KR900009051B1 - Process for preparing 2-keto -l-gulonic acid - Google Patents

Abstract

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Description

[Name of invention]

Method for preparing 2-keto-L-gulonic acid

Detailed description of the invention

The present invention relates to the preparation of 2-keto-L-gulonic acid by microbiological (fermentative or enzymatic) reduction of 2,5-diketo-D-gluconic acid. In particular, (i) the use of newly induced variants with 5-keto-D-gluconic acid metabolic defects or intermediates in the reduction process, (ii) the variants themselves used to implement the process, (iii) the reducing system A fermentation broth used as a raw material to be reduced or an intermediate product thereof by addition of nitrate and hydrogen donor, and (iv) fermentation broth or a variant cultured with 2,5-diketo-D-gluconic acid producing microorganism as a surface active agent. It relates to a method of sterilization.

The present inventors have already described various microorganisms capable of producing 2-keto-L-gulonic acid from 2,5-diketo-D-gluconic acid (hereinafter abbreviated as 2-keto-L-gulonic acid producing strain (I)). And a method for preparing 2-keto-L-gulonic acid by microorganisms (see US Pat. Nos. 3,922,194 (RE 30,872), 3,959,076 and 3.963.574). Each of these strains (I) produces the main product 2-keto-L-gulonic acid as well as the undesirable byproduct 2-keto-D-gluconic acid from 2,5-diketo-D-gluconic acid. We also invented a mixed culture method to prevent undesirable accumulation of 2-keto-D-gluconic acid in the medium (see US Pat. No. 3,998,697).

Nevertheless, there has been a need to establish a more commercially viable method for preparing 2-keto-L-gulonic acid. There is also a need for the production of 2-keto-L-gulonic acid from 2,5-diketo-D-gluconic acid and the development of its molar yield.

Therefore, an object of the present invention is to use a newly generated variant from 2,5-diketo-D-gluconic acid to minimize the accumulation of 2-keto-D-gluconic acid, which is undesirable for fermentation broth. To provide an improved microbial method for preparing 2-keto-L-gulonic acid, and to provide variants that can be used to carry out the method.

Another object of the present invention is to improve the process by adding nitrate and hydrogen donor to the fermentation medium containing the variant and / or 2,5-diketo-D-gluconic acid to be supplied to the medium as a starting material of the present invention. .

Another object of the present invention is to use a 2,5-diketo-D-gluconic acid fermentation broth as the starting material of the present invention, which is sterilized with a surface active agent in advance so that the 2,5-diketo present significantly in the broth. The method is improved by reducing the number of cells of the -D-gluconic acid producing microorganism.

According to the present invention, 2-keto-L-gulonic acid is prepared from 2,5-diketo-D-gluconic acid in the presence of live or processed microorganisms capable of producing 2-keto-L-gulonic acid. Provide an improved method. This method is characterized by the use of variants as (1) 5-keto-D-gluconic acid metabolic defects as said microorganisms, and (2) practically not capable of producing 2-keto-D-gluconic acid. The variants themselves used to carry out the method are also provided.

Throughout this specification and claims, "living or processed microorganisms" include processed products obtained by treating microbial cells as well as growth or resting microorganisms. This processed product is exemplified as a cell suspension, cell extract, cell mash obtained from crude or purified enzyme or microorganism. All acids involved in the present invention are to be understood to include salts or esters.

According to an aspect of the present invention, there is provided a further improved process wherein the growth of the above-described variants and the conversion of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid are carried out in the presence of a hydrogen donor or nitrate to provide. The hydrogen donor is added with 2,5-diketo-D-gluconic acid to be supplied to the medium or separately to the medium. Nitrate is added to the medium at the beginning of the culture or at the beginning or at any time when 2,5-diketo-D-gluconic acid is supplied.

According to another aspect of the invention, the fermentation broth in which 2,5-diketo-D-gluconic acid fish microorganism (III) is cultured is used as 2,5-diketo-D-gluconic acid to be supplied to the medium, An even more improved method of sterilizing viable cells of microorganism (III) that lived in this broth with a surface active agent prior to feeding is provided.

(1) Induction of variant (II)

The inventors of the present invention have directed a microbial strain (parent (I)) capable of producing 2-keto-L-gulonic acid from 2,5-diketo-D-gluconic acid by treating the parent (I) by a known method for mutation. Variant (II) derived from This variant (II) was chosen to grow at all or little at 5-keto-D-gluconic acid but grows at D-gluconic acid and was found to be unable to produce 2-keto-D-gulonic acid. Therefore, variant (II) will be referred to as a variant that has a metabolic defect of 5-keto-D-gluconic acid and cannot produce 2-keto-D-gluconic acid, ie, variant (II) may Upon contact with, 5-diketo-D-gluconic acid, 2-keto-L-gulonic acid is produced and accumulates in the contact mixture without incidental accumulation of undesirable 2-keto-D-gluconic acid.

Any known method for variation is effectively used to obtain variant (II) from (I). This method includes exposure to ultraviolet radiation or X-ray radiation and treatment with mutants such as N-methyl-N'-nitro-N-nitrosoguanidine (NTG), acriflavin, ethyl methanesulfonate, and the like. .

In a specific example of the invention, the suspension of treated strain (I) is optionally cultured and then diluted to a suitable concentration using sterile physiological saline, and a portion (0.5-1 ml) of the diluted suspension is 0.5-2% Scatter and spread therein in minimal (agar) medium containing D-gluconic acid (also containing vitamins and trace elements necessary for the growth of microorganisms). The colonies grown in the medium were transferred to another minimal (agar) medium containing 0.5 to 2% of 5-keto-D-gluconic acid by plate repetition, and then unable to grow like 5-keto-D-gluconic acid. Select a colony. The cells in the selected colonies have no ability to utilize the 5-keto-D-gluconic acid that the parent strain (I) had, or this ability is reduced to less than 1/10 compared to the parent strain (I).

It is clear that each of the variants (II) produces 2-keto-L-gulonic acid without producing the byproduct 2-keto-D-gluconic acid in the presence of 2,5-diketo-D-gluconic acid in culture conditions. .

Variants (II) obtained in the above-described method were derived from parent (I), Corynebacterium sp.FERM-P 2770, ATCC No. 31,090 (KAIST 830526-08611) derived variant FERM-BP 108 (KAIST 830526-09811), Another strain (I), Corynebacterium sp.FERM-P 2687, variant FERM-BP 107 (KAIST 830526-09711) derived from ATCC No.31,081 (KAIST 830526-08411) and the like can be exemplified (Table 1). One). Detailed taxonomic descriptions of this parent strain (I) are described in US Pat. Nos. 3,959,076 and 2,998,697.

[Variants (mutants) having 2-keto-L-gulonic acid producing microorganisms (parents) and 5-keto-D-gluconic acid metabolic defects derived therefrom and which are not able to actually produce 2-keto-D-gluconic acid]

TABLE 1

The above-mentioned fact that the loss or significant reduction of 5-keto-D-gluconic acid producing activity is caused by the loss of 5-keto-D-gluconic acid metabolic activity from the parent strain is indicated by coryneform bacteria (Bergey's manual common to all 2-keto-L-gulonic acid producing microorganisms belonging to) according to the definition described in the 8th edition of Determinative Bacteriology. Another variant or variant of the above-mentioned variant (II) may also be used to embody the present method as long as it has these properties. Moreover, any microorganism which incorporates a gene expressing this property of variant (II) prepared by gene staining, transformation or cell fusion can be used.

(2) conversion of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid in the presence of variant (II)

Any known conditions used to cultivate parent (I), process the cultured broth and obtain 2-keto-L-gulonic acid from this broth, as described in US Pat. No. 3,922,194, 3,959,574, can be used as variants (II). Incubating and performing the steps accordingly can be applied with minor changes.

No particular restriction is imposed on the composition of the medium used to culture the variant (II). For example, sugars such as glucose, sucrose and molasses and polyhydric alcohols such as glycerol can be used as carbon atoms at a concentration of 0.5 to 5%, corn steep liquor, peptone, meat extract, ammonium, nitrate, etc. The same known nitrogenous material can be used as the nitrogen source at a concentration of 0.5 to 5%. Other inorganic salts (eg, salts of calcium, magnesium, potassium, zinc, manganese and iron) or any factor that can promote the production of the final product can be added to the composition. The composition may vary depending on the nature of the strain used, the raw material, the amount of 2,5-diketo-D-gluconic acid and other incidental conditions.

A saline solution of 2,5-diketo-D-gluconic acid can also be used as a raw material. Or belonging to the genus Erwinia or Gluconobacter, which can produce 2,5-diketo-D-gluconic acid from D-glucose (according to the definition in Buggy's Manual, 8th edition, and accordingly acetomonas described in 7th edition). It is also suitable to use a fermentation broth obtained by culturing microorganisms, including acetobacter and gluconobacter, in a cell-free filtrate or a solution sterilized with a chemical (eg sodium dodecyl sulfonate).

The 2,5-diketo-D-gluconic acid fish microorganism (III) used to embody the method of the invention belongs to the genus Gluconobacter or Erwinia and is a microorganism which can be exemplified as described in Table 2 below.

TABLE 2

Detailed categorical description of this strain is described in US Pat. No. 3,998,697 or EP 0046284.

Samples of this microorganism strain as well as those described above have been deposited with the Fermentation Research Institute (FRI) in Yadabe, Japan and / or the American Type Culture Collection (ATCC) in Maryland, USA, and from this depository, as defined by the Butafest Treaty. It is available. Microorganisms with an IFO number can be used from a fermentation laboratory in Osaka, Japan.

The amount and the method of supplying 2,5-diketo-D-gluconic acid to be supplied to the medium vary depending on the microorganisms and the culture medium and the culture conditions, but the above-mentioned acid is usually cultured or cultured so that the total concentration is 1 to 10%. The broth is applied intermittently or in portions. The amount of acid partially added is preferably adjusted to a concentration of 0.05 to 2% relative to the total amount of medium or culture broth at the time of feeding.

The pH of the medium or culture broth is usually maintained at 5.0-9.0, preferably 6.0-8.5, during the conversion by the addition of an alkaline substance such as calcium carbonate from time to time. Alternatively, a suitable buffer may be added to the medium at the beginning of the culture.

This condition can be applied to other embodiments of the present invention by slight modifications.

(3) Use of nitrates and hydrogen donors in culture

In the specific procedure for preparing 2-keto-L-gulonic acid from 2,5-diketo-D-gluconic acid without incidental accumulation of 2-keto-D-gluconic acid by using variant (II), The inventors have added nitrates to broths in which the variant (II) grows and / or medium in which the variant (II) acts on 2,5-diketo-D-gluconic acid, and the hydrogen donor to the 2,5-diketo- The addition of D-gluconic acid to the broth simultaneously increases the production of 2-keto-L-gulonic acid from 2,5-diketo-D-gluconic acid significantly and moles of 2-keto-L-gulonic acid It was found that the yields were significantly improved, and they completed the second aspect of the present invention based on this finding.

When variant (II) is inoculated and incubated in medium supplemented with 0.1-0.5% nitrate, the growth of variant (II) reaches its maximum 10-24 hours after the beginning of the culture. Furthermore, nitrate is added to the medium at a concentration of 0.05-0.5% within 10 hours of growth or in this state of growth. Nitrates that can be used in this process include alkali metal salts such as potassium nitrate and sodium nitrate, alkaline earth metal salts such as calcium nitrate and magnesium nitrate and manganese nitrate.

Nitrate can be added to the medium early in the culture of variant (II). The addition of nitrates may be carried out simultaneously when one or partly feeds 2,5-diketo-D-gluconic acid. Preferably at the beginning of the culture and at the beginning of the 2,5-diketo-D-gluconic acid feed. The benefit caused by the addition of nitrates is also significantly increased by adding the hydrogen donor to the medium at the same time as the 2,5-diketo-D-gluconic acid feed. That is, the production of 2-keto-L-gulonic acid can be significantly increased by the addition of hydrogen donors.

As the hydrogen donor any carbohydrate and polyhydric alcohol can be used as long as the strain can utilize them. The hydrogen donor is preferably added at the same time as the supply of 2,5-diketo-D-gluconic acid in combination with 2,5-diketo-D-gluconic acid. The concentration of the hydrogen donor can vary depending on the conditions under which 2,5-diketo-D-gluconic acid is applied and the conditions in which the variant or variant is cultured, but the hydrogen donor is usually supplied with a 2,5-dike It is added at a concentration ranging from 5% to 50% of the amount of to-D-gluconic acid.

The advantage of adding nitrates according to the invention is the fact that nitrates participate in the metabolism of carbohydrates or organic acids added as hydrogen donors by microbial cells. In addition, it activates a reduction system for reducing 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid in microbial cells, and effectively supplies hydrogen released from a hydrogen donor to the reduction system. The addition of nitrate alone without the addition of these hydrogen donors results in less production of 2-keto-L-gulonic acid; In other words, the advantage of nitriding addition to increase the production and yield of 2-keto-L-gulonic acid is significantly improved by the addition of a hydrogen donor.

In addition to the advantages described above, nitrate addition can also maintain the broth pH value (6.5-8.0) for a long time, which is useful for the preparation of 2-keto-L-gulonic acid from 2,5-diketo-D-gluconic acid. desirable.

Nitrate improves the metabolism of hydrogen donors by microorganisms and is also believed to be used to activate and stabilize the reducing system rather than by microorganisms as a nitrogen source.

In general, the effect of nitrogen containing compounds that can be used as nitrogen sources is assessed by increasing cell concentration. Among the similar inorganic compounds, ammonium salt shows a much larger increase effect than nitrate. That is, ammonium salts increase cell concentration at the beginning of culture or upon broth upon feeding 2,5-diketo-D-gluconic acid, but show little effect on increasing the production of 2-keto-L-gulonic acid.

In contrast, nitrates show little effect in increasing cell concentration with ammonium salts, but significantly increase the production of 2-keto-L-gulonic acid. This effect will be explained in more detail in the Examples.

(4) Sterilization of 2,5-diketo-D-gluconic acid fermentation broth by surface active agent

In the specific process of commercially producing 2-keto-L-gulonic acid, the present inventors prepared 2,5-produced by culturing strain (III) (containing (III) / ml of 10 ^{8-10 10} viable cells). An attempt was made to effectively prepare 2-keto-L-gulonic acid by feeding the diketo-D-gluconic acid fermentation broth directly to the broth cultured with variant (II).

In the course of the trial, it was found that, unlike the case of parent (I), the production of 2-keto-L-gulonic acid was markedly hindered when variant (II) was used in this method.

As a result of extensive research, it has been found that the disturbance phenomenon occurs when strain (III) grows above 10 ⁵ viable cells / ml.

As a method of sterilizing strain (III) in a 2,5-diketo-D-gluconic acid fermentation broth, sterilization by high temperature sterilization and filtration is used. However, the thermal instability of 2,5-diketo-D-gluconic acid and the sterilization by filtration are expensive when used for commercial scale production, so the establishment of another method of sterilizing strain (III) was made by the inventors. It is necessary to specify the commercial production of 2-keto-L-gulonic acid. As a result of various studies, the third aspect of the present invention has been completed by developing a safe and reliable method for sterilizing strain (III) with a surface active agent.

The third aspect of the present invention does not require any special design to sterilize the process and does not interfere with the production of 2-keto-L-gulonic acid by variant (II), since the surface active agent can be easily separated. The surface active agent itself has the advantage of being inexpensive and readily available. This advantage facilitates large scale production using this method.

Sodium dodecyl sulfate (SDS) of the surface active agents studied is particularly preferred.

The method of the present invention is briefly explained by the following.

First, 2,5-diketo-D-gluconic acid-producing strain (III) is inoculated into a medium containing a substrate, nitrogen source such as D-glucose, corn steep liquor and inorganic salt, and incubated in air with stirring at 28 ° C. .

The fermented broth prepared as described above is divided into two parts, and one part is sterilely added with a surface active agent to a final concentration of 0.01 to 0.25 w / v%, and then left at 5 to 28 ° C. for several hours with occasional shaking. . Another portion without addition of the surface active agent is left for several hours at 5- to 28 ° C.

During surface active agent treatment, for example with sodium dodecyl sulfate (SDS), the number of viable cells of strain (III) in the treated broth is reduced to less than 1 / 1,000,000 of the untreated broth viable cells. However, this reduction may vary depending on the strain and the surface active agent used. Some of the untreated 2,5-diketo-D-gluconic acid fermentation broth is sterilized by filtration. As a hydrogen donor, D-glucose was aseptically added to each of the surface active treatment broth, the sterilized broth, and the untreated 2,5-diketo-D-gluconic acid fermentation broth, and the final concentration was 1-10%. After that, the broth is fed to the culture of variant (II).

Separately, variant (II) is appropriately incubated with 2,5-diketo-D-gluconic acid fermentation broth during surface active treatment. The 2,5-diketo-D-gluconic acid fermentation broth prepared above was supplied to the culture of the mutant (II) in 15 to 120 minutes, and the 2,5-diketo-D- remaining on the broth was supplied. The concentration of 2,5-diketo-D-gluconic acid is 0.05-2.0% while controlling the amount of gluconic acid, and the culture is continued for 48 hours from the beginning of feeding. When sterilized by surface active agent treatment or filtration, 2,5-diketo-D-gluconic acid is readily converted to 2-keto-L-gulonic acid. However, without sterilization, the cell number of strain (III) increases to 10 ⁵ cells / ml or more, and finally production of 2-keto-L-gulonic acid is stopped.

As will be explained later in the Examples, sterilization may be carried out by surface active agent treatment or filtration. However, sterilization in filtration of broth is only possible for small scale treatments, so if broth contains high concentrations of 2,5-diketo-D-gluconic acid, it is impossible to incorporate it into commercial scale production at a price. Therefore, it is more advantageous to sterilize with the surface active agent according to the invention.

The invention is explained in more detail through the following examples.

The content and order are as follows:

Example 1

Induction of Variants (II)

Preparation A, 2,5-diketo-D-gluconic acid fermentation broth

Preparation B. Solution of Ca 2,5-diketo-D-gluconate.

Example 2

Use of variant (II) in 2-keto-L-gulonic acid production by fermentation

Example 3

Use of Cell Suspensions and Cell Extracts of Variant (II) in 2-keto-L-gulonic Acid Production by Contact

Example 4

Addition of Nitrate and Hydrogen Donor to Fermentation Broth

Example 5

Sterilization of 2,5-diketo-D-Gluconic Acid Fermentation Broth by Surfactant

Analysis method

(i) 2-keto-L-gulonic acid, 2-keto-D-gluconic acid and 2,5-diketo-D-gluconic acid.

(a) gas-liquid chromatography

Column: SE 52 (5%)

Sample: Trimethylsilylated Sample

Carrier Gas: Helium

Temperature: 160 ~ 210 ℃

(b) paper or thin layer chromatography

Carrier: Toyo Rossi 50 or TLC Aluminum Sheet Cellulose (available from Toyo Rossi K.K or Merc A.G)

Electric solvent: Phenol: Formic acid: Water = 75: 4: 25

Color development: Sprayed with AHF solution (prepared by dissolving 0.93 g aniline and 1.66 g phthalic acid in n-butanol saturated in 100 mL water) and then heated at 105 ° C. for 2 minutes.

(ii) D-glucose

"Glucose B test" available from Wako Junyaku Kogyo Kabushiki Kaisha

Example 1

(Induction of variant (II))

(1) liquid medium

The aqueous solution containing 1.0% of lacto beef extract (Difco), 1.0% of bacto peptone (Difco) and 0.5% of NaCl was adjusted to pH 7.2. 50 ml of this solution is charged into a 500 ml conical flask and sterilized at 115 ° C. for 20 minutes.

(2) minimum (agar) badge

The medium of the following composition is adjusted to pH 7.2 and sterilized for 15 minutes at 115 ℃.

NH ₄ Cl 0.5%

NH ₄ NO 0.1%

NaSO ₄ 0.2%

MgSO ₄ 7H2O 0.1%

CaCO ₃ 0.0001%

KH ₂ PO ₄ 0.3%

K ₂ HPO ₄ 0.1%

Trace element solution ^* 0.1%

Vitamin solution ^** 0.1% and

Agar 2.0%

^* Trace element solution (component per liter)

_{Na 2 B 4 O 7 · 10H} 2 O 88㎎

(NH ₄ ) ₆ MO ₇ O ₂₄ 4H ₂ O 37 mg

FeCl ₃ · 6H ₂ O 970 mg

ZnSO ₄ 7H ₂ O 88 mg

270 mg of CuSO ₄ H ₂ O and

MnCl ₂ 4H ₂ O 27 mg

^** Vitamin solution (component per liter)

Thiamine 1.0mg

Pantothenic Acid 10mg

Niacin 10 mg and

Biotin 0.1mg

(3) a solution of D-gluconic acid and 5-keto-D-gluconic acid

Each of sodium D-gluconate and aqueous sodium 5-keto-D-gluconate solution (10%) was adjusted to pH6.8-7.2 and sterilized by filtration.

(4) How to isolate variants

1 loop each of Corynebacterium sp.FERM-P 2770 (KAIST 830526-08611) and Corynebacterium sp.FERM-P 2687ST 830526-08411) was inoculated into the liquid medium (1) and incubated for 8 hours at 28 ° C. do. To each culture medium, pre-sterilized 0.2% N-methyl-N'-nitro-N-nitrosoguanidine aqueous solution was added until the final concentration was 0.02%, and the culture was continued for another 30 minutes. The cultured medium is collected by centrifugation (10,000 rpm for 15 minutes). Collected cells are washed three times with sterile physiological saline and suspended in each 10 ml saline. Each 1 ml suspension is inoculated in medium (1) and incubated at 28 ° C. for 15 hours. Using the sterilized culture medium saline 10 ^{2-10 3} alive cells was diluted with / ㎖. Then, 0.5-1 ml of diluted culture solution is spread over a flat medium prepared by mixing D-gluconic acid solution (3) and at least agar medium (2) in a ratio of 1: 9 and grown at 28 ° C. for 3-5 days. .

A colony of grown cells (strain (1) in Table 3 grown in D-gluconic acid medium) was velvet cloth with a 5-keto-D-gluconic acid solution (3) and a minimum of agar medium (2) at a ratio of 1: 9. Transfer to plated media prepared by mixing. After culturing for 3 to 5 days, colonies of cells grown in D-gluconic acid medium but not growing in 5-keto-D-gluconic acid medium are collected and cultured in D-gluconic acid medium.

Selected variants (strain (2) in Table 3, which cannot grow on 5-keto-D-gluconic acid medium) were inoculated in medium (1) containing 1% 5-keto-D-gluconic acid and 24 at 28 ° C. Incubate for hours. The remaining 5-keto-D-gluconic acid is detected by paper chromatography to confirm that this strain (2) cannot utilize 5-keto-D-gluconic acid.

The medium 2 to be described in Example 2 was inoculated with one loop of variant (strain (3) in Table 3) found to be unable to use 5-keto-D-gluconic acid and incubated for 20 hours. To this culture medium, calcium 2,5-diketo-D-gluconate solution to be described in Preparation B is added to a final concentration of 1.0% and further incubated for 24 hours.

The amount of 2-keto-D-gluconic acid and 2-keto-L-gulonic acid in the cultured media is determined by paper chromatography. The results are shown in Table 3 below.

The preferred variant of 308, which has a 5-keto-D-gluconic acid metabolic defect and is unable to produce 2-keto-D-gluconic acid, has a mutation in 84,520 colonies of Corynebacterium sp.FER-P 2770 (KAIST 830526- 08611), and 11 variants were obtained from Corynebacterium sp.FER-P 2687 (KAIST 830526-08411) of a colony of 29,1000 colonies.

TABLE 3

Preparation A (2,5-diketo-D-gluconic acid fermentation broth)

(i) species

D-glocose 1.0%, corn steep liquor (CSL) 5.0%, potassium monophosphate (KH ₂ PO ₄ ) 0.1%, magnesium sulfate (mgSO ₄ · 7H ₂ O) 0.02% and calcium carbonate (CaCO ₃ ) The aqueous solution containing 0.5% was adjusted to pH6.8-7.0 with 10% NaOH solution, divided into 50 ml each and filled into 500 ml conical flasks. This seed medium is sterilized at 120 ° C for 20 minutes.

(ii) species culture

The flask was inoculated with the seed medium (i) in the 1-loop Erwinia vanktata FER-P 5452 (KAIST 830526-09111) shown in Table 1 and under stirring (amplitude 71 m. 270 r.pm) at 28 ° C. for 8 to 11 hours. Incubate. Finish the cultivation at the point where the optical density reaches about 8 (end point).

(iii) fermentation medium

Aqueous solution containing 20% D-glucose, 3% CSL, 0.1% KH ₂ PO ₄ , CaCO ₃ and 0.01% antifoam polypropylene glycol (P-2000) was adjusted to pH6.8-7.0 and 20 minutes at 120 ° C. After sterilization, each 455 ml each is filled with a sterilized 1 l fermentor and 450 ml of the seed culture solution (ii) are added.

(iv) fermentation

The fermentation is carried out for 20-30 hours with stirring at 1740 r.p.m, in a stream of 600 Nml / min, at 28 ° C. (final concentration of 2,5-diketo-D-gluconic acid is about 19 w / v%). The broth is centrifuged to remove cells and the supernatant is sterilized by filtration. This is used as 2,5-diketo-D-gluconic acid fermentation broth.

(v) endpoint

Fermentation completes when the pink spot of 2-keto-L-gulonic acid disappears from the thin layer chromatography of broth.

Manufacture B

(Calcium 2,5-diketo-D-gluconate solution)

Aqueous solution containing 5% concentration of 5% powdered calcium 2,5-diketogluconate is sterilized by filtration.

Example 2

(Use of variant (II) in the production of 2-keto-L-gulonic acid by fermentation)

(1) Seed badge (common to the examples below)

D-Glocose 1.0%, Bactose yeast extract (Difco) 0.5%, Bactopeptone (Difco) 0.5% Potassium phosphate (KH ₂ PO ₄ ) 0.1%, and magnesium sulfate (MgSO ₄ · 7H 2 O) 0.02% One aqueous solution is adjusted to pH 7-7.2 with 10% NaOH solution. 50 ml each is charged into a 500 ml conical flask and sterilized for 20 minutes at 115 ° C.

(2) fermentation medium

Aqueous solutions containing 1.0% D-glocose, 3.0% corn steep liquor (CSL), 0.1% KH ₂ PO ₄ , and 0.02% mgSO ₄ · 7H ₂ O are adjusted to pH 7-7.2 with 10% NaOH solution. . Each 50 ml is charged into a 500 ml conical flask and sterilized at 115 ° C. for 20 minutes.

(3) Preparation of 2-keto-L-gulonic acid

Seed medium (1) was inoculated into the 2-keto-L-gulonic acid producing microorganism (Parent (1)) or 1 loop of variant (II) derived from (1) shown in Table 4, and the medium was incubated at 28 ° C. Incubate for hours. Then, 5 ml of each of these species cultures is inoculated into the fermentation broth 2 and cultured. One of the 2,5-diketo-D-gluconic acid solutions (Preparation A and B) was added to the fermentation medium at the beginning of the fermentation within 16 hours after the fermentation time so that the final concentration was 2% for 48 hours and then for 48 hours. Continue further fermentation.

Gas-liquid chromatography results of this product are shown in Table 4 below. According to Table 4, 2-keto-D-gluconic acid is not detected from the broth of variant (II), but a significant amount of 2-keto-D-gluconic acid is detected from the parent strain (I).

[The amount of 2-keto-L-gulonic acid and 2-keto-D-gluconic acid remaining in the broth examined at 48 hours after the start of culture after culturing each of the parent strain (I) and the variant (II) ( Mg / ml)]

TABLE 4

2KLG: 2-keto-L-gulonic acid, 2DLG: 2-keto-D-gluconic acid

Example 3

(Use of cell suspensions and cell extracts in the production of 2-keto-L-gulonic acid by contact)

(1) Species Culture

The seed medium (1) described in Example 2 had Corynebacterium sp.FER-P 2770 (KAIST 830526-08611) (1) or 5-keto-D-gluconic acid metabolic defect and 2-keto-D-glu One loop of the variant FER-P 108 (KAIST 830526-09811) (II) which cannot produce cholic acid is inoculated and incubated for 24 hours with stirring at 28 ° C.

(2) culture to prepare cell suspension or extract

The fermentation exhaust (455 ml) described in Example 2, which further contained 0.01% foam suppression, polypropylene glycol 2000 (P 2000), was aseptically filled into a 1-l fermenter and inoculated with 45 ml of culture broth (1). It is fermented for 16 hours in an air stream at 600 Nml / min with stirring at 28 ° C. at 17 r · p.m.

(3) Preparation of Cell Suspension

The cultured broth 2 is collected by centrifugation (15,000 r.p.m) and washed twice with physiological saline. The washed cells are suspended in 0.05M Tris buffer (p.H 7.5) to prepare a suspension of OD660 nm = 12.

(4) Preparation of Cell Extracts

Cells washed in the same manner as described in (3) above are suspended in 0.05M Tris buffer (pH7.8) to make a suspension of OD660 nm = 100 which is passed through a French pressurized selfless at 1,000 kg / cm. Complete cells and cell debris are removed by centrifugation at 20,000 G for 30 minutes. Supernatants are dialyzed in 0.05M Tris buffer (pH 7.5) for 15 hours and used as cell extracts.

(5) Production of 2-keto-L-gulonic acid using narrow suspension (3)

8 ml of cell suspension 3 is combined with 2 ml of Ca 2,5-diketo-D-gluconate solution (Preparation B) and the mixture is reacted under stirring at 30 ° C. for 15 hours. The reaction mixture is then centrifuged (15,000 G.15 min) to remove cells and analyzed by 2-keto-L-gulonic acid and 2-keto-D-gluconic acid by gas liquid chromatography.

The results of the quantitative analysis are shown in Table 5 below.

TABLE 5

(6) Production of 2-keto-L-gulonic acid using cell extract (4)

Cell extract 4 (0.5 mL) was added 2.5 mL of 0.1 M Tris containing 74 μmol Ca 2,5-diketo-D-gloconate and 15 μmol NADPH (reduced nicotine amide adenine dinucleotide phosphate) It is added to buffer (pH 7.5) and reacted at 30 ° C. for 16 hours. Quantitative analysis of this reaction mixture is shown in Table 6 below.

TABLE 6

From the results described in Tables 5 and 6 above, it is certain that 2-keto-L-gulonic acid is produced in the reaction using the cell suspension 3 or the cell extract 4.

In both cases, the product prepared from the parent 2-keto-L-gulonic acid producing microbial strain FERM-P 2770 (KAIST 830526-08611) (I) is not only 2-keto-D-gulonic acid but also 2-keto-D -Gulonic acid is also produced, but the product prepared from variant (II) produces 2-keto-L-gulonic acid and no 2-keto-D-gulonic acid.

Example 4

(Addition of Nitrate and Hydrogen Donor to Fermentation Broth)

(1) 2,5-diketo-D-gluconic acid fermentation broth

The fermentation broth formulated according to Preparation A is centrifuged (10,000 r.p.m. 15 minutes) to remove cells and the supernatant is sterilized by filtration (2,5-diketo-D-gluconic acid concentration; 19%).

The sterile fermentation broth is mixed with sterile aqueous solution of D-glucose (50%) to bring the final concentration of hydrogen donor, ie, D-glucose, to the broth to 3.8%.

(2) Fermentation batches (common to the examples below for the preparation of 2-keto-L-gulonic acid)

Aqueous solution containing 2.0% of D-glocose, 3.0% of CSL, 0.1% of KH ₂ PO ₄ , 0.02% of MgSO ₄ · 7H ₂ O and 0.01% of foam inhibiting polypropylene glycol (P-1000) was prepared at pH7.0 to 7.2 ml and sterilize 450 ml at 115 ° C. for 20 minutes and fill the sterilized 1 l fermentor.

(3) Preparation of nitrogen compound adduct

Sodium nitrate, potassium nitrate, sodium nitrite and ammonium chloride were each dissolved in water to form a 10% aqueous solution sterilized by filtration and the adduct was added to the medium at the beginning of culture and at the beginning of 2,5-diketo-D-gluconic acid feed. Add to

(4) fermentation (for preparing 2-keto-L-gulonic acid)

Corynebacterium sp.FERM-P 2770.ATCC No.31090, which has a 5-keto-D-gluconic acid defect in the seed medium (1) described in Example 2 and cannot produce 2-keto-D-gluconic acid One loop of the variant FERM-BP 108 (KAIST 830526-09811) derived from (KAIST 830526-8611) is inoculated and the medium is incubated at 28 ° C. for 20 to 24 hours with stirring. 50 ml of this seed culture solution was inoculated into the fermentation broth (2), and various nitrogen compound adducts prepared according to the above (3) were aseptically added to obtain a final concentration of 0.25%. Incubate for 10-16 hours at pm.

After confirming the disappearance of D-glucose by the above-described quantitative analysis method, nitrogen compound adduct 3 is added again so that the final concentration is 0.1%. Also, 3.8% D-glucose containing 2,5-diketo-D-gluconic acid fermentation broth is added so that the final concentration of glucose is 0.2%.

Subsequently, 2,5-diketo-D-gluconic acid is partially supplied to the culture every 15 to 120 minutes so that the concentration after feeding is about 0.2%, during which the disappearance of the acid is examined from the broth.

The 2,5-diketo-D-gluconic acid feed stops 45 hours after the start of the feed and the incubation continues for another 3 hours (total culture time since start; 48 hours).

After incubation, the broth was gas chromatographed to analyze 2-keto-L-gulonic acid, 2-keto-D-gluconic acid and 2,5-diketo-D-gluconic acid, respectively.

The results confirmed that 2-keto-D-gluconic acid was not detected in the medium. The accumulation of 2-keto-L-gulonic acid in broths containing various nitrogen compound adducts is shown in Table 7 below.

[Advantages of Various Nitrogen Compound Adducts on Accumulation of 2-keto-L-gulonic Acid in Fermentation Medium at the Beginning of 2,5-diketo-D-Gluconic Acid]

TABLE 7

(The figures in the table indicate the accumulation of 2-keto-L-gulonic acid in the medium, and the values in parentheses indicate the molar yield of 2-keto-L-gulonic acid from the precursor obtained by the following general formula: -Keto-L-gulonic acid, mole) ÷ consumed 2,5-diketo-D-gluconic acid, mole) × 100)

As shown in Table 7, nitrate was added to the medium or broth at the beginning of 2,5-diketo-D-gluconic acid feed and at the beginning of fermentation, and D-glucose as 2,5-diketo-D- as a hydrogen hydrogen conjugate. Compared with the addition of gluconic acid, the accumulation of 2-keto-L-gulonic acid in broth increased from 8.2 mg / ml to 40 mg / ml (approximately 5 times increase) in broth. , The yield (mol%) of 2-keto-L-gulonic acid was confirmed to increase from 41% to 93%.

On the other hand, cell concentration by optical density (O.D.) when D-glucose disappears from the broth is measured as described in Table 8 below, indicating that nitrate does not act as a nitrogen nutrient. As shown in Table 8, the increase in cell concentration by addition of nitrate is limited to only 11.6%, while the increase by addition of ammonium salt is about 40%. This fact indicates that the effect of nitrates as a nitrogen source is less than that of ammonium salts.

From this fact, the addition of nitrates to the medium has little effect on the increase in cell concentration, but increases production of 2-keto-L-gulonic acid and 2-keto 2,5-diketo-D-gluconic acid. It can be seen that it has a great advantage of improving the molar yield for conversion to -D-gulonic acid.

TABLE 8

[Effects of Adducts Applied at the Beginning of Culture on Microbial Growth]

(The figures in the table represent the cell concentration by optical density (O.D) measured at 660 nm when D-glucose was lost).

Example 5

(Sterilization of 2,5-diketo-D-gluconic acid fermentation broth with surface active agent)

The broth prepared according to Preparation A is separated into several parts immediately after incubation.

A designated surface active agent is aseptically added to each part of the broth and the final concentration is brought to 0.025 w / v%. The surfactants used are listed in Table 9 below.

Treatment of the surface active agent with SDS in this 2,5-diketo-D-gluconic acid fermentation broth for 6 hours with occasional stirring at 28 ° C. resulted in 2-keto-L-gulonic acid fermentation with variant (II) to be described below. Used for

TABLE 9

[Sterilization Effect of Various Surfactants on Strain (III) FERM-P 5452]

(1) Addition of D-Glocose to 2,5-diketo-D-gluconic acid fermentation broth

Immediately before feeding broth to the cultured broth of variant (II), an aqueous solution of D-glucose (50%) was added to each of the SDS-treated fermentation broth, the sterilized fermentation broth and the untreated broth to give a final concentration of 3.8%. To be This 2,5-diketo-D-gluconic acid fermentation broth is hereinafter referred to as "substrate solution" and is used as a raw material for 2-keto-L-gulonic acid production.

(2) Preparation of 2-keto-L-gulonic acid from 2,5-diketo-D-gluconic acid by variant (II))

(i) Species Culture

Species medium (1) described in Example 2 had 5-keto-D-gluconic acid metabolic defects and was unable to produce 2-keto-D-gluconic acid and also corynebacterium sp. Inoculate 1 loop of variant (II) FERM-BP 108 (KAIST 830526-09811) derived from FERM-P 2770, ATCC No. 31090 (KAIST 830525-08611) and stir at 28 ° C. for 24 hours (amplitude 71 mm, 270 r. Incubate under pm).

(ii) fermentation (a)

The fermentation broth (2) described in Example 4 was further inoculated with 50% of the seed culture solution by further containing 0.2% sodium nitrate, and stirred for 10 to 16 hours while stirring (1740 rpm) at a flow rate of 600 Nml / min at 28 ° C. Incubate for a while.

(iii) fermentation (b)

D-glucose was consumed to variant (II). After removal from the culture solution of FERM-BP 108 (KAIST 830526-09811), the substrate solution prepared in (1) was separated and added to the culture agent to give a 2,5-diketo-D-gluconic acid concentration of 0.2%. .

The substrate solution is fed to the culture every 15 to 120 minutes while checking for a decrease in the amount of 2,5-diketo-D-gluconic acid in the culture so that the concentration after every feed is about 0.2%.

The substrate solution is finished at 45 hours and the incubation is continued for another 3 hours so that the total culture time is 48 hours after the start of supply.

Fermentation broth is aseptically sampled at designated intervals to analyze 2,5-diketo-D-gluconic acid and 2-keto-L-gulonic acid and the viable cells are counted in the sample (measurement (3)).

The results of the analysis and measurement are listed in Table 10.

TABLE 10

^* 25 D㎏: 2,5- diketo-gluconate -D-

^** 2 KLG: 2-keto-L-gulonic acid

(3) Counting Viable Cell Number of Strain (III)

The sampled fermentation broth is diluted with sterile physiological saline, and the glycerine is spread over bouillon agar medium and incubated at 28 ° C. for 24 to 36 hours. The viable cells in the medium are counted by colony.

Note: Since the growth rate of strain (III) is greater than that of variant (II), cells of strain (III) mixed with cells of variant (II) can be counted without substantial difficulty.

From the results of this experiment, the following facts are confirmed.

Namely, (1) Among various surface active agents proposed and evaluated as a fungicide for 2,5-diketo-D-gluconic acid-producing broth strain (III), SDS (sodium dodecyl sulfate) is the best in bactericidal effect. (See Table 9).

(2) In the results of the 2-keto-L-gulonic acid production experiment in which three substrate solutions were used under the above conditions (see Table 10): i) In the fermentation experiment in which an untreated substrate solution was used, the strain ( The number of viable cells of III) reached 10 ^{5-10 7} cells / ml at 24 hours after the start of the addition of the substrate solution, whereas the accumulation of 2-keto-L-gulonic acid increased little after 24 hours compared to the other two experiments. And, ii) in the fermentation experiments in which the substrate solution treated with SDS was added, the increase in viable cell number of strain (III) was adjusted to more than 10 ⁴ cells / ml even after 48 hours after the start of the addition of the substrate solution, 2 The accumulation of keto-L-gulonic acid reaches about 27 mgml, and iii) for ii) the accumulation of 2-keto-L-gulonic acid is dependent on the accumulation of fermentation experiments with sterile substrate solution applied by filtration. Iv) in the last fermentation experiment with sterile substrate solution applied by filtration, the viable cells of strain (III) It turns out that not even continue the production of 2-keto acid -L- cave without difficulty.

Claims (19)

A method for preparing 2-keto-L-gulonic acid from 2,5-diketo-D-gluconic acid in the presence of living or processed microorganisms capable of producing 2-keto-L-gulonic acid, the 5-keto An improved method characterized by using bacterial variants of the Coryneform group that have actual binding to -D-gluconic acid metabolism and are not able to actually produce 2-keto-D-gluconic acid. The method of claim 1, wherein said variant belongs to the genus Corynebacterium. The method of claim 1, wherein the variant is Corynebacterium sp.FERM-BP 107 (KAIST 830526-09711). The method of claim 1, wherein the variant is Corynebacterium sp.FERM-BP 108 (KAIST 830526-09811). The method of claim 1, wherein the variant is cultured in a water-soluble nutrient medium fed with 2,5-diketo-D-gluconic acid. The 2,5-diketo-D-gluconic acid according to claim 5, wherein the 2,5-diketo-D-gluconic acid producing microorganism belonging to the genus Gluconobacter or Erwinia contains D-glucose in the form of fermentation broth. Produced by culturing in one nutrient medium. The microorganism of claim 6, wherein the microorganism is acetomonas albosesameia ATCC 21998 (KAIST 830526-08111), Gluconobacter melanogenum IFO 3293, Gloconobacter rubicinosus IFO 3244, Erwinia citruus ATCC 31623 KAIST 830526-08811), Erwinia funktatar ATCC 31626 (KAIST 830526-09111), Erwinia funktatar var. ATCC 31624 (KAIST 830526-08911), Erwinia funktatar var. ATCC 31625 (KAIST 830526-09011), Erwinia funktatar var. ATCC 31627 (KAIST 830526-09211), Erwinia Tereus ATCC 31628 (KAIST 830526-09311), Erwinia Tereus var. ATCC 31629 (KAIST 830526-09411), Erwinia Tereus var. ATCC 31630 (KAIST 830526-09511) and Erwinia Tereus var. ATCC 31631 (KAIST 830526-09611). The method of claim 1, wherein the conversion of the growth 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid is performed in the presence of a nitrate or hydrogen donor. The method of claim 8, wherein the nitrate is a salt of an alkali metal or an alkaline earth metal. The method of claim 8 wherein said nitrate is sodium nitrate or potassium nitrate. The method of claim 8, wherein the concentration of the nitrate is 0.05 to 0.5w / v%. 9. The method of claim 8, wherein said hydrogen donor is selected from the group consisting of carbohydrates and polyhydric alcohols. The method of claim 8, wherein the hydrogen donor is selected from the group consisting of glycerol, glucose, fructose, sucrose and molasses. 9. The method of claim 8, wherein the hydrogen donor is 5-50% of the 2,5-diketo-D-gluconic acid. 7. The method of claim 6 wherein the 2,5-diketo-D-gluconic acid fermentation broth is sterilized with a surface active agent before being fed. The method of claim 15, wherein said surface active agent is sodium dodecyl sulfate (SDS). The method of claim 16 wherein the SDS is added at a concentration of 0.01 to 0.25 w / v%. The method of claim 1, characterized in that it is in the form of a cell suspension of the microorganism. The method of claim 1, characterized in that it is in the form of a cell extract of the microorganism.