JPH03164166A - 5-keto-d-gluconic acid metabolism deficient variant - Google Patents

Abstract

NEW MATERIAL:A 5-keto-D-gluconic acid metabolism deficient variant by inducing the variant from a strain belonging to the genus Corynebacterium, capable of producing 2 keto-L-gulonic acid. EXAMPLE:Corynebacterium sp. (FERM BP-1079). USE:Producing 2-keto-L-glonic acid from 2,5-diketo-D-gluconic acid without accu mulating 2-keto-D-gluconic acid in a medium. PREPARATION:A strain [e.g. Corynebacterium sp. (FERM P-2770)] belonging to the genus Corynebacterium, capable of producing 2-keto-L-gulonic acid, is varied by ultraviolet irradiation, etc., and a bacterium not growing in a medium of 5-ketc-D-gluconic acid is selected.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the production of 2-keto-L-gulonic acid.
Keto-D-gluconic acid does not accumulate in the medium <,2
．． This invention relates to a mutant microorganism used for carrying out a production method for microbially obtaining 21-keto L-gulonic acid from 5-diketo D-gluconic acid. The present inventors first investigated a number of microorganisms (2-keto-L-gulonic acid producing strains) that can produce 21-keto L-gulonic acid from 2,5-diketo p-gluconic acid.
2-keto-L- using the heading I)
Invented a method for producing gulonic acid. (Tokuko Showa 50-215
No. 59, Special Publication No. 53-25033, and Special Publication No. 59
(Refer to Publication No. 6-15877). In both cases (I), the main product 2-keto-L-gulonic acid is produced from the raw material 2,5-diketo-D-mono-gluconic acid, and 2-keto-D-glunic acid is produced as a by-product. this unwanted 2
-A mixed culture method was invented to prevent keto-D-gluconic acid from accumulating in the medium. (Refer to Japanese Patent Publication No. 54-19468) This time, the present inventors have discovered that 2-
Keto-L-gulonic acid producing strain (I) was subjected to mutation treatment using a conventional method, and among the mutated strains (I), a mutant strain that did not grow on 5-keto D-gluconic acid but grew on D-gluconic acid (this The mutant strain was 5-keto D-gluconate metabolism deficient mutant strain (n
), and this mutant strain (n) was shown to have the property of not producing 2-keto-D-gluconic acid. I found it. Furthermore, when (n) is cultured and brought into contact with 2,5-diketo-D-gluconic acid, 2-keto-L-
We discovered that gulonic acid accumulates, and based on this knowledge, we completed the invention as described in the claims at the beginning. That is, according to the present invention, a 5-keto-D-gluconic acid metabolism-deficient mutant strain (II) derived from a 2-keto-L-gulonic acid producing strain (I) is provided. This (I[) or its treated product was brought into contact with a medium containing 2,5-diketo D-gluconic acid or its salts, and 21-keto L was added to the medium.
- By accumulating gulonic acid and collecting it, 2-
Keto-L-gulonic acid can be produced. In order to obtain (I[) more efficiently than (I), ultraviolet irradiation,
Treatments such as X-ray irradiation or contact with mutagenic agents such as N-methyl-N'12-N-nitrogueanidine (NTG), acrifurpine, and ethylmethane sulfonic acid are adopted. It will be done. Mutation treated (I)
Dilute to an appropriate concentration, or re-incubate and dilute to an appropriate concentration, and add a portion (0.5 to 1 ml) of the diluted solution to 0.5 to 2%
Grow on a minimal agar medium containing D-gluconic acid (contains vitamins and trace elements necessary for growth). The grown bacterial colonies are transferred to a minimal agar medium containing 0.5 to 2% of 5-keto-D-gluconic acid (Repnoka method), and colonies that do not grow on the 5-keto-D-gluconate medium are selected. The selected mutant strain has lost the metabolic activity of 5-keto D-gluconic acid or I/, of the parent strain (I). (The mutant strain obtained in this way is referred to as the 51-keto D-glufonic acid metabolism-defective mutant strain (I)). (n
) was cultured and brought into contact with 2.5-diketo D-gluconic acid. It was confirmed that it generates . As an example of (II) obtained by the above method, (I) is Corynebacterium sp.
m sp. FERM-P 2770,
ATCC No. 31090) (FERM-BP-108) and Corynebacterium sp.
rium sp. FERM-P26
87, ATCC No. 31081) (FeRM-BP-107). (Margins below) The above fact, that is, the fact that the ability to produce 21-keto D-gluconic acid could be lost or significantly weakened by deleting the ability to metabolize 5-keto-D-gluconic acid, is the fact that Phrineform. This is common to all 21-keto L-gulonic acid producing bacteria belonging to the group (according to the definition in Pursey's Manual Determinative Bacteria 8th Edition). There are no particular restrictions on the medium used for culturing (I[). For example, as a carbon source, glufus,
Sugars and polyhydric alcohols such as sucrose, glycerin, and blackstrap molasses are used, and commonly used nitrogen compounds as nitrogen sources, such as corn stew liquor,
Pebtone, meat extract, ammonium salts, nitrates, etc. are used. Inorganic salts (for example, salts II of calcium, magnesium, potassium, zinc, manganese, iron, etc.) and factors that promote the production of the target substance may be added. Culture medium composition, characteristics of the strain used, and raw material 2,5-diketo D
-Varies depending on the amount of gluconic acid used and other conditions. As the raw material 2.5-diketo D-gluconic acid, 2.
An aqueous solution of 5-diketo D-gluconate can be used, or an aqueous solution of 5-diketo D-gluconate can be used. 2.5-diketo-D- from D-glucose, which belongs to the genus Acetobacter, Acetomonas, and Gruffnobacter according to the 7th edition of the same edition.
It is also possible to use a filtered and sterilized solution of the fermentation liquor obtained by culturing microorganisms capable of producing gluconic acid, or a solution treated with sterilization using chemicals (for example, sodium dodecyl sulfate). The conditions for adding 2.5-diketo D-gluconic acid vary depending on the strain and culture conditions, but usually 1 to 10% of 2.5-diketo D-gluconic acid is added at once or intermittently in small amounts. ．． It is preferable to add 2.5-diketo D-gluconic acid at the start of culture, or within 10 hours before and after the end of bacterial growth. The culture time varies depending on the addition conditions of the raw material 2,5-diketo D-gluconic acid, but it is usually cultured for 24 to 96 hours after addition of 2,5-diketo D-gluconic acid. The end point of the culture is the point at which the acid disappears. 2-keto L-gulonic acid, 2-keto-D-gluconic acid, and 2,5-diketo D-gluconic acid in the fermentation liquid can be separated and quantified by common means for analyzing sugars and related substances. ．． For example, gas chromatography, paper chromatography, thin layer chromatography, etc. are used. These can be used in various ways depending on the purpose. The present invention will be explained in more detail using Examples below. 1 bunch 1 batch 2 (manufacturing of 2-keto-L-gulonic acid) D-Gulfus 1.0% Bacto Yeast Extract (Difco) 0.5%
Bact Bepton (Difco) 0.
5% potassium monophosphate (KH.PO.)
0.1% magnesium sulfate (MgSOv7H10)
0. 02% (pH 7 with I0% NaOH)
Adjust to ~7.2 and add 50ml to a 500ml Erlenmeyer flask.
Dispense into recipients and sterilize at 115℃ for 20 minutes. ) and 1.0% D-Gulfus 1.0% CSL 3.0%x
u*poa O. 1
%MgSOv7H*O O
．． 11
Sterilize at 5°C for 20 minutes. ) Powdered calcium 2,5-diketo D-gluconate
．． Make a 0% aqueous solution, and filter and sterilize this aqueous solution using a pre-sterilized filter. (2,5-diketo D-glucon Erwinia bunctata (FERM-P5452,
ATCC No. 31626) and seed medium (I%D-
Gurufus, 5% CSL, 0.1% KH. Poi 0.0
2%MgSOa 4H*O-0. 5% calcium carbonate (CaCOs), pH 6.8-7.0, 50
Dispense 50 ml into a 0 ml Erlenmeyer flask, sterilize at 115°C for 20 minutes), inoculate one platinum loop, and culture with shaking at 28°C for 8 to 11 hours. (Amplitude 7 1 mIII, 2 7 0 r.
p. m. ,same as below). 45ml of this seed culture
ml of 2.5-diketo D-gluconic acid fermentation medium (20
% D-glucose, 3% CSL, 0.1% KHmPO.
, 6.3% (acos, pH 6.8 ~ 7.0, 115
℃, sterilized for 20 minutes) and then sterilized in a pre-sterilized 12 fermenter at 28℃ for 17 to 30 hours under aeration of 600 Nml/min to 174 Or. p. m. Ferment while stirring. (2,5-diketo D-gluconic acid 19
w/v%). This fermentation liquid is centrifuged to remove bacterial cells,
The supernatant is filtered and sterilized using a pre-sterilized filter. This is called the 2,5-diketo D-gluconic acid fermentation liquid. Gas chromatography and paper chromatography are used to quantify 2-keto-L-gulonic acid and 2-keto-D-gluconic acid. Gas chromatography conditions Column: Silicon gum SE52 (5%) Carrier gas: Helium Column temperature = 160°C to 210°C Sample: Trimethylsilylated paper Chromatography conditions Support: Toyo Roshi No. 50 Developing solution: Phenol: Formic acid dihydrate - 75:4:2S Color development: AHF # solution (0.93 g of 72'J and 1.66 g of tar acid dissolved in 100 ml of water-saturated n-butanol) ) and let it develop color at 105℃ for 2 minutes. (6) Preparation of 2-keto-L-gulonic acid Add 1 platinum loop of the 2-keto-L-gulonic acid producing strains listed in Table 1 or the 5-keto-D-gluconic acid metabolism deficient mutant strain to the seed medium of ■. After inoculating and culturing with shaking at 28°C for 24 hours, 5 ml of this seed culture was inoculated into the main fermentation medium and cultured with shaking. Acid solution (a3) or (ω) was added to the final concentration of 2% at the start of culture or 16 hours after the start of culture, and cultured for 4.8 hours.
2-Keto-L-gulonic acid and 2-keto-D-gluconic acid were determined by gas chromatography, and the results shown in Table 2 were obtained. 5-keto-p-gluconic acid metabolism defective mutant strain (I
) 2-keto-D-gluconic acid was not detected in the culture solution. Furthermore, phenol: formic acid: water -75:4:
Paper chromatography was performed using 25 to detect 2-keto D-gluconic acid, but 5-
2-Keto-D-gluconic acid was not detected in the culture solution of the keto-D-gluconic acid metabolism-deficient mutant strain (I[). (Qt bottom margin) House'sei 4 2 (Induction of mutated microorganisms (I[)) Chikuichi Kisei Bakuto Beef Extract (Difco) 1.
0% Bact Pebton (Difco)
1.0%NaCl
0. 5% (pH 7.2, 5 oat/500 m
(L Erlenmeyer flask, sterilized at 115°C for 20 minutes) C.L.O.
5%NH, No.
0.1%Na*soa
0.2%11gSO4・7H,O
O. L%CaCO.
0. 0001%KH. P0.
0.3% Lupoa
0. IN trace element liquid (Note 1)
0. L-horn vitamin liquid (Note 2)
0.1 square agar
2. 0511 (pH 7.2, 1 1 5
℃, sterilized for 15 minutes) (Note 1) Trace element solution (IN containing the following ingredients: NaJaOy'lOH10
88mg(NHa')*MOtOs
14}1to 37■FaC
116HaO 97
0ml<ZnSO44H*0
8.8a@CuSOv5H. 0
270g MtlC114HsO
72■ (Note 2)
Vitamin liquid (IN contains the following ingredients) Thiamine
1.0@bantothenic acid
10mg nicotinamide
LOmg bisaichin 0.
1+I@p-sodium gluconate, 5-keto-D-
Make each sodium gluconate into a 10% aqueous solution and adjust the pH to 6.
．． After confirming that it was 8 to 7.2, it was sterilized by filtration and prepared. Corynebacterium sp.
bactarium sp. FERM-P
2 7 7 0) or Corynebacterium
Species ( Corynabacterium s
p. One platinum loop of each of FERM-P2687) was inoculated into the liquid medium (I), and cultured with shaking at 28°C for 8 hours. 0.2% N, pre-sterile filtered
-Methyl-N-nitro-N-nitroguanidine solution was added to a final concentration of 0.02%, incubated for 30 minutes, and centrifuged for 1 minute. ! (1 0.00O r.p.
m. After 15 minutes), the bacterial cells were collected, washed three times with sterile physiological saline, and suspended in 10 ml of sterile physiological saline.
1 ml of this suspension was inoculated into the medium (I) and incubated for 15 hours for 2 hours.
Cultured with shaking at 8°C. This culture solution was diluted with sterile physiological saline to a concentration of 101 to 10" viable bacteria/ml.
Next, 0.5 to 1 ml of this diluted solution was applied to a plate medium containing a 1:9 ratio of D-gluconic acid aqueous solution (1) and minimal agar medium (2), and cultured at 28°C for 3 to 5 days. A colony of grown bacteria (strain ■ growing on D-gluconic acid medium in Table 3) was mixed with the 5-keto D-gluconic acid aqueous solution of ■■.
A replica was made using velvet cloth, etc. on a plate culture medium containing 1:9 of the minimal medium. After culturing this for 3 to 5 days, the above D-
A colony of bacteria that grows on gluconic acid medium but not on 5-keto-D-gluconic acid medium was added to D-gluconic acid medium and allowed to grow. This selected mutant strain (strain ■ that does not grow on the 51-keto D-gluconic acid medium in Table 3) was inoculated into the medium (■) containing 1% of 51-keto D-gluconic acid and shaken at 28°C for 24 hours. Cultivated. This culture solution was subjected to paper chromatography as described in Example 1 to quantify 5-keto-D-gluconic acid, and it was confirmed that this strain (2) did not assimilate 5-keto-D-gluconic acid. A mutant strain confirmed not to assimilate 5-keto-D-gluconic acid (51-keto D-gluconic acid non-assimilating strain ■ in Table 3) was cultured in the medium described in Example 1. After inoculating one platinum loopful of bacteria into a cell and culturing for 20 hours, 2.5 described in Example 1, (3)
-Diketo D-calcium gluconate aqueous solution was added to the final concentration of 1.0%, and cultured for an additional 24 hours.
The 2-keto-D-gluconic acid and 2-keto-L-gulonic acid contained in this culture solution were quantified by paper chromatography, and the results shown in Table 3 were obtained. In this way, Corynebacterium sp.
Corynebacterium sp.
11 strains of 51-keto D- from 29,100 mutant-treated strains of
A mutant strain defective in gluconate metabolism was obtained. (Margins below) - (Production of 2-keto-L-gulonic acid from bacterial cell suspension and extract) 1: In the seed medium of Example 1, Corynebacterium sp. ．． F
One loopful of ERM-P 2770 (I) or the mutant strain FERM-BP 108 (It) derived from the strain was inoculated, and cultured with shaking at 28°C for 24 hours. Tonsheng 1: Medium of Example 1, ■ (However, it contains 0.01% polypropylene glycol P-2000 as an antifoaming agent)
Pour 500 ml into a sterilized 12-volume fermentation bowl, inoculate with 50 ml of the seed culture obtained in (I), and incubate at 1740 r. p. m.
stirring at 28°C under aeration of 600 Nml/min.
Culture was performed for 16 hours. O> Bacteria. (The culture solution obtained was centrifuged (15.00 O
r. p. m. ．． After 20 minutes), the bacterial cells were collected and washed twice with physiological saline. This washed bacterial body is 0.05M Tris Butts? One (p
H7.5) OD. ■． -12. Muciloid (I1): The washed bacterial cells obtained in the above (■) were added to 0.05M Tris● buffer (pH 7.8) at OD.●@..- 1 0 0
Suspend it so that it becomes
1000 kg/am") to disrupt the bacterial cells. From this disruption, centrifugation (20,000 kg/am", 30 minutes)
After removing the bacterial cells (bacterial cells and debris), the supernatant was dialyzed (15 hours) against 0.05M Tris buffer (pH 7.5), and this was used as a bacterial cell extract. ■ Sensitive suspension of 2-keto-L-gulone prepared by ■■ 8 ml of 2.5-diketo p-gluconate calcium solution (Example 10)) 2 m
This mixture was reacted with shaking at 30°C for 15 hours. After the reaction was completed, the bacterial cells were removed by centrifugation (Is.oooc, 15 minutes), and the 21-ketoL in the supernatant was removed.
-Gulonic acid and 2-keto-D-gluconic acid in Example 1
(It was quantified by gas chromatography as described in Section El, and the results shown in the following Table 4 were obtained. 0.5 ml of the fourth surface extract (4) was 75 μmol
2. Containing 2.5-diketo D-calcium gluconate and 15 μl of NADPH (reduced diphthinamide adenine dinucleotide phosphate). 5
The mixture was added to 0.1M Tris buffer (pH 7.5) and reacted at 30°C for 16 hours. The quantitative results of this reaction solution are shown in Table 5 below. Table 5 From the results shown in Tables 4 and 5 above, the bacterial suspension solution ■
It was confirmed that 21-keto L-gulonic acid was produced by reaction using a bacterial cell extract (gradient).

Claims (1)

[Scope of Claims] 1) A 5-keto-D-gluconate metabolism-deficient mutant derived from a 2-keto L-gulonic acid producing strain (I) belonging to the genus Corynebacterium as a parent strain. Stock (II). 2) The mutant strain (II) according to claim 1, which substantially does not produce 2-keto-D-gluconic acid. 3) Corynebacterium as described in claim 1)
Species FERMBP-107. 4) Corynebacterium as described in claim 1)
Species FERMBP-108.