JP3160384B2 - Pyrrole-2-carboxylic acid decarboxylase and process for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a pyrrole-2-carboxylic acid decarboxylase, a method for producing the enzyme, and a new bacterial strain belonging to the genus Bacillus that produces the enzyme. Pyrrole-2-carboxylic acid decarboxylase, a novel enzyme of the present invention,
Pyrrole-2-carboxylic acid to generate the decarboxylated pyrrole and HCO ₃ ^- pyrrole in the presence of generating the substrate as pyrrole-2-carboxylic acid.

[0002]

2. Description of the Related Art Aromatase decarboxylase (hereinafter also referred to as "decarboxylase") is known to widely exist in animals, plants and microorganisms. Histidine decarboxylase, tyrosine decarboxylase, aromatic-L-amino acid decarboxylase, etc., which are decarboxylase substrates, have also been purified and their properties have been investigated in detail (Rosenthaler, Girard ( BMGuirard), Chang (GWChang), Snell (EESne)
II), Proc. Natl. Acad. Sci. USA, 54 , 152 (196
5); Chang, Snell, Biochemistry, 7 , 2005 (1
968); Ips (Epps, HMR), Biochem. J., 38 ,
242 (1944); GALancaster, TLSourkens, Can. J. Biochem., 50 , 79.
1 (1972); Nakazawa, H.Kum
agai), Yamada (H.Yamada), Biochem.Biophys.Res.Commu
n., 61 , 75 (1974)).

With respect to decarboxylase using a substrate other than an amino acid, there have been reports on phosphoribosylaminoimidazole carboxylase, orotidine-5'-phosphate decarboxylase, aminobenzoate decarboxylase, and the like (LNLukens, Buchanan).
(JMBuchanan), J. Biol. Chem., 234 , 1799 (1
959); Brody (RSBrody), Westheimer (F.
H. Westheimer), J. Biol. Chem., 254 , 4238 (19
79); BelBerser, JRWild, M
eth. Enzymol., 51 , 135 (1978);
(WGMcCullough), JTPiligian, IJDaniel, J. Amer. Chem. Soc., 79 , 628 (1
957)), however, the nature of microorganisms, especially bacterial enzymes, has not been clarified because of their instability. Above all,
Pyrrole-2-carboxylic acid decarboxylase has not been reported at all through animals, plants and microorganisms.

[0004]

Under the above circumstances, the present inventors have conducted intensive studies on useful enzymes produced by microorganisms, and as a result, a novel enzyme having an ability to produce pyrrole-2-carboxylic acid decarboxylase has been obtained. The present inventors have found a bacterium and succeeded in isolating pyrrole-2-carboxylate decarboxylase from the bacterium, thereby completing the present invention. That is,
The present invention, the pyrrole-2-carboxylic acid is decarboxylated to produce a pyrrole, also HCO ₃ ^- presence, pyrrole to produce a pyrrole-2-carboxylic acid pyrrole as a substrate for -
It is intended to provide a 2-carboxylic acid decarboxylase. The present invention also relates to a pyrrole belonging to the genus Bacillus.
A bacterium capable of producing 2-carboxylic acid decarboxylase is cultured in a pyrrole-2-carboxylic acid decarboxylase-inducing medium, and then pyrrole-2-carboxylic acid decarboxylase is isolated from the cells. A method for producing pyrrole-2-carboxylic acid decarboxylase is also provided. Pyrrole-2- of the present invention
Carboxylic acid decarboxylase is a novel enzyme which has not been reported at all in animals, plants and microorganisms, and has characteristics unprecedented in its properties. The enzyme of the present invention makes it possible to carry out a decarboxylation reaction of pyrrole-2-carboxylic acid or a carboxylation reaction of pyrrole instead of an organic synthesis method. The use of the enzyme of the present invention makes it possible to produce the desired product efficiently under mild conditions without the need for a special device or equipment because the reaction is an enzymatic reaction, and without producing by-products. .

[0005] The pyrrole-2-carboxylate decarboxylase of the present invention can be obtained by culturing a bacterium belonging to the genus Bacillus and capable of producing pyrrole-2-carboxylate decarboxylase. Examples include Bacillus
And Bacillus megaterium PYR2910 strain. Bacillus megaterium PYR29
The ten strains were isolated and identified by the present inventors from nature, and were sent to the Institute of Microbial Industry and Technology by the National Institute of Advanced Industrial Science and Technology on September 8, 1992.
45). The mycological properties of this microorganism are as follows.

(A) Morphology (meat agar medium, cultured at 30 ° C. for 24 hours) Cell shape and size: Bacillus, 1.2-1.5 μm ×
2 to 5 μm 2. Cell polymorphism: +3. Motility: -4. Spores: +5. Gram stainability: +6. Acid resistance:-

(B) Growth conditions in each medium (30 ° C., 24
Time) 1. Gravy agar plating: colonies 1.5 to 2.0 in diameter
mm round, regular, pale yellow in color, granular in surface, opaque with no luster, low convex Gravy agar slope culture: Medium growth, pale yellow,
2. Surface is granular, opaque with no gloss Broth liquid culture: The culture broth is uniformly turbid and the color is pale yellow

(C) Physiological properties 1. Spore shape: oval or cylindrical Spore polymorphism: -3. 3. Location of spores: at or near the end Intracellular microspheres: +5. Anaerobic growth:-Growth at 6.5% NaCl: (+) Slight growth 7.7% NaCl growth: + 8.10% NaCl growth:-9. Growth at pH 5.7: +

[0009] 10. Generation of acid from glucose: +11. Production of gas from glucose: -12. VP test: -13. Egg yolk reaction: -14. Casein degradation: +15. Gelatin degradation: +16. Degradation of tyrosine: +17. Hydrolysis of starch: -18. NO ₃ ^- to NO ₂ ^- :-

19. Use of citric acid (Coser's medium):
+20. Arginine dihydrolase [Moller's medium]:-21. PH of VP medium: 5.2 22. Use of propionic acid (Coser's medium): -23. Hydrolysis of esculin: +24. Urea hydrolysis: +25. ONPG: +

[0011] Based on the above mycological properties, Bergie's Manual of Systematic Bacteriolog
y). This microorganism is an aerobic, gram-positive bacillus, has spores, and has other physiological properties, so that it is Bacillus megateriu (Bacillus megateriu).
m).

<Culture conditions> It is not necessary to use a special medium as a medium for the above-mentioned strain, and a normal medium may be used. As a carbon source, glucose, lactose, starch, dextrin, sugars such as maltose, amino acids such as glutamic acid, or organic acids such as fumaric acid, malic acid, polypeptone as a nitrogen source, yeast extract, soybean powder hydrolyzate, etc. Use of natural nitrogen source, inorganic salts such as NaCl, potassium phosphate, magnesium sulfate, etc., and trace metal components such as calcium chloride, boric acid, copper sulfate, potassium iodide, ferrous sulfate, manganese sulfate, zinc sulfate, etc. Can be.

[0013] The pyrrole-2-carboxylic acid decarboxylase of the present invention is an inducing enzyme. Enzyme-inducing compounds must be added as inducers. A preferred example of the medium is shown in Table 1 below. In Table 1, the composition of the metal solution is as shown in Table 2 below.

[0014]

TABLE 1 pyrrole-2-carboxylic acid 1.5g fumaric acid 10g polypeptone 10g Yeast extract _{0.5g MgSO 4 · 7H 2 O 0.5g} K 2 HPO 4 1g metal solution 5 ml 1L (pH 7.0)

[0015]

Table 2 Table _{2 CaCl 2 · 2H 2 O 400mg} H 3 BO 3 500mg CuSO 4 · 5H 2 O 40mg KI 100mg FeSO 4 · 7H 2 O 200mg MnSO 4 · 7H 2 O 400mg ZnSO 4 · 7H 2 O 400mg H 2 _{MoO 4 · 2H 2 O 200mg HCl} (12N) 10ml 1L

The culture pH is about 6.5 to about 8.5, preferably 7.0, the culture temperature is about 25 ° C. to about 40 ° C., preferably 28 ° C., and aerobically stirred or shaken for about 24 hours. While culturing. The pyrrole-2-carboxylic acid decarboxylase of the present invention can be obtained by isolating the enzyme from the bacteria cultured in this manner.

<Enzyme collection method> In order to isolate the enzyme of the present invention from the above culture solution, known purification methods can be used alone or in combination. For example, the culture is centrifuged to collect the cells, the cells are disrupted by sonication, and the cell-free extract is obtained by centrifugation. This is subjected to ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, hydroxylapatite chromatography, gel filtration chromatography, and the like to purify the enzyme of the present invention. An example of the purification method is shown below.

(1) A cell-free extract is obtained from the ultrasonically crushed cells by centrifugation. (2) Ammonium sulfate fractionation is performed, and the fraction having an ammonium sulfate concentration of 45% to 70% is suspended in a 20 mM potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol. (3) After dialysis against the same buffer, the mixture was subjected to DEAE Sephacel chromatography, and 20 mM potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol and KCl
To elute with a gradient elution method from 0.1M to 0.3M KCl. (4) 20% saturated ammonium sulfate was added to the active fraction, followed by octyl sepharose CL-4B chromatography, and 20 mM potassium phosphate buffer containing 1 mM dithiothreitol.
(pH 7.0) and ammonium sulfate, eluted with a concentration gradient elution method of 20% to 0% ammonium sulfate.

(5) Then, the mixture was subjected to butyl toyopearl column chromatography in the same manner as in step (4) to give 1 mM
Elution is carried out with a 20 mM potassium phosphate buffer solution (pH 7.0) containing dithiothreitol and ammonium sulfate by a concentration gradient elution method of 16% to 0% ammonium sulfate. (6) The active fraction was 20 m containing 1 mM dithiothreitol.
After dialysis against M potassium phosphate buffer (pH 7.0),
Hydroxyapatite chromatography
Potassium phosphate buffer containing mM dithiothreitol
(pH 7.0) and elute with a concentration gradient elution method from 20 mM to 200 mM potassium phosphate. (7) After concentration of the active fraction, TOYOPEARL HW-5
The mixture is subjected to 5 gel filtration chromatography and eluted with 100 mM potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol and 0.2 M NaCl.

<Activity measurement method> Bacillus megaterium
(Bacillus megaterium) Pyrrole-2-carboxylate decarboxylase obtained from PYR2910 has a markedly reduced activity when the cells are disrupted, and it is difficult to measure the activity, so that the enzyme cannot be purified. In order to solve this problem, the present inventors have studied an enzyme stabilizing agent and an activity measuring method. As shown in Table 3 below, the present inventors have found that the present enzyme exhibits its activity in the presence of an organic acid. I found it.

[0021]

TABLE 3 organic acid relative activity (%) formic acid 43.7 acetic 100 propionic acid 131 butyric acid 135 valeric 34.5 caproic acid 23.4 enanthic acid 33.1 caprylic 11.2 pelargonic acid 6.1 capric Acid 1.7

Pyruvic acid 10.7 2-Chloropropionic acid 78.6 Lactic acid 0.0 Isovaleric acid 22.9 DL-2-methylbutyric acid 91.7 Isocaproic acid 7.6 2-Methylvaleric acid 29.3 Oxalic acid 0.0 Malonic acid 0.0 Succinic acid 0.0 Glutaric acid 0.0 Adipic acid 29.9 Pimelic acid 177

Benzenecarboxylic acid 9.7 3-Phenylpropionic acid 44.1 4-Phenylbutyric acid 31.8 5-Phenylvaleric acid 0.0 Acrylic acid 51.3 Propiolic acid 94.7 Methacrylic acid 75.8 Crotonic acid 0 0.0 Maleic acid 0.0 Fumaric acid 0.0

50 mM of each organic acid was added to the reaction solution, and the amount of pyrrole produced from pyrrole-2-carboxylic acid was compared. In this case, the amount of pyrrole produced when 50 mM of acetic acid was added was taken as 100%, and the ratio to the amount was shown. Organic acids having 1 to 10 carbon atoms, especially acetic acid,
Propionic acid, butyric acid, pimelic acid and the like are particularly effective, and many other organic acids have also exhibited activity. When these organic acids were not added to the reaction solution, no activity was exhibited.
The concentration of each organic acid in the reaction solution varies, for example, about 4 mM for pimelic acid and about 100 mM for acetic acid were appropriate. Based on this, the method for measuring the activity of pyrrole-2-carboxylic acid decarboxylase was determined as follows.

A 100 mM potassium phosphate buffer (pH 7.
0), 20 μl of an enzyme solution of unknown activity was added to a reaction solution comprising 100 mM ammonium acetate and 100 mM pyrrole-2-carboxylic acid as a substrate, and the total volume was 2.0 ml.
And This is incubated at 30 ° C. for 10 minutes, the reaction is stopped by adding 0.5N NaOH (0.5 ml), and the pyrrole formed is measured by HPLC. Under these conditions, the enzyme activity producing 1 μmol of pyrrole per minute was defined as 1 unit. The HPLC conditions are shown below. Column: M & S PACK C18 (4.6 × 150 mm) Solvent: 20 mM KH ₂ PO ₄ (pH 2.5) / CH ₃ CN (8
/ 2) Detection: absorbance at 210 nm Flow rate: 1.0 ml / min Temperature: room temperature (20-25 ° C)

<Properties of enzyme> The physicochemical properties of the enzyme of the present invention are shown below. 1. Action and specificity Decarboxylate pyrrole-2-carboxylic acid to produce pyrrole. The Km value for pyrrole-2-carboxylic acid is 2
3.8 × 10 ^-3 M, Vmax is 217.4 μmol / min / mg
Met. The HCO ₃ ^- presence, to produce a pyrrole-2-carboxylic acid from pyrrole.

2. Optimum pH and stable pH range Using the enzyme of the present invention, an enzymatic reaction was carried out under the respective pH conditions in the range of pH 3.5 to 10.0 in accordance with the method of the above activity measurement method. However, the buffer used was 100 mM acetate buffer, pH 3.5 in the range of pH 3.5 to 6.0.
100 mM citrate buffer in the range from to 6.5,
In the range of pH 5.5 to 7.0, 100 mM 2- (N-morpholino) ethanesulfonic acid (MES) buffer, pH
100 mM potassium phosphate buffer in the range of 6.0 to 8.0, 100 mM triethanolamine buffer in the range of pH 6.5 to 8.5, 100 mM N-2-hydroxylethyl piperazine in the range of pH 7.0 to 8.0. -N'-2-ethanesulfonic acid (HEPES) buffer, 100 mM Tris-HCl buffer in the range of pH 7.0 to 9.0, 100 mM borate buffer in the range of pH 8.0 to 10.0, pH 8.5 to 10 in the range of 2.0 a 100mM NH ₄ OH-NH ₄ Cl buffer. FIG. 1 shows the relative activities when the buffers at each pH were used. From the results shown in FIG. 1, it can be seen that the optimum pH of the enzyme of the present invention is about 6.5 at 30 ° C.

Further, the enzyme of the present invention was dissolved in a buffer having a predetermined pH, kept at 50 ° C. for 10 minutes, and the residual activity was measured. However, the buffer used was 100 mM acetate buffer, pH 3.5 in the range of pH 3.5 to 6.0.
100 mM citrate buffer in the range of 5 to 6.5, and 100 mM 2- (N in the range of pH 5.5 to 7.0.
-Morpholino) ethanesulfonic acid (MES) buffer,
100 mM potassium phosphate buffer in the range of pH 6.0 to 8.0, 100 m in the range of pH 6.5 to 8.5
M triethanolamine buffer, pH 7.0 to 9.
In the range of 0, it is 100 mM Tris-HCl buffer. Fig. 2 shows the relative activities when using buffers at each pH.
Shown in The results shown in FIG. 2 indicate that the enzyme of the present invention is relatively stable in the pH range of 6 to 9.

3. Range of Suitable Temperature for Action and Temperature Stability Using the enzyme of the present invention, an enzyme reaction was carried out in the range of 10 ° C. to 60 ° C. according to the above-mentioned activity measurement method. The result is shown in FIG. As can be seen from the results in FIG.
To 60 ° C, and the optimum temperature was 45 ° C. To examine the stability of the enzyme of the present invention, the enzyme was treated at pH 7.0 at each temperature of 0 ° C. to 75 ° C. for 10 minutes, and the residual activity was examined. FIG. 4 shows the results. As is clear from the results of FIG. 4, no decrease in the activity was observed in the temperature treatment from 0 ° C. to 45 ° C., and the activity was stable, and about 60% of the residual activity was observed in the temperature treatment at 50 ° C.

4. Effect of Additive (Inhibitor) Using the enzyme of the present invention, an enzymatic reaction was carried out in a solution containing 1 mM of various additives according to the above-mentioned activity measurement method. The results are shown in Table 4 below.

Table 4 Additives (1 mM) No relative activity (%) 100.0 LiCl, NaCl, BaCl ₂ , CaCl ₂ , MnCl ₂ , MgCl ₂ , PbCl ₂ , ZnCl ₂ , CoCl ₂ , FeSO ₄ , 85. _{2~113.3 SnCl 2, NiCl 2, CdCl} 2, AlCl 3, FeCl 3 CuCl 2 3.3 HgCl 2 0.0 AgNO 3 0.0 iodoacetic acid 100.3 N-ethylmaleimide 88.4

5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) 59.6 p-chloromercuribenzoic acid (PCMB) 25.2 hydroxylamine 80.3 phenylhydrazine 15.7 semicarbazide 106.5 cysteamine 75 .1 aminoguanidine 97.5 D, L-penicillamine 108.5 D-cycloserine 106.4 3-methyl-2-benzothiazolinone hydrazone (MBTH) 73.6 NaN ₃ 142.6

O-Phenanthroline 42.8 8-Hydroxyquinoline 65.7 Ethylenediaminetetraacetic acid (EDTA) 105.0 2,2′-Bipyridyl 75.0 Tiron (disodium catechol-3,5-disulfonate) 99 .5 Diethyldithiocarbamic acid 91.8 KCN 22.0

From the results shown in Table 4, it is expected that the enzyme of the present invention has cysteine at the active center since its activity is inhibited by metals such as Cu, Hg and Ag, and SH reagents such as DTNB and PCMB. Was. In addition, the activity was inhibited by a chelating agent such as o-phenanthroline, 8-hydroxyquinoline, and 2,2′-bipyridyl, suggesting that the enzyme may contain Fe. further,
This enzyme was found to be pyridoxal 5'-phosphate (PLP) because no inhibition was observed with many carbonyl reagents.
Was also found to be a decarboxylase that was not required as a coenzyme.

5. Molecular weight The molecular weight determined by SDS electrophoresis was about 50,000. In addition, TSK-Gel G3000SW by HPLC
The molecular weight by gel filtration chromatography using a column was calculated to be 98,000. From this, it is considered that the enzyme of the present invention is a dimer enzyme having the same subunit.

[0035]

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples. Example (a) Culture of bacteria Bacillus megaterium PYR2910 using the medium (20 liters) shown in Table 1 above.
Was shake-cultured at 28 ° C. for 24 hours. (b) Collection and Purification of Enzyme The bacterial cell culture solution (20 liters) obtained in the above step (a) is centrifuged to collect cells, washed with physiological saline, washed with 20 mM phosphoric acid containing 1 mM dithiothreitol. Potassium acid buffer
(pH 7.0) (500 ml) and a cell-free extract was prepared by sonication (step 1).

The obtained cell-free extract was diluted to a protein concentration of about 10 mg / ml and salted out with ammonium sulfate (saturation 45-70%). The obtained precipitate was suspended in 20 mM potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol (step 2), dialyzed against the buffer, and then subjected to DEAE Sephacel column chromatography (column: 4). 0.5cm x 30cm)
20 mM potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol and KCl with 0.1M to 0.3M.
Elution was performed by the concentration gradient elution method of MKCl (step 3).
20% saturated ammonium sulfate was added to the active fraction, and octyl sepharose CL-4B column chromatography (column: 2.
(6 cm × 18 cm) and eluted with 20 mM potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol and ammonium sulfate in a concentration gradient elution method of 20% to 0% ammonium sulfate (step 4).

Next, the active fraction was subjected to butyl toyopearl column chromatography (column: 2 cm × 14 cm), and 16% to 0% with 20 mM potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol and ammonium sulfate.
Elution was performed by the ammonium sulfate concentration gradient elution method (step 5). The active fraction was dialyzed against 20 mM potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol, and then subjected to hydroxylapatite chromatography (column: 1.2 cm ×
8 cm) and 5 mM to 3 mM using potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol.
Eluted with a concentration gradient elution method of 00 mM potassium phosphate
(Step 6). Finally, TOYOPEARL HW-5
The gel was subjected to 5 gel filtration chromatography and eluted with a 100 mM potassium phosphate buffer (pH 7.0) containing 1 mM dithiothreitol and 0.2 M NaCl, and the active fraction was collected to obtain a purified enzyme (step 7). ).

Table 5 below shows the degree of purification of the enzyme in each step when purified by the above method. In addition, the enzyme activity in Table 5 is a value measured by the above activity measurement method. As shown in Table 5, in Step 7, 989 units / m
g protein purified enzyme (about 120 compared to the original enzyme solution)
Twice purified).

[Table 5] Table 5 Step Total protein Total activity Specific activity Purification factor Activity recovery rate (mg) (unit) (unit / mg protein) (×) (%) 1 12100 99900 8.26 1 100 2 5680 88500 15.6 1.89 88.6 3 1260 62100 49.3 5.97 62.2 4 411 38800 94.4 11.4 38.8 5 84.2 36700 436 52.8 36.7 6 47.0 23600 502 60.8 23.6 7 17.6 17400 989 120 17.4

[Brief description of the drawings]

FIG. 1 is a graph showing the relative activity of the enzyme of the present invention at each pH value.

FIG. 2 shows the enzyme of the present invention dissolved in various buffers,
The graph which shows the relative activity in each pH value after holding at 10 degreeC for 10 minutes.

FIG. 3 is a graph showing the relative activity of the enzyme of the present invention at each temperature from 0 ° C. to 60 ° C.

FIG. 4 is a graph showing the relative activity after treating the enzyme of the present invention at pH 7.0 at each temperature of 0 ° C. to 75 ° C. for 10 minutes.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields surveyed (Int. Cl. ⁷ , DB name) C12N 9/02 C12N 1/20 BIOSIS (DIALOG) JICST file (JOIS) MEDLINE (STN) WPI (DIALOG)

Claims (9)

(57) [Claims] 1. A pyrrole-2-carboxylic acid decarboxylase having the following properties; Action and Substrate Specificity (a) Pyrrole-2-carboxylic acid is decarboxylated to produce pyrrole. (b) HCO ₃ ^- presence, to produce a pyrrole-2-carboxylic acid pyrrole as a substrate for. 2. Optimum pH: 6.5 at 30 ° C 3. Stable pH: 6.0-9.0 4. 4. Range of optimal temperature for operation: optimal temperature is 45 ° C. Thermal stability: Stable up to 45 ° C. when kept at pH 7.0 for 10 minutes. 6. Molecular weight: The molecular weight of the subunit by SDS electrophoresis is 50,000, and the molecular weight by TSK-Gel G3000SW gel filtration column is 98,000. 7. Michaelis constant Km value for pyrrole-2-carboxylic acid: 23.8 × 10 ⁻³ M. 2. The enzyme according to claim 1, which is obtained from a bacterium belonging to the genus Bacillus. 3. The method according to claim 2, wherein the bacterium is Bacillus megaterium (B.me
gaterium). 4. The enzyme according to claim 2, wherein the bacterium is Bacillus megaterium PYR2910 strain deposited with the Institute of Microbial Industry and Technology, National Institute of Industrial Science and Technology as No. 13145. 5. A bacterium having the ability to produce pyrrole-2-carboxylate decarboxylase according to claim 1, which belongs to the genus Bacillus, and cultured in a pyrrole-2-carboxylate decarboxylase-inducing medium. From the cells, the pyrrole-2-
A method for producing pyrrole-2-carboxylic acid decarboxylase, comprising isolating carboxylic acid decarboxylase. 6. The method according to claim 5, wherein the pyrrole-2-carboxylic acid decarboxylase is isolated from the cells in the presence of an organic acid having 1 to 10 carbon atoms. 7. The pyrrole-2- belonging to the genus Bacillus.
The method according to claim 5 or 6, wherein the bacterium capable of producing carboxylate decarboxylase is Bacillus megaterium. 8. The pyrrole-2- belonging to the genus Bacillus.
A bacterium capable of producing carboxylate decarboxylase has been deposited in Bacillus megaterium PYR29 deposited with the National Institute of Microbial Industry as National Institute of Industrial Science No. 13145.
The method according to claim 5, 6 or 7, wherein the number of strains is 10. 9. Bacillus megaterium PYR2910
No. 13145 of the microbes.