JPS58162296A - Preparation of 2-keto-l-gulonic acid - Google Patents

Abstract

PURPOSE:To prepare 2-keto-L-gulonic acid, in high efficiency, by culturing a 5-keto-D-gluconic acid metabolism-deficient mutant derived from a 2-keto-L- gulonic acid-producing strain in a medium containing 2,5-diketo-D-gluconic acid. CONSTITUTION:A 5-keto-D-gluconic acid metabilism-deficient mutant (e.g. Corynebacterium sp. FERM-BP No.107) is obtained by the mutagenic treatment of a 2-keto-L-gulonic acid-producing strain (e.g. Corynebacterium sp. FERM-P No.2770) by ultraviolet irradiation, etc., and the mutant is cultured in a medium added with 2,5-diketo-D-gluconic acid. Only 2-keto-L-gulonic acid is accumulated in the medium without producing unnecessary 2-keto-D-gulconic acid, and the accumulated 2-keto-L-gulonic acid is separated from the medium in high efficiency.

Description

DETAILED DESCRIPTION OF THE INVENTION In particular, it is possible to eliminate the accumulation of l-D-gluconic acid in the medium. 2,3;-diketo D-gluconic acid 2
- A method for producing keto L-gulonic acid microbially and a mutant microorganism used for carrying out the method.

The present inventors first discovered that many microorganisms (referred to as niketo-shi-gulonic acid-producing bacterial strains (1)) that can produce euke-1--L-gulonic acid from 2,3-diketo-D-gluconic acid.
We have discovered a method for producing λ-keto L-gulonic acid. (Special public Shojθ-2lL1, Special public Sho 5
3-. 2. , tθ33.

and Tokuko Shoj Otsu-/jg No. 77). In both cases (1), the main product 2-keto L-gulonic acid is produced from the raw material, j-diketo D-gluconic acid, and suketo D-gluconic acid is produced as a by-product. In order to prevent this undesired 2-keto D-gluconic acid from accumulating in the medium, a mixed culture method was invented. (Refer to the Tokukosho!'l--/9Getsu g issue) This time, the present inventors mutated (1) using the usual method, and selected j-ke1-- from the mutated (1). D
- A mutant strain that does not grow on gluconic acid but grows on D-gluconic acid (this mutant strain is called a j-keto-loguconate metabolism defective mutant strain (1)) is induced, and this mutant strain (1) grows on D-gluconic acid.
, 3-diketo-D-gluconic acid has a property of not producing 2-ke1-D-gluconic acid.

Also, culture (1) and 2. ! Based on this finding, we have found that when contacted with diketo D-gluconic acid, euke-L-gulonic acid accumulates without substantially accumulating undesirable 2-keto D-gluconic acid in the medium. The invention has been completed as described in the claims at the beginning.

That is, according to the present invention, Suke 1. -J-kel--D-gluconic acid metabolism\deficient mutant strain (1) derived from L-gulonic acid producing strain (1) or processed products thereof. 2,
! ;S. A method for producing nor-keto-L-gulonic acid is provided, which comprises bringing su-keto-L-gulonic acid into contact with a medium containing X-diketo-D-gluconic acid or a salt thereof to accumulate su-keto-L-gulonic acid in the medium and collecting the same.

According to another aspect of the present invention, the microorganism used for carrying out the above production method, that is, the norketo-L-gulonic acid producing strain (1) belonging to the coryneform group, is used as a parent strain and is derived from this. Also provided is a mutant strain (1) deficient in j-keto-low gluconate metabolism.

−　μm (1) To efficiently obtain good (1), use ultraviolet irradiation.

Treatments such as X-ray irradiation or contact with mutagenic agents such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG), acriflavine, and ethylmethanesulfonic acid are adopted. . Mutation treatment was applied (
Dilute 1) to an appropriate concentration, or culture it again and then dilute it to an appropriate concentration, and add 1 part (0.t~/πt) of the diluted solution to (2!;
-Grow by coating on a minimal agar medium containing 2% D-gluconic acid (containing vitamins and trace elements necessary for growth).

The grown bacterial colony is θj~. Transfer to a minimal agar medium containing 5-keto D-gluconic acid (replica method),
Colonies that do not grow on D-gluconic acid medium are selected. The selected mutant strain has either lost its metabolic activity of 1-keto D-gluconic acid or has decreased it to less than ゛'//θ of the parent strain (1) (the mutant strain obtained in this way is - Rho gluconate metabolism defective mutant strain (referred to as It)).

(I) was cultured and brought into contact with 2,3-diketo-D-gluconic acid. It has been confirmed that it produces As an example of (II) obtained by the above method, (I) is Corynebacterium sp.
m sp. FERM-P277θ, ATCC NI13
109θ) mutant strain (FERM-BP-10
Corynebacterium sp.
P26g7, ATCC Nα3/θzal) mutant strain (
Examples include FERM-BP-/θ7).

(Margins below) -ri - The above fact, that is, the fact that the ability to produce 2-keto D-gluconic acid could be lost or significantly weakened by deleting the ability to metabolize j-keto D-gluconic acid, indicates that coryneform This is common to all nyketo-L-gulonic acid producing bacteria belonging to the group (according to the definition in the Phys. Manual of Determinative Bacteriology, G Edition).

There are no particular restrictions on the medium used for culturing (1). For example, sugars and polyhydric alcohols such as glucose, sucrose, chrycerin, and waste sugar t are used as carbon sources, and commonly used nitrogen compounds are used as nitrogen sources, for example.

Corn steep liquor, peptone, meat extract, ammonium salts, and nitrates are used.

Inorganic salts (e.g. calcium, magnesium).

Salts such as potassium, zinc, manganese, iron, etc.) or factors that promote the production of the target substance may be added. The medium composition depends on the characteristics of the strain used and raw materials 2. It varies depending on the amount of t-diketo-D-gluconic acid used and other conditions.

As the raw material 29.5-diketo-D-gluconic acid.

An aqueous solution of 2.3-diketo-D-gluconate may also be used, or Erwinia sp.

The genus Gluconobacter (the genus Gluconobacter here refers to
It is based on the G edition of Bacteriology and belongs to the genus Acetobacter, Acetomonas, and Gluconobacter in the 7th edition. 2. From D-glucose. j
It is also possible to use a filtered and sterilized solution of a fermentation liquor obtained by culturing a microorganism capable of producing -diketo-D-gluconic acid, or a solution treated with a sterilization agent (for example, sodium dodecyl sulfate).

2. The conditions for adding 3-diketo-D-gluconic acid are that it is added intermittently.

What is the timing of addition of s,j-dikel--D-gluconic acid?

Add it at the start of culture or when the growth of bacteria ends. It is desirable to add within 10 hours before and after the end of the treatment.

The culture time is 2. j-dikel--It varies depending on the addition conditions of D-gluconic acid, but it is usually
After adding D-gluconic acid, culture for 2 hours to 9 hours, 2.
The end point of the culture is defined as the point in time when 3-dikel---D-gluconic acid disappears L7.

2-1--L-gulonic acid 9.2-keto-D in the fermentation liquid
- Curconic acid, osdiketo D-gluconic acid,
Common sugars and related substances can be separated and quantified by analytical means. For example, gas chromatography,
Paperk O? )')” Laughee.

Thin layer chromatography is used. these are,
It can be used in various ways depending on the purpose.

The present invention will be explained in more detail using Examples below.

Example / (Production of 2-keto L-gulonic acid) (1)
Seed medium D-glucose/θ-related bacterium
Yeast Extra Lac L- (Dir co)
05% Bacto Peptone (Dirco)
03% potassium monophosphate (KH2PO,)
θ/magnesium sulfate (MgSO, 7H20)
0θ2 ratio (pH 7-72 with 70% NaOH)
Pour enough θml into a θθml Erlenmeyer flask. ``Sterilize for 0.2θ minutes.'' (2) Main culture medium D-glucose/θ-related corn・
Stape Liquor (C8L) 3θ KH2PO
4,θ/CHMgSO, ・7H,20θ02CH (adjusted to pH 7-7.2 with 10tIONaOH, jθ
Pour enough jθπ into a θml Erlenmeyer flask//K”0.
Sterilize for 2θ minutes. ) Powdered calcium 1,5-diketo D-gluconate is made into an aqueous solution of a very high temperature, and this aqueous solution is filtered and sterilized using a filter that has been sterilized in advance.

(4) Preparation of 2.3-diketo-D-ri')LrJ acid fermentation broth Erwinia punctata (FERM-P3
4132, kTcc 111 [L3/Otsu λOtsu] in 9 kinds of media (/Kariichi Glucose, j%C3L, θ/%DenchiP)
to, 0021 Mg5O, -7H2O, 03% calcium carbonate (CaCO3).

pH g ~ 7θ, jθθynl dispense jθν into Erlenmeyer flask, //! Sterilize for 20 minutes) and inoculate it with a platinum loop.
Incubate with shaking at °C for ~ // hr. (amplitude 717LW
I, 27Or, P, m, the same applies hereafter). Use this seed culture solution! r! ;ttrl Tj-diketo D-
Glucon °C 92θ min sterilization ) plus pre-sterilized /! Fermentation is carried out in a fermenter for 2f'C, /7 to 3θ hours while stirring at /7'lθr, p0m under aeration of 2f'C, /7 to 3θ hours. (2,3-diketo-D-gluconic acid/
9w/vchi). Bacterial cells are removed from this fermentation liquid using a centrifuge, and the resulting serum is filtered and sterilized using a pre-sterilized filter.

This is 2. J--diketo-D-gluconic acid fermentation liquid.

(5) Analytical method Quantification of ne-keto-d-gulonic acid and 2-keto-D-gluconic acid is carried out using gas chromatography and paper chroma 1.
- Use graphics.

Gas chromatography conditions Column: Silicon gum sEt, 2 (5%) Carrier gas: Helium Calano temperature: /O θ0C~, 2/θ0C Sample:
Conditions for carrying out trimethylsilylated paper chromatography Support: Toyo P paper Nn3θ Developing solution: Phenol: Formic acid: Water = 7j Nige:, 2j Color development: AHF solution (aniline θ939 and phthalic acid/Otsug with water saturated n-butanol/θθml (dissolved in).

/θ3”C for 2 minutes to develop color.

(6) Production of 2-keto L-gulonic acid Using the λ-keto L-gulonic acid producing strains listed in Table 1 or the j-keto-L-gluconic acid metabolism deficient mutant strain (1)
After inoculating 1 platinum loop into the seed medium of (2) and culturing with shaking at 21"C for a period of time, this seed culture solution, !;ysl, was inoculated into the main fermentation medium of (2) and cultured with shaking.

2. For this main fermentation medium. A j-diketo-D-gluconic acid solution ((3) or (4)) was added at the start of the culture or 76 hours after the start of the culture so that the final concentration was 2, and the culture was continued for a period of time. When the obtained culture solution was subjected to gas chromatography as described in (5), no neiketo-L-acid was detected. Furthermore, paper chromatography was performed using phenol:formic acid:water = 7-fNige:2j, and a2
- Keto D-gluconic acid was detected, but λ-keto D-gluconic acid was not detected in the culture solution of the j-keto-lo gluconic acid metabolism defective mutant strain (I).

(Left space below) Example 2 (Induction of mutant microorganism (1)) (1) Liquid medium Bacto-beefextract (pifco)
lθ Bacto Peptone (Dirco) lθ Chi NaC7iOJ NH1/, C103 'NH, No30/Chi Nar so op -21M
g5O, ・7H, 20θ/Chi CaCO3θθ0θ/Chipo θ3Chi+2 g, 2HPO, θ/Chi trace element solution (Note 1) 0/Tivitamin solution (Note 2) θl Chi agar
2. θ悌CpH72, //! ;”C,
/3.
IIO□-/θH, OfFM! (Involvement) tMOyO,
! 4.・ψchi0 37daFeCp, -6H20
97θ da Zn5O11, -7H2 OK Zada Cu SOII-jH, 20-27θ da MnCl, ”
#H2072ml (Note 2) Contains the following ingredients in vitamin liquid Cal) Thiamin / θ Dapantothenic acid 10 ~ Nicotinic acid amide / θ ~ Hyotine θ / Danic acid solution Sodium D-gluconate, Sodium j-keto D-gluconate After confirming that the pH was 72g to 72, the mixture was prepared by sterilization by filtration.

(4) Mutation induction method Corynebalathylium sp.
acteriurn Sp, FERM-P 277θ)
or.

Corynebacterium sp. (Coryne-1)
+acteriom sp, FERM-P a2 f7) were each inoculated into the liquid medium of (1) by platinum loop,
2. ! Shaking culture was carried out at r\°C for ff hours. N-methyl-N'-2I-rhoN- of θ2 which has been sterile-filtered in advance
Add nitrosoguanidine solution to a final concentration of 0θ2ch, continue incubation for 3θ minutes, and centrifuge (/4θθθr).
, p0m.

After 13 minutes), the bacterial cells were collected, washed three times with sterile physiological saline, and suspended in sterile physiological saline/θml.

This suspension/ml was inoculated into the medium of (1)/j hours,
The cells were cultured with shaking at 2°C. This culture solution was diluted with sterile physiological saline so that the number of viable bacteria was 10 to 1θ cells/tnl. Next, this diluted solution was added to a plate medium containing the D-gluconic acid aqueous solution of (3) and the minimal agar medium of (2) at a ratio of θS
~/Mt was coated and cultured at 2°C for 3~j days.

A colony of grown bacteria (strain (1) growing on D-gluconic acid medium in Table 3) was added to l:9 of the j~keto-D-gluconic acid aqueous solution of (3) and the minimal medium of (2). A replica was made on a flat plate using Herod's cloth. This was grown for 3~/f-, but bacterial colonies that did not grow on the j-keto D-gluconic acid medium were added to the D-gluconic acid medium and grown.

This selected mutant strain (j-kel--D-1'' in Table 3)
The non-growing strain (2)) on the gluconic acid medium was inoculated into the medium (1) containing 5-keto-D-gluconic acid/%, and the strain was grown for 2 hours.
The culture solution was described in Example 1, and j-keto D-gluconic acid was quantified by paper chromatography. It was confirmed that no acid was assimilated.

A mutant strain confirmed not to assimilate this j-keto D-gluconic acid (3-keto D-gluconic acid non-assimilating strain (3) in Table 3) was used in Examples/( 2) Inoculate a platinum loop into the described medium and after culturing for θ hours, carry out 11 fills/,
(3) 2. An aqueous solution of calcium j-diketo-D-gluconate was added at a final concentration of 10, and the mixture was further cultured for 2j hours. This culture solution was subjected to paper chromatography to quantify the amounts of keto-D-gluconic acid and keto-gulonic acid, and the results shown in Table 3 were obtained. In this way, Corynebacterium sp. (FERM-P277θ) was mutated and grown from the llA32θ strain. (r7) 11 mutant strains deficient in !-keto D-gluconic acid metabolism were obtained from the mutant-treated strain 29/θθ strain.

(Leaving space below) Procedural amendment (spontaneous) 1985, French Month 2, 1, Indication of the case: 1982 Patent Application No. 35452 2, Title of the invention 2 -Keto-■, -Process for producing gulonic acid 3, Relationship with the case of the person making the amendment Patent applicant 4, agent address 5-12-4 Sagikai, Fukushima-ku, Osaka 553 Shionogi & Co., Ltd. Patent Department 1) Add the following sentence to the end of the specification.

"Example 3 (Production of 2-keto L-gulonic acid using bacterial cell suspension and extract) (1) Seed culture: In the seed medium of Example/, (1), Corynebacterium sp, FERM-P 277θ(1) Or,
Mutant strain F'ERM-BP 10J derived from the strain
(1) respectively.

One platinum loopful was inoculated and cultured with shaking at 2f'C for 2 hours.

(2) Main culture: Example/, (2) medium (contains θθ/4 of polypropylene glycol P-2θ0θ as an antifoaming agent)
Pour 3θθml into a sterilized 1 liter fermenter.

Inoculate the seed culture solution jθml obtained in (1), /7'lθr
, p.

m, stirring, θθN ml/min of aeration, 21°
C.

Culture was carried out for 1 hour.

(3) Preparation of bacterial cell suspension: =2- The culture solution obtained in (2) was centrifuged (lmOθθr, pom
,.

After 20 minutes), the bacterial cells were collected and washed twice with physiological saline.

This washed bacterial body was washed with 00. tM) Squirrel Buffer (
It was suspended in pT('Z5) so that OD=/2.

10 nm (4) Preparation of bacterial cell extract: The washed bacterial cells obtained in (3) above were
'ODAAOnm=/θθ in buffer (pH 7g)
The cells were suspended and subjected to a French press (pressure lθθθkQ/d) to crush the bacterial cells. After removing cells (cells and debris) from this crushed solution by centrifugation (20, θθθG, 30 minutes), the supernatant was
Dialysis against 3M Tris buffer (pH 7, t)
73 hours) This was used as a bacterial cell extract.

(5) Production of 2-kel-L-gulonic acid using suspension (3) Suspension (3) The mixture was reacted at 3g°C for 13 hours. After the reaction is completed, centrifugation (lmu θθθG,
15 minutes) to remove the bacterial cells, and quantify 2-keto-L-gulonic acid and λ-kel-monocurconic acid in the supernatant by gas chromatography as described in Example 1 (5). The results shown in Table 1 below were obtained.

The third surface extract (4) θ3tttl, 737zrnoles
25-diketo-D-calcium gluconate and 73
7zmoles NADPH (reduced nicotinamide
2.3 ml of θ containing (adenine dinucleotide phosphate)
/M+-lith buffer (pH 75), and this was reacted at 3θ°C for 16 hours. The quantitative results of this reaction solution are shown in Table 5 below.

From the results shown in Table 5 and Table 5, it was found that the reaction using bacterial cell suspension (3) and bacterial cell extract (4)
-Keto L-gulonic acid was confirmed to be produced.

In both reactions, 2-ke 1. of the parent strain. - All treated products obtained from the L-gulonic acid producing bacterium (FERM-P 277θ) co-produced 2-keto L-gulonic acid and su-keto-D-gluconic acid, whereas the mutant strain The treated product (II) produced only suketo-L-gulonic acid and did not co-produce 2-keto-I--D-gluconic acid. "that's all

Claims (1)

[Scope of Claims] /) The j-keto D-gluconic acid metabolism-defective mutant strain (1) derived from the 2-keto L-gulonic acid producing strain (1) or a processed product thereof; 2. Contact with a medium containing S-diketo-D-gluconic acid or a salt thereof to accumulate norketo-L-gulonic acid in the medium. A method for producing norketo-L-gulonic acid, which comprises collecting the same. 2) The method according to claim 1, wherein the mutant strain (It) does not substantially produce norketo-D-gluconic acid. 3) The 2-keto-L-gulonic acid producing strain (1) is -3
1J Coryneform Group
The method according to claim 1, characterized in that the bacterial strain belongs to Bacteria.uporBacteria. 11) The method according to claim 1), wherein the 2-keto L-gulonic acid producing strain belongs to the genus Corynebacterium. j) It is characterized by being derived from the parent strain Nyketo-L-gulonic acid producing strain (1) belonging to the coryneform group! -Kel--D-gluconic acid metabolism defective mutant strain (1). B) The mutant strain (11) according to claim 3), wherein the 2-keto L-gulonic acid producing strain (1) belongs to the genus Corynebacterium. 7) Corynebacterium sp. FERMBP-/θ7 g) Corynebacterium sp. FERMBP-/θg according to claim j).