JPH03247292A - Production of 2,3-dihydroxy-p-toluic acid and microorganism used therefor - Google Patents

Abstract

PURPOSE:To efficiently produce the subject toluic acid useful as an antioxidant, etc., on a practical level by culturing a specific microorganism using p-xylene, etc., as a single carbon source in a p-xylene-containing culture medium. CONSTITUTION:A 2,3-dihydroxy-p-toluic acid-producing microorganism, belonging to the genus Arthrobacter and capable of growing using p-xylene or p-toluic acid as a single carbon source is cultured in a culture medium containing the p-xylene or p-toluic acid to collect the objective p-toluic acid from the resultant culture. Furthermore, the aforementioned producing microorganism is preferably Arthrobacter.sp. MX-3 deposited as FERM P-11173.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention provides a method for producing 2.
．． A method for producing 3-dihydroxy-p-toluic acid,
and regarding the microorganisms used in the production method.

[Problems to be solved by conventional technology and invention] 2.3
-Bicidroxy-p-toluic acid has two hydroxyl groups, a methyl group, and a carboxyl group adjacent to each other in its molecule, and its structure makes it an extremely effective antioxidant and preservative.
Although it has been thought to be useful as a bactericidal agent, a raw material for chemical synthesis, etc., chemical synthesis is extremely difficult due to its special structure, which has as many as three types and four substituents on the benzene nucleus.

Therefore, although it is possible to synthesize it as a reagent using extremely complicated techniques such as protection of hydroxyl groups and separation of oxidation products, a synthetic method that can withstand industrial practical use has not yet been developed.

On the other hand, p-
It has been known for a long time that this compound exists as an intermediate in the xylene metabolic system (APPLIED MICRO
BIOLOGY, June 1969. P, 85
3856).

Research is also underway to produce this compound using microorganisms, but the microorganisms have low tolerance to p-xylene, and none of them have reached a practical level (U.S. Pat. No. 3,592,845).
No. Therefore, a practical method for producing 2,3-dihydroxy-p-toluic acid has been desired.

[Means to solve the problem]

Under these circumstances, the present inventors investigated using various microorganisms in order to solve the above-mentioned problem, and found that 2,3-dihydroxy-p-toluyl alcohol can be produced by using a certain type of microorganism belonging to the genus Arylobacter. The present invention was completed by discovering that it can be produced on a practical level.

That is, the present invention relates to a 2,3-dihydroxy-p-toluic acid-producing bacterium that belongs to the genus Slobacter and can grow using p-xylene or p-4 toluic acid as a sole carbon source.
2,3-dihydroxy-p-)ruyl, which is characterized by culturing in a medium containing p-xylene or I)-toluic acid, and collecting 2゜3-dihydroxy-p-toluic acid from the culture. Method for producing acid; enzyme derived from resting cells or cells of 2,3-dihydroxy p-toluic acid-producing bacteria belonging to the genus Arilobacter and capable of growing using p-xylene or p-toluic acid as a single carbon source A process for producing 2,3-dihydroxy-p-toluic acid characterized by contacting p-xylene or p-toluic acid with p-xylene or p-toluic acid, and The present invention provides a 2,3-dihydroxy-p-toluic acid-producing bacterium that can be grown as a source of 2,3-dihydroxy-p-toluic acid.

The 2,3-dihydroxy-p-toluic acid-producing bacterium of the present invention (hereinafter referred to as "the microorganism of the present invention") belongs to the genus Arylobacter and can grow using p-xylene or p-toluic acid as a sole carbon source. and 2,3-dihydroxy=
As long as it has the ability to produce p-toluic acid, its origin does not matter. For example, the microorganism of the present invention can be grown in a medium to which only p-xylene or p-toluic acid is added as a carbon source, and tested to determine whether it has the ability to produce 2,3-dihydroxy-p-toluic acid. It can be separated by screening.

Details of the separation method are illustrated below. That is, first, a medium (hereinafter referred to as a medium) that does not contain a carbon source among the components necessary for the growth of microorganisms is dispensed into a test tube or the like, sterilized, and then soil collected in advance is added. P-xylene or p-toluic acid is added to this, and culture is performed using a test tube shaker or the like. This culture solution was subcultured using a platinum loop into another test tube, etc., which had been previously dispensed and sterilized, and then p
- Add xylene or p-toluic acid and further incubate on a test tube shaker. After applying this culture solution or a culture solution diluted with sterile water etc. to an agar medium etc., p.
- Add xylene or p-toluic acid and further culture. The microorganism □ of the present invention can be selected by isolating colonies obtained by culturing and confirming the ability of each strain to produce 2<3>-dihydroxy-p-toluic acid.

General culture medium components other than the carbon source can be used for the culture medium used in the separation operation. That is, nitrogen sources include, for example, inorganic nitrogen compounds such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium carbonate, ammonium acetate, ammonium nitrate, sodium nitrate, urea, yeast extract, dried yeast, peptone,
Organic nitrogen sources such as meat extracts, cornstarch liquor, casamino acids, etc. can be used. Examples of inorganic salts that can be used include potassium, sodium, iron, magneum, manganese, copper, calcium, and cobalt salts. In addition, the culture conditions in the separation operation may generally be those that do not kill the microorganisms, such as aerobic culture at a pH of about 5 to 9 and a temperature of about 20 to 40° C. for about 1 to 30 days.

To confirm the ability of the obtained strains to produce 2,3-dihydroxy-p-toluic acid, each strain was injected with p-xylene or p-toluic acid alone into the same medium as used in the above separation procedure. The resulting culture solution was separated by centrifugation, etc., and the supernatant was analyzed using an appropriate analytical method, such as high-performance liquid chromatography (HP liquid chromatography).
LC), gas chromatography (GC), nuclear magnetic resonance spectroscopy (NMR), infrared absorption spectrum (IR
), ultraviolet absorption spectrum (UV), etc. may be used for analysis.

In the separation operation, 2,3-dihydroxy-p-
If toluic acid-producing bacteria cannot be obtained or the productivity is low, mutate the obtained strain that is able to assimilate and tolerate p-xylene or p-toluic acid using a general mutation method. It is effective to use the same method described above for confirming the ability to produce 2,3-dihydroxy-p-toluic acid for mutant strains.

All general mutation methods for microorganisms can be used as mutation methods.For example, methods for using physical mutagens such as ultraviolet rays and ionizing radiation as mutagens and irradiating microorganisms on an agar medium include ultraviolet irradiation and cobalt-60 irradiation. There are laws etc. Methods using chemicals as mutagens include ethyl methanesulfonate (EMS), N-methyl-N'
- Chemical mutagens such as alkylating agents such as N-nitrosoguanidine (NTG) and ethylnitrosouric acid (BNII) and base analogs such as bromodeoxyuridine (BrdUrd) are added to microorganisms in a buffer solution. A method of culturing can be mentioned. Further, examples of methods using biological mutagens include transboson mutagenesis.

The microorganisms obtained by the above-mentioned isolation procedure, the x-37 strain, which is an example of its mutant strain, and the similar mutant strain MX-3 strain were analyzed using the bacterial classification and identification method [Purgation Aids Manual of Microorganisms].
Systematic Bacteriology (Bergey
s Manual of SystematicBac
Identification tests were performed according to the 1st edition, 1984]. As a result, X-37 strain and MX-
The three strains have the following mycological properties.

(a) Morphology ■ Cell shape and size Coccus (diameter 1.2-1.7 μm)... Dormant bacilli (0,
6-1. OX2-8μm)・Proliferation phase ■Cell pleomorphism
Yes ■ Mobility No ■ Spores present None ■ Durham staining positive ■ Anti-acidity No b) Growth status in each medium ■ Broth agar plate medium Good growth, orange (shape), round (bulge), navel-shaped ( Peripheral) Golden rim (Surface) Rough surface (glossy) Dull luster ■ Meat juice agar slant medium Good growth, orange (ridged) thin film-like to plate-like ■ Meat juice liquid culture Good growth Thin film with floating matter, sediment ■ Meat juice gelatin puncture Do not liquefy cultured gelatin ■ Lidomus milk alkaline ■ BCP milk alkaline (C) Physiological properties ■ Reduction of nitrate ■ Nitrogen reaction ■ MR test ■ vP test ■ Formation of indole ■ Formation of hydrogen sulfide ■ Hydrolysis of starch ■ Utilization of citric acid (Properties) (+) (-) (-) (-) (=) (-) (-) Buttery Koser's medium Christensen's medium to be used ■ Use of inorganic nitrogen Nitrate to be used Ammonium salt to be used ■ Pigments to be used Formation of (-) ■Urease (-) ■Oxidase (-) 0 Catalase (+) ■Growth range temperature Does not grow at 10℃.Grows at 26.5-37.0℃.Does not grow at 45.0℃.pH4, Does not grow at 20 Grows at 5.23-9.45 Does not grow at 10,00 ■ Attitude towards oxygen Aerobic @ 0-F test Negative ■ Nitrogen fixation (=) ■ Utilization of carbohydrates Generation of acid production gas ( 1) L-arabinose (+>(->(
2) D-xylose (+) (-) (3
) D-glucose (+>(-) (4) D-mannose (+) (-) (5) D-fructose (+) (-) (6) D-galactose (+)
(-) (7) Maltose (+)
(-) (8) Sucrose (+)
(-) (9) Lactose (+) (
-) αQ trehalose (+) (-)
Ql) D-Sorvit (+>(-) ■D-Mannit (+) (-) α Inosit
(+) (->α glycerin
(+> (-)α starch (+
) (-) From the above test results, x-37 strain and MX-3
The strain is recognized as belonging to the genus Arilobacter. However, conventional bacteria belonging to the genus Arilobacter do not have resistance to aromatic hydrocarbons. In order to confirm this, we used Arthrobacter globiformis (Arthrobacter globiformis), a species of the genus Arthrobacter.
lobiformis) IFO12137, Arithrobacter viscosus (＾rthrobacterv
iscosus) IFO13497 and X-37 and M
The resistance of X-3 to each solvent was investigated. The results are shown in Table 1.

Table 1: No resistance, +: Resistance*3 Arthrobacter sp.
) was recognized as a new strain belonging to Arithlobacter sp. X-37 and Arilobacter sp. MX.
It was named -3. These strains were submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute No. 11174 (FBRM P
-11174) and FBR No. 11173 (FBR
It has been deposited as M P-11173).

Note that x-37 strain and MX-3 strain are MX-3 strain 2.
It has the ability to produce 3-dihydroxy-p-toluic acid, and
The difference is that 37 stocks do not have this.

In order to oxidize p-xylene or p-toluic acid to produce 2,3-dihydroxy-p-toluic acid using the microorganism of the present invention, the microorganism of the present invention is cultured in a medium containing p-xylene or p-toluic acid. Alternatively, a resting cell of the microorganism or an enzyme derived from the cell and p-xylene or p-
Toluic acid may also be contacted.

Next, a method for producing 2,3-dihydroxy-p-toluic acid using, for example, Arylobacter sp. MX-3 strain (hereinafter referred to as ``synthesis'') as the microorganism of the present invention will be described. There are no particular restrictions on the medium to be used when cultivating the medium, as long as it contains p-xylene or 1)-toluic acid, as long as the bacteria grow well.

That is, as a nitrogen source, for example, ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium carbonate, ammonium nitrate, sodium nitrate,
Inorganic nitrogen compounds such as urea and organic nitrogen sources such as yeast extract, dried yeast, peptone, meat extract, cornstarch liquor, and casamino acids can be used.

Examples of inorganic salts include potassium, sodium, iron,
Various salts such as magnesium, manganese, copper, calcium, and cobalt can be used.

As a carbon source, in addition to p-xylene or p-toluic acid, benzoic acid, phthalic acid, salicylic acid, glucose, fructose, starch hydrolyzate, meat extract, etc., which can be assimilated and multiplied by the phlegm, are added as co-oxidation substrates. It is preferable to do so.

The culture conditions may be as long as the cells do not die and can proliferate; for example, the culture temperature is about 20 to 37°C, more preferably about 25°C.
Preferably, the culture is carried out at ~37°C, the pi of the medium is about 5.2-9.4, more preferably about 6.0-8.0, and in an aerobic atmosphere for about 1-30 days.

In the present invention, 2,3-dihydroxy-p-toluic acid can be produced from either p-xylene or p-toluic acid, but it is desirable to use p-xylene from the viewpoint of raw material cost. The raw material p-xylene may be added from the beginning of the culture or during the culture. If the amount of p-xylene added is significantly increased, the growth of the cells will be lowered, so when performing conversion in a growth system, the amount of p-xylene added should be 0.01 to 10 wt%, more preferably 0.01 to 10 wt%, based on the medium. 1-5
It is desirable that the content be within the range of wt%. Since p-xylene tends to evaporate during culture, it is desirable to provide a cooling trap or the like or to continuously add p-xylene to prevent evaporation. Furthermore, since the pH in the medium decreases with the production of 2,3-dihydroxy-p-toluic acid, it is desirable to adjust the pH with an alkaline solution such as sodium hydroxide, ammonia, potassium hydroxide, or the like. When using p-toluic acid as a raw material, it is necessary to neutralize it with an alkaline solution such as sodium hydroxide or potassium hydroxide beforehand after adding it to the culture medium. In this case as well, the amount added is 0.01 to 10 wt%, more preferably 0.1 to 10 wt%, based on the medium.
It is preferably within the range of 5 wt%. Cultivation can be carried out under aerobic conditions, such as in a Sakalo flask or jar fermentor.
can be used. Production can be done either batchwise or continuously using a bioreactor or the like.

Next, 2.
When producing 3-dihydroxy-p-toluic acid, the bacterial cells used can be produced under the same culture conditions as described above. The obtained bacterial cell culture can be used as it is as an enzyme source, but the microbial cells obtained by solid-liquid separation by operations such as centrifugation are washed with a solution such as phosphate buffer, and the solution is It can also be used suspended in. Furthermore, enzymes derived from bacterial cells can be purified by conventional methods. That is, for example, a suspension of microbial cells is crushed using ultrasonic waves, a French press, a high-pressure homogenizer, etc., the resulting crushed bacterial cells are subjected to solid-liquid separation by centrifugation, etc., and then subjected to column purification and electrolysis. A purified enzyme can be obtained using general purification techniques such as electrophoresis. Also,
It is also possible to use immobilized microbial cells and enzymes, and by using immobilized ones, the reaction can be carried out efficiently. This immobilization can be carried out by a conventional method such as a calcium alginate method, a polyacrylamide gel method, a polyurethane resin method, or a photocrosslinking resin method.

Using these bacterial cells or bacterial cell-derived enzymes, 2.3
-To produce dihydroxy-p-toluic acid,
The bacterial cells or enzymes derived from the bacterial cells are treated with p-xylene or p-xylene.
- The reaction may be caused by contacting with toluic acid in a phosphate buffer or the like. The reaction temperature and pH are preferably the same as those for the culture conditions of the present bacteria described above, and the reaction is preferably carried out in an aerobic atmosphere. In this reaction, it is preferable to appropriately add an electron donor such as methanol, ethanol, hydrogen, nicotinamide adenine dinucleotide (NAD), formaldehyde, or formic acid as an energy source.

2,3 in the culture solution or reaction solution obtained as above.
-Dihydroxy-p-toluic acid can be purified by conventional methods. That is, filtering the culture solution or reaction solution,
After processing by centrifugation etc., the obtained solution is made acidic by adding an acid solution such as hydrochloric acid or sulfuric acid,
Dihydroxy-p-toluic acid can be recovered. It is also possible to recover by methods such as solvent extraction, and known purification methods such as column chromatography can be used in combination as appropriate.

〔Effect of the invention〕

According to the present invention, p-xylene or p-toluic acid is grown as the sole carbon source, 2,3-dihydroxy-p-
2,3-dihydroxy-p from p-xylene or p-toluic acid using a novel microorganism capable of producing toluic acid, its dormant cells, or an enzyme derived from the cells.
- Toluic acid can be produced efficiently.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to these examples.

Example 1 The culture medium components shown in Tables 2 and 3 were each dissolved in tap water 1β to prepare a culture medium (the pH of each culture medium was 7.0).

Margin Table 2 Below Margin Table 3 Below Margin Table 3 10 rnl of the culture medium shown in Table 2 was placed in a test tube with an outer diameter of 21 mm and sterilized at 121° C. for 15 minutes. After cooling, 5 was collected at an oil refinery facility in the Goi region of Chiba Prefecture and the Yokkaichi region of Mie Prefecture.
0.5 g of each of the 000 types of soil were added, and 100 μl of separately filter-sterilized p-xylene was added to each, and cultured with reciprocal shaking at 30° C. and 25 Orpm using a test tube shaker for 7 days. Ten volumes of the culture medium shown in Table 2 were dispensed into separate test tubes and sterilized.

Three platinum loops of the above culture solution were inoculated into this, and 100 μl of p-xylene was added to each. After the addition, culture was performed using a test tube shaker at 30° C. and 25 Orpm for 7 days with reciprocal shaking. If growth is observed, use sterile water for 10 minutes.
After diluting in a range of 4 to 10 times, the agar medium shown in Table 3 prepared earlier was applied to the agar plate medium placed in a Petri dish, and p-xylene was added in 1-by-1 increments to the lid side of the Petri dish. Afterwards, the cells were cultured at 30°C for 7 days. The resulting colonies were isolated and subcultured into test tubes prepared by adding 2, 3, 4, 5, and 10 wt % of p-xylene to the medium shown in Table 2, respectively, and cultured. As a result, Arylobacter sp.
-37 stocks were selected.

2. of the obtained Arilobacter sp. X37 strain.
5fi production capacity of 3-dihydroxy-p-toluic acid
gB, Arylobacter sp. X-37
When the culture solution of the strain was analyzed by HPLC, good results were not obtained.
37 strains were subjected to mutation treatment using the nitrosoguanidine method shown below, and 2,3-dihydroxy-p-
We decided to select a strain with high toluic acid production ability.

That is, 10 m of the culture medium in Table 2 was placed in a test tube with an outer diameter of 21 mm.
1! After cooling, 100 μl of separately sterilized p-xylene was added (medium 1). One platinum loop of Arithlobacter sp.
Culture was performed with reciprocal shaking at pm for 3 days. This culture solution was centrifuged at 15,000 rpm for 30 seconds for washing, the supernatant was discarded, and the suspension was suspended in the same amount of Tris-maleic acid buffer (pt16.0) as the culture solution. This washing operation was repeated two more times. After this, 3 again at 15000 rpm.
Centrifuge for 0 seconds, discard the supernatant, and add 100 μg/N-methyl-N'-nitro N-nitrosoguanidine (NTG) to Tris-maleic acid buffer (pH 6,0).
Add the solution dissolved at the concentration of - to the original culture solution and add 3
It was incubated for 1 hour at 0°C and 25 Orpm.

The agar medium shown in Table 3 was sterilized at 121°C for 15 minutes and spread on petri dishes to prepare an agar plate medium. After washing the bacterial cells incubated for 1 hour as described above three times in the same manner as described above, a solution diluted 1,000 times the original culture solution with the same buffer was applied to this Petri dish, and a plating solution was applied to the lid side of this Petri dish. -Xylene 1- was added and cultured at 30°C for 4 days.

Each obtained colony was inoculated into medium 1 with a platinum loop,
Culture was performed using a test tube shaker at 30° C. and 250 rpm for 3 days with reciprocal shaking. As a result of analyzing the culture solution, 2,3-
Ninety-eight bacterial strains producing dihydroxy-p-toluic acid were obtained. Among these 98 strains, the most 2,3-dihydroxy-p-
The mutant strain that produced a high amount of toluic acid was named Arilobacter sp. M13.

Example 2 10 ml of the culture medium shown in Table 2 prepared in Example 1 was placed in a test tube with an outer diameter of 21 mm and sterilized at 121 t' for 15 minutes. After cooling, one platinum loop of Arithlobacter sp. MX-3 was transplanted, and 100μ of separately filter-sterilized p-xylene was added!
and incubate at 30°C 25 Orp using a test tube shaking incubator.
Culture was performed at m for 3 days with reciprocal shaking. After centrifuging the culture solution, quantitative analysis of the supernatant by IIPLc revealed that the amount of 2,3-dihydroxy-p-toluic acid produced was 15
mg/j! Met.

In addition, the produced 2,3-dihydroxy-p-toluic acid was extracted with diethyl ether, separated and purified by thin layer chromatography (TLC), and structurally analyzed by NMR and MS. dihydroxy-p-
It was confirmed that it was toluic acid.

Example 3 The same procedure as in Example 2 was carried out, except that 100 mg of p-toluic acid was used instead of p-xylene as the substrate to be added, and the mixture was added to the medium and then neutralized to pH 7.0 with an aqueous sodium hydroxide solution. , the generated 2゜3-dihydroxy-p
-Toluic acid was 9/1.

Example 4 Add the substrate to 100 mg of p-toluic acid and add 10
μ! The procedure was carried out in the same manner as in Example 3 except that p-xylene was added, resulting in 2,3-dihydroxy-p
-Toluic acid was 30 μ/l.

Example 5 500mj! In a Sakalo flask, the second sample prepared in Example 1 was added.
200 d of the medium shown in the table was added and sterilized at 121°C for 15 minutes. After cooling this, culture solution 10- prepared in the same manner as in Example 2 was inoculated thereto. Furthermore, add 1 ml of sterilized p-xylene to this! and sodium benzoate 50
mg was added. Incidentally, an apparatus as shown in FIG. 1 was used. That is, the liquid 1 containing the above-mentioned culture liquid etc. is located at the bottom of the Sakaro flask 2, and the upper part of the flask 2 is provided with a jacket 3 whose top is open. -Xylene (1-) 4 is contained, and the flask is sealed with a silicone stopper 5.

Using such an apparatus and flask shaking culture solution, 30
When cultured at 115 rpm for 10 days, 2,3-
Dihydroxy-p-toluic acid is 350mg/j! Obtained.

Example 6 Yeast extract 1 g/l was added to the medium shown in Table 2 prepared in Example 1.
The culture medium 11 containing the above was placed in a 21-jar fermentor equipped with a cooling trap and sterilized at 121°C for 15 minutes. After cooling, p-xylene was mixed with 10- and sodium benzoate 500
mg was added. Furthermore, culture solution 100- prepared in the same manner as in Example 2 was inoculated, and 2N hydroxylated)
30°C while adjusting p) to 16.8 with Jum aqueous solution.
When cultured for 5 days under the condition of 500 rpm aeration 1[2β/min, 3200 μ/1 of 2,3-dihydroxy-p-toluic acid was obtained.

Example 7 One platinum loop of Arilobacter sp. This culture solution was washed at 15,000 rpm for 3 minutes.
The mixture was centrifuged for 0 seconds, the supernatant was discarded, and the mixture was suspended in the same amount of Tris-maleic acid buffer (pH 6.0) as the culture solution. This washing operation was repeated two more times. Next, 0.1wt% sodium benzoate was added to the culture medium shown in Table 3 obtained according to Example 1, sterilized at 121°C for 15 minutes, and 20rrLl was placed in a petri dish to prepare an agar plate medium. 100 μl of cloudy bacterial cells were added and spread using a spreader.

After directly irradiating the petri dish with a 15 watt ultraviolet lamp from a distance of 30 cm for 1 minute, culturing was carried out at 30° C. for one week, and the colonies that appeared were isolated. Each of the obtained colonies was inoculated into medium 1 using a platinum loop, and cultured with reciprocating shaking for 3 days at 30° C. and 25 Orpm using a test tube shaker. As a result of analyzing the culture solution, 2,3-dihydroxy-p-
Eleven strains producing 4-ruylic acid were obtained.

Comparative Example 1 The strain to be inoculated is Arilobacter sp.
Microtechnology Research Institute No. 11174 (FBRM P-11174
), Arilobacter globiformis IFO12
137, Arilobacter viscosus IPO134
97, Pseudomonas fluorescens (Pseud
omonasfluorescens) IFO392
4. Pseudomonas putida
When the same procedure as in Example 2 was carried out except that Pseudomonas putida ATCC 17484 and IFO 12996 were used, 2,3-dihydroxy p-toluic acid was not produced at all.

[Brief explanation of drawings]

FIG. 1 shows a Sakalo flask with a jacket installed inside, which was used in Example 5 and used in the method of the present invention. 1...Prepared medium 2...Sakaro flask 3...Jacket 4...P-xylene 5...Silicone stopper and above Figure 1 Procedure amendment (voluntary) 1゜2゜Description of the case 1990 Patent Application No. 44332 Name of the invention 2. Process for producing 3-dihydroxy-p-toluic acid and its use Address 1-3-6 Nihonbashi Ningyocho, Chuo-ku, Tokyo (103)
Kyodo Building Tel: (669) 0904 (Main) (687)
0) Patent attorney Miyuki Ariga 1st -m:-
7 (7756) Patent Attorney Toshio Takano 1st -m:-=(967
3) In the "Detailed Description of the Invention" section of the specification subject to the amendment by Patent Attorney Toshio Nakajima, the fourth line from the bottom of the second page of the amended specification contains "2.3-bicidroxy-p-toluic acid.""2,3-dihydroxy-p-toluicacid" Correct. (2) Page 8, line 14 r (BrdUrd) J and Aruo “(Brdurd) J” is corrected. Page 10, line 7 from the bottom, “■Nitrogen reaction” is replaced with “■Denitrification reaction” I am corrected. On page 13, line 4, rX-37 and MX-L+ are corrected to "rX-37 strain and MX-3 strain." (5) In the third line from the bottom of page 13, the text r*3 X-37J is corrected to read "*3 X-37 stocks." (3) (4) (1) and later revision (6) On page 13, line 2 from the bottom, the text "r*4 MX-3J" is corrected to "r*4MX-3 stock." (Power, page 24, lines 7-8 and page 28, lines 6-8 from the bottom)
Line 5 says ``Arythrobacter sp. :correct. (8) In the third line from the bottom of page 25, the text ``Arithlobacter sp. MX-3J'' has been corrected to read ``Arithlobacter sp. MX-3 strain''. (9) In the fifth line from the bottom of page 27, the statement ``With a silicone plug 5'' is corrected to ``With a breathable silicone plug 5.'' α-口 Page 29, line 2, "Continuing Example 1" is corrected to "Next Example 1."

Claims (1)

[Claims] 1. Belongs to the genus Arilobacter, and contains p-xylene or p-xylene.
- 2,3-dihydroxy-p-toluic acid producing bacteria that can grow using toluic acid as the sole carbon source are cultured in a medium containing p-xylene or p-toluic acid, and the 2,3- A method for producing 2,3-dihydroxy-p-toluic acid, which comprises collecting dihydroxy-p-toluic acid. 2. Belongs to the genus Arilobacter and contains p-xylene or p
- A feature of the invention is that a resting bacterial cell or an enzyme derived from a 2,3-dihydroxy-p-toluic acid producing bacteria that can grow using toluic acid as a sole carbon source is brought into contact with p-xylene or p-toluic acid. 2,3-dihydroxy-p-
Method for producing toluic acid. 3. Belongs to the genus Arilobacter and contains p-xylene or p
-Can be grown using toluic acid as the sole carbon source2,3
-Dihydroxy-p-toluic acid producing bacteria. 4. FERMP-1117 No. 11173 (FERMP-1117)
3) Aristlobacter sp. M deposited as
2,3-dihydroxy-p according to claim 3, which is X-3
- Toluic acid producing bacteria.