly Lys Xaa Leu Val Pro Gln Thr Lys 225 230 235 240 His Tyr Ser Trp Met Lys Ala Ala Asp Val Val Gly Ile Gly Leu Asp                 245 250 255 Gln Val Val Pro Ile Pro Val Asp Asp Asn Tyr Arg Leu Asp Val Asn             260 265 270 Glu Leu Glu Lys Thr Ile Asn Lys Leu Val Ala Glu Lys Thr Pro Ile         275 280 285 Leu Gly Val Val Ala Val Ala Gly Ser Thr Glu Glu Gly Ala Val Asp     290 295 300 Pro Val Asp Lys Val Val Glu Leu Arg Asn Lys Leu Met Lys Gln Gly 305 310 315 320 Thr Tyr Phe Tyr Leu His Val Asp Ala Ala Tyr Gly Ala Tyr Ala Arg                 325 330 335 Ala Leu Phe Leu Asp Glu Asp Gly Asn Phe Ile Pro Trp Asp Lys Leu             340 345 350 Gln Glu Val His Gln Lys Tyr Gly Asp Phe Gln Glu Asn Lys Glu Tyr         355 360 365 Ile Ser Lys Asp Thr Tyr Asn Ala Phe Lys Ala Ile Ser Glu Ala Glu     370 375 380 Thr Val Thr Ile Asp Pro His Lys Met Gly Tyr Val Pro Tyr Ser Ala 385 390 395 400 Gly Gly Ile Ala Ile Arg Asn Ile Thr Met Arg Asn Ile Ile Ser Tyr                 405 410 415 Phe Ala Thr Tyr Val Phe Asp Lys Arg Ser Lys Ala Pro Ser Met Leu             420 425 430 Gly Ala Tyr Ile Leu Glu Gly Ser Lys Ala Gly Ala Thr Ala Ala Ala         435 440 445 Val Trp Thr Ala His Arg Val Leu Pro Leu Asn Val Ala Gly Tyr Gly     450 455 460 Lys Leu Ile Gly Arg Ser Ile Glu Ala Ala Asn Arg Phe Tyr Asp Phe 465 470 475 480 Leu Asn Asn Leu Ser Phe Asn Ile Asn Gly Gln Glu Ile Glu Val Asn                 485 490 495 Pro Leu Thr Lys Pro Asp Phe Asn Met Val Asp Trp Thr Phe Asn Ile             500 505 510 Lys Gly Asn Lys Asp Xaa Ala Lys Met Asn Asp Leu Asn Leu Ala Phe         515 520 525 Tyr Gln Glu Ala Ser Phe Val Lys Gly Pro Ile Tyr Asn Asn Asp Phe     530 535 540 Ile Thr Ser His Thr Asp Phe Ala Gln Asp Gly Tyr Gly Asp Ser Pro 545 550 555 560 Lys Ala Tyr Val Lys Ser Leu Gly Phe Thr Asp Ala Asp Trp Gln Lys                 565 570 575 Glu Lys Ala Val Gly Ile Leu Arg Ala Cys Val Leu Thr Pro Leu Leu             580 585 590 Tyr Asp Lys Asp Asn Phe Ala Glu Tyr Ala Asp Lys Xaa Lys Ser Ala         595 600 605 Met Thr Glu Lys Leu Thr Lys Ile Val Asp Glu Lys Val Leu Gln Thr     610 615 620 Asn 625 <210> 2 <211> 1878 <212> DNA <213> Tetragenococcus halophilus <400> 2 atgactgaca atatcagtaa gaacgatgat ttaaatctta acgcactttt tatcggtgat 60 aaggccgaaa atgctgattt attcaaatca acattggtca aattggttaa tgaacacatg 120 ggctggcgta aaaattatat gccacaagat aagccattaa tctctgagga cgaaaaaacg 180 tccaaatcat ttacaaatac tgtaaataaa atggacgaag ttttggatga attatcagta 240 cgcctacgta caaattcagt tccatggcat tcacctcgtt atttcggaca tatgaattcc 300 gaaacattga tgccatcaat tcttgcatat acatacgcaa tgttatggaa tggtaacaac 360 gttgcatatg aatcatcacc aggtacttct caaatggaag aagaagttgg taaggacttc 420 gctaagctat tcaacttcga tcatggttgg ggccatatcg ttgctgatgg atcattagca 480 tgtatggaag gcctatggta tgcaagaaat gttaagagtt taccattagc aatgaaagaa 540 gtaaagccag aactagttgc tggtaagagc gattgggaat tactaaatat gcccgytgat 600 gaaatcatgg acatccttaa tagccaagat gatgacacaa ttgatgctat taaagctcac 660 tcagctcgtg gtggcatgga tctttctaaa cttggtaaat gkttagtacc acaaacaaaa 720 cactatagtt ggatgaaagc tgcagatgtt gtcggtattg gtttggatca agttgttcct 780 attccagttg acgataacta tagacttgat gttaacgagc ttgaaaagac aattaacaag 840 ttagttgctg aaaagactcc tatccttggt gttgttgctg ttgctggttc aactgaagag 900 ggtgccgttg atcctgttga taaggtcgtt gaattacgta ataagttgat gaaacaagga 960 acatatttct atctacatgt tgatgctgca tatggtgctt atgcacgtgc actattctta 1020 gacgaagatg gaaactttat cccttgggac aaactacaag aagtacatca aaaatatggt 1080 gacttccaag aaaacaaaga atatatttca aaagatactt ataatgcatt taaagctatt 1140 tccgaagcag aaacagtaac aattgatcct cataagatgg gatatgtacc ttactctgct 1200 ggtggtattg caatccgtaa tattacaatg cgtaatatca tttcatactt tgctacttat 1260 gtatttgata aacgttcaaa ggctccttca atgttaggtg cttatattct tgaaggttca 1320 aaggccggtg ctactgctgc agctgtctgg actgcacata gagtattgcc attgaatgtc 1380 gctggctatg gtaaattgat cggtcgttca atcgaagctg ctaaccgctt ctatgacttc 1440 ttgaacaact tgagctttaa catcaatggt caagaaattg aagtaaatcc attgacaaag 1500 cctgatttca atatggtcga ttggacattt aatattaaag gaaataaaga cyttgctaag 1560 atgaatgatt tgaatcttgc attttatcaa gaagcatcat tcgttaaggg tcctatctat 1620 aacaatgact ttattacatc acacactgac tttgcacaag atggttatgg tgatagtcct 1680 aaagcatacg ttaagagcct tggatttacg gatgctgatt ggcaaaagga aaaggcagtc 1740 ggaattctac gtgcatgtgt attgacacca cttctatacg acaaagataa ttttgctgaa 1800 tatgcagaca agrttaagag tgcaatgact gaaaaattaa ctaagattgt tgatgaaaaa 1860 gttctacaaa caaactag 1878 <210> 3 <211> 625 <212> PRT <213> Tetragenococcus halophilus <400> 3 Met Thr Asp Asn Ile Ser Lys Asn Asp Asp Leu Asn Leu Asn Ala Leu                   5 10 15 Phe Ile Gly Asp Lys Ala Glu Asn Ala Asp Leu Phe Lys Ser Thr Leu              20 25 30 Val Lys Leu Val Asn Glu His Met Gly Trp Arg Lys Asn Tyr Met Pro          35 40 45 Gln Asp Lys Pro Leu Ile Ser Glu Asp Glu Lys Thr Ser Lys Ser Phe      50 55 60 Thr Asn Thr Val Asn Lys Met Asp Glu Val Leu Asp Glu Leu Ser Val  65 70 75 80 Arg Leu Arg Thr Asn Ser Val Pro Trp His Ser Pro Arg Tyr Phe Gly                  85 90 95 His Met Asn Ser Glu Thr Leu Met Pro Ser Ile Leu Ala Tyr Thr Tyr             100 105 110 Ala Met Leu Trp Asn Gly Asn Asn Val Ala Tyr Glu Ser Ser Pro Gly         115 120 125 Thr Ser Gln Met Glu Glu Glu Val Gly Lys Asp Phe Ala Lys Leu Phe     130 135 140 Asn Phe Asp His Gly Trp Gly His Ile Val Ala Asp Gly Ser Leu Ala 145 150 155 160 Cys Met Glu Gly Leu Trp Tyr Ala Arg Asn Val Lys Ser Leu Pro Leu                 165 170 175 Ala Met Lys Glu Val Lys Pro Glu Leu Val Ala Gly Lys Ser Asp Trp             180 185 190 Glu Leu Leu Asn Met Pro Ala Asp Glu Ile Met Asp Ile Leu Asn Ser         195 200 205 Gln Asp Asp Asp Thr Ile Asp Ala Ile Lys Ala His Ser Ala Arg Gly     210 215 220 Gly Met Asp Leu Ser Lys Leu Gly Lys Trp Leu Val Pro Gln Thr Lys 225 230 235 240 His Tyr Ser Trp Met Lys Ala Ala Asp Val Val Gly Ile Gly Leu Asp                 245 250 255 Gln Val Val Pro Ile Pro Val Asp Asp Asn Tyr Arg Leu Asp Val Asn             260 265 270 Glu Leu Glu Lys Thr Ile Asn Lys Leu Val Ala Glu Lys Thr Pro Ile         275 280 285 Leu Gly Val Val Ala Val Ala Gly Ser Thr Glu Glu Gly Ala Val Asp     290 295 300 Pro Val Asp Lys Val Val Glu Leu Arg Asn Lys Leu Met Lys Gln Gly 305 310 315 320 Thr Tyr Phe Tyr Leu His Val Asp Ala Ala Tyr Gly Ala Tyr Ala Arg                 325 330 335 Ala Leu Phe Leu Asp Glu Asp Gly Asn Phe Ile Pro Trp Asp Lys Leu             340 345 350 Gln Glu Val His Gln Lys Tyr Gly Asp Phe Gln Glu Asn Lys Glu Tyr         355 360 365 Ile Ser Lys Asp Thr Tyr Asn Ala Phe Lys Ala Ile Ser Glu Ala Glu     370 375 380 Thr Val Thr Ile Asp Pro His Lys Met Gly Tyr Val Pro Tyr Ser Ala 385 390 395 400 Gly Gly Ile Ala Ile Arg Asn Ile Thr Met Arg Asn Ile Ile Ser Tyr                 405 410 415 Phe Ala Thr Tyr Val Phe Asp Lys Arg Ser Lys Ala Pro Ser Met Leu             420 425 430 Gly Ala Tyr Ile Leu Glu Gly Ser Lys Ala Gly Ala Thr Ala Ala Ala         435 440 445 Val Trp Thr Ala His Arg Val Leu Pro Leu Asn Val Ala Gly Tyr Gly     450 455 460 Lys Leu Ile Gly Arg Ser Ile Glu Ala Ala Asn Arg Phe Tyr Asp Phe 465 470 475 480 Leu Asn Asn Leu Ser Phe Asn Ile Asn Gly Gln Glu Ile Glu Val Asn                 485 490 495 Pro Leu Thr Lys Pro Asp Phe Asn Met Val Asp Trp Thr Phe Asn Ile             500 505 510 Lys Gly Asn Lys Asp Phe Ala Lys Met Asn Asp Leu Asn Leu Ala Phe         515 520 525 Tyr Gln Glu Ala Ser Phe Val Lys Gly Pro Ile Tyr Asn Asn Asp Phe     530 535 540 Ile Thr Ser His Thr Asp Phe Ala Gln Asp Gly Tyr Gly Asp Ser Pro 545 550 555 560 Lys Ala Tyr Val Lys Ser Leu Gly Phe Thr Asp Ala Asp Trp Gln Lys                 565 570 575 Glu Lys Ala Val Gly Ile Leu Arg Ala Cys Val Leu Thr Pro Leu Leu             580 585 590 Tyr Asp Lys Asp Asn Phe Ala Glu Tyr Ala Asp Lys Ile Lys Ser Ala         595 600 605 Met Thr Glu Lys Leu Thr Lys Ile Val Asp Glu Lys Val Leu Gln Thr     610 615 620 Asn 625 <210> 4 <211> 1878 <212> DNA <213> Tetragenococcus halophilus <400> 4 atgactgaca atatcagtaa gaacgatgat ttaaatctta acgcactttt tatcggtgat 60 aaggccgaaa atgctgattt attcaaatca acattggtca aattggttaa tgaacacatg 120 ggctggcgta aaaattatat gccacaagat aagccattaa tctctgagga cgaaaaaacg 180 tccaaatcat ttacaaatac tgtaaataaa atggacgaag ttttggatga attatcagta 240 cgcctacgta caaattcagt tccatggcat tcacctcgtt atttcggaca tatgaattcc 300 gaaacattga tgccatcaat tcttgcatat acatacgcaa tgttatggaa tggtaacaac 360 gttgcatatg aatcatcacc aggtacttct caaatggaag aagaagttgg taaggacttc 420 gctaagctat tcaacttcga tcatggttgg ggccatatcg ttgctgatgg atcattagca 480 tgtatggaag gcctatggta tgcaagaaat gttaagagtt taccattagc aatgaaagaa 540 gtaaagccag aactagttgc tggtaagagc gattgggaat tactaaatat gcccgctgat 600 gaaatcatgg acatccttaa tagccaagat gatgacacaa ttgatgctat taaagctcac 660 tcagctcgtg gtggcatgga tctttctaaa cttggtaaat ggttagtacc acaaacaaaa 720 cactatagtt ggatgaaagc tgcagatgtt gtcggtattg gtttggatca agttgttcct 780 attccagttg acgataacta tagacttgat gttaacgagc ttgaaaagac aattaacaag 840 ttagttgctg aaaagactcc tatccttggt gttgttgctg ttgctggttc aactgaagag 900 ggtgccgttg atcctgttga taaggtcgtt gaattacgta ataagttgat gaaacaagga 960 acatatttct atctacatgt tgatgctgca tatggtgctt atgcacgtgc actattctta 1020 gacgaagatg gaaactttat cccttgggac aaactacaag aagtacatca aaaatatggt 1080 gacttccaag aaaacaaaga atatatttca aaagatactt ataatgcatt taaagctatt 1140 tccgaagcag aaacagtaac aattgatcct cataagatgg gatatgtacc ttactctgct 1200 ggtggtattg caatccgtaa tattacaatg cgtaatatca tttcatactt tgctacttat 1260 gtatttgata aacgttcaaa ggctccttca atgttaggtg cttatattct tgaaggttca 1320 aaggccggtg ctactgctgc agctgtctgg actgcacata gagtattgcc attgaatgtc 1380 gctggctatg gtaaattgat cggtcgttca atcgaagctg ctaaccgctt ctatgacttc 1440 ttgaacaact tgagctttaa catcaatggt caagaaattg aagtaaatcc attgacaaag 1500 cctgatttca atatggtcga ttggacattt aatattaaag gaaataaaga ctttgctaag 1560 atgaatgatt tgaatcttgc attttatcaa gaagcatcat tcgttaaggg tcctatctat 1620 aacaatgact ttattacatc acacactgac tttgcacaag atggttatgg tgatagtcct 1680 aaagcatacg ttaagagcct tggatttacg gatgctgatt ggcaaaagga aaaggcagtc 1740 ggaattctac gtgcatgtgt attgacacca cttctatacg acaaagataa ttttgctgaa 1800 tatgcagaca agattaagag tgcaatgact gaaaaattaa ctaagattgt tgatgaaaaa 1860 gttctacaaa caaactag 1878 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <400> 5 actgacaata tcagtaagaa cgatgattta aa 32 <210> 6 <211> 32 <212> DNA <213> Artificial Sequence <400> 6 tcttcagttg aaccagcaac agcaacaaca cc 32 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <400> 7 acwgayaaya tttcwaaraa ygaygayyta aa 32 <210> 8 <211> 32 <212> DNA <213> Artificial Sequence <400> 8 tcttcwgtwg awccwgcaac wgcaacaacw cc 32 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <400> 9 acaaawgawg cttcttgata aaawgctara tt 32 <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <400> 10 Thr Asp Asn Ile Ser Lys Asn Asp Asp Leu Asn Leu Asn Ala Leu Phe                   5 10 15 Ile  17 <210> 11 <211> 15 <212> PRT <213> Artificial Sequence <400> 11 Thr Pro Ile Leu Gly Val Val Ala Val Ala Gly Ser Thr Glu Glu                   5 10 15 <210> 12 <211> 15 <212> PRT <213> Artificial Sequence <400> 12 Met Asn Asp Leu Asn Leu Ala Phe Tyr Gln Glu Ala Ser Phe Val                   5 10 15

[Brief description of drawings]

FIG. 1 is a graph showing the salt concentration stability of the tyrosine decarboxylase of the present invention.

FIG. 2 is a graph showing the pH stability of the tyrosine decarboxylase of the present invention.

FIG. 3 is a graph showing the temperature stability of the tyrosine decarboxylase of the present invention.

FIG. 4 is a graph showing the influence of the tyrosine decarboxylase of the present invention on the stability of pyridoxal phosphate.

FIG. 5 is a graph showing the effect of tyrosine decarboxylase of the present invention on the stability of 2-mercaptoethanol.

FIG. 6 is a graph showing the salt concentration dependence of the tyrosine decarboxylase of the present invention.

FIG. 7 is a graph showing the pH dependence of the tyrosine decarboxylase of the present invention.

FIG. 8 is a graph showing the temperature dependence of the tyrosine decarboxylase of the present invention.

FIG. 9 is a photograph (drawing substitute photograph) instead of a drawing of agarose gel electrophoresis showing the result of amplification of a DNA fragment derived from the tyrosine decarboxylase gene of the present invention by a PCR method using PCR primers TDCF and TDCR. Double underline indicates a strain having tyrosine decarboxylase activity, and M is λ
HindIII and arrows indicate the positions of the amplified bands.

FIG. 10 shows the amino acid sequence of the tyrosine decarboxylase derived from Tetragenococcus halophilus of the present invention.

FIG. 11 shows the nucleotide sequence of the tyrosine decarboxylase gene derived from Tetragenococcus halophilus of the present invention.

FIG. 12 shows the continuation of the above.

FIG. 13 shows the continuation of the above.

FIG. 14 Tetragenococcus halophilus HSC
1 shows the amino acid sequence of tyrosine decarboxylase derived from C809 strain.

FIG. 15 shows the continuation of the above.

FIG. 16: Tetragenococcus halophilus HSC
1 shows the base sequence of the tyrosine decarboxylase gene derived from the C809 strain.

FIG. 17 shows the continuation of the above.

FIG. 18 shows the continuation of the above.

FIG. 19 shows the base sequence of the primer TDCF for detecting the tyrosine decarboxylase gene of the present invention.

FIG. 20 shows the base sequence of the primer TDCR for detecting the tyrosine decarboxylase gene of the present invention.

FIG. 21. Oligonucleotide PCR primer TDF
The base sequence of is shown.

FIG. 22. Oligonucleotide PCR primer TDR
1 shows the base sequence of 1.

FIG. 23. Oligonucleotide PCR primer TDR
2 shows the base sequence of 2.

FIG. 24 shows the N-terminal amino acid sequence.

FIG. 25 shows the amino acid sequence of Internal Peptide 1.

FIG. 26 shows the amino acid sequence of internal peptide 2.

Claims (8)

[Claims] 1. A tyrosine decarboxylase TDCTH having the following properties. (1) Action: It acts on tyrosine to produce tyramine. (2) Optimum pH: When using Mr. Mcllvaine's buffer containing 0.4 mM pyridoxal phosphate 5.5 (17)
% NaCl present). (3) pH stability: most stable at pH 5.5 (17% Na
In the presence of Cl). (4) Stabilization: Relatively stable in the presence of 1.0 M ammonium sulfate. Very stable in the presence of pyridoxal phosphate and 2-mercaptoethanol. (5) Molecular weight: The molecular weight measured by SDS polyacrylamide gel electrophoresis is about 70.2 KDa. 2. Tyrosine decarboxylase TDCTH, which is the following protein (a) or (b): (A) A protein having the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. (B) A protein having an amino acid sequence in which a part of amino acid residues is substituted, deleted, inserted or added in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing and having tyrosine decarboxylase activity. 3. A DNA of the tyrosine decarboxylase TDCTH gene comprising the DNA encoding the protein according to claim 2. 4. The DNA of the tyrosine decarboxylase TDCTH gene according to claim 3, which comprises the following DNA (a) or (b): (A) DN consisting of the nucleotide sequence shown in SEQ ID NO: 2 in Sequence Listing
A. (B) DN consisting of the nucleotide sequence shown in SEQ ID NO: 2 in Sequence Listing
A DNA which hybridizes with A under stringent conditions and which encodes a protein having tyrosine decarboxylase activity. 5. Tetragenococcus having the ability to produce tyrosine decarboxylase TDCTH
ccus ) microorganisms are cultivated in a medium, and a protein having tyrosine decarboxylase activity is collected from the obtained culture, and a method for producing the protein. 6. A microorganism belonging to the genus Tetragenococcus is Tetragenococcus halophilus ( Tetrag).
Enococcus halophilus ). 7. A tyrosine decarboxylase TDCTH gene, which is characterized by a PCR method using an oligonucleotide prepared based on a continuous partial sequence of the nucleotide sequence according to claim 3 or 4 as a PCR primer. To detect the DNA of the gene to be used. 8. A tyrosine decarboxylase TDCTH activity characterized by a PCR method using an oligonucleotide prepared on the basis of a continuous partial sequence of the nucleotide sequence according to claim 3 or 4 as a PCR primer. Method for detecting microorganisms.