JP2675074B2 - 2-Keto-L-glaric acid and a method for producing the same, and a method for producing L-grosaccharoascorbic acid and 2,3-O-acetal or ketal using the acid as a raw material - Google Patents

Description

TECHNICAL FIELD The present invention relates to a synthetic raw material for an antioxidant, a reagent for sugar metabolism research,
2-Keto-L- useful as a starting material for the synthesis of optically active compounds
Gularic acid (2-keto-L-Gularica fcid, L-xylo-2
-Hexulosalic acid: L-xylo-2-Hexulosaric acid, formula 1) and a method for producing the glamic acid by fermentation, and an antioxidant starting from this, and a raw material for an optically active compound Useful L-grosaccharoascorbic acid (L-Gulosacc
haroascorbic acid, L-threo-hexa-2-enalo-
1,4-lactone, L-threo-Hexo-2-enaro-1,4-lact
one) and a method for producing a 2,3-O-acetal or ketal of 2-keto-L-glaric acid, which is useful as a raw material for an antioxidant starting from 2-keto-L-gularic acid.

Conventional Technology and Problems 2-Keto-L-glaric acid is a novel compound first discovered in the present invention.

L-grosaccharoascorbic acid is vitamin C (L-
It has been reported to be a metabolite of ascorbic acid) in rats and monkeys [Biochemical and Biophysical Research Communications (Bioche
mical and Biophysical Research Communication
s), 71, 1004-1009 (1976)], and their physical properties and physiological activities have also been studied [vitamins, 56, 11].
7-131 (1982)]. Two methods have been known so far as a method for synthesizing L-grosaccharoascorbic acid.

The first method is 2,3-O-isopropylidene-α-L-
Hydroxymethyl groups at the 1- and 6-positions of sorbose were catalytically air-oxidized in the presence of a noble metal catalyst to give 2,3-O-isopropylidene-2-keto-L-glaric acid (2,3-O-isoprop
ylidene-α-L-xylo-2-hexulosaric acid) followed by acid treatment to produce L-grosaccharoascorbic acid [US Pat. No. 2,428,43].
8 go, No. 2,483,251; Carbohydrate research, 60, 251-258 (197)
8)].

The second method is to oxidize the hydroxymethyl group at the 6-position of L-ascorbic acid. Before the oxidation, the hydroxyl group at the 2-position is sulfonated to protect the enediol group at the 2- and 3-positions, and then a noble metal catalyst is used. The 6-position hydroxymethyl group is converted to a carboxyl group by catalytic air oxidation in the presence,
Finally, the protecting group at the 2-position is deprotected by the action of an acid to obtain L-grosaccharoascorbic acid [Carbohydrate Research, 60 , 251-258 (19).
78); Vitamins, 56 , 117-131 (1982)].

In the first method, 2,3-O-isopropylidene-α-L-sorbose, which is a starting material compound, is used in two steps to obtain L-
Although grosaccharoascorbic acid has been derived, this method is used for the oxidation of 2,3-O-isopropylidene-α-L-sorbose to 2,3-O-isopropylidene-2-keto-L-glaric acid. In the process, since it is necessary to use an expensive noble metal catalyst, and the liquidity of the reaction liquid is kept near neutral, the reaction operation is complicated and the reaction yield is not satisfactory.

In the second method, the number of steps is long from L-ascorbic acid, which is a raw material compound, to 3 steps, and in addition, the step of converting the 6-hydroxymethyl group of L-ascorbic acid-2-O-sulfonate to a carboxyl group is described above. It is necessary to use the above-mentioned expensive noble metal catalyst, and since the liquidity of the reaction liquid is kept near neutral, the reaction operation is complicated and the yield is not satisfactory.

That is, the above-mentioned conventional method has a long reaction step and requires the use of an expensive noble metal catalyst, and the reaction operation is complicated, so it cannot be said to be an industrial method. Further, although L-grosaccharoascorbic acid was already synthesized in 1947, as a derivative, a salt of L-grosaccharoascorbic acid has only been synthesized so far.
It is considered that the conventional method has proved to be difficult as a practical manufacturing method.

2,3-O-acetal or ketal of 2-keto-L-glaric acid is used as a synthetic intermediate of L-grosaccharoascorbic acid described above as 2,3-O-isopropylidene-
2-keto-L-glaric acid (2,3-O-isopropylidene-
Only α-L-xylo-2-hexulosaric acid) is known [U
SP, No.2,428,438; USP, No.2,483,251; Carbohydrate Res
earch 60 , 251-258 (1978)], but as described in the known synthesis method of L-grosaccharoascorbic acid, 2,3,: 4, obtained by reacting L-sorbose with acetone. Hydroxymethyl groups at the 1- and 6-positions of 2,3-O-isopropylidene-O-sorbose derived by partial hydrolysis reaction of 6-di-O-isopropylidene-L-sorbose were contacted with air in the presence of a noble metal catalyst. Oxidation synthesizes 2,3-O-isopropylidene-2-keto-L-glaric acid.

In the oxidation step, an expensive noble metal catalyst must be used, and the reaction operation is complicated because the liquidity of the reaction solution is kept near neutral, and the reaction yield is not always satisfactory.

Means for Solving Problems The present inventors are studying the oxidation mechanism of monosaccharides by microorganisms, and the bacteria isolated from soil data, K591s strain, 12-5 strain, 12
-15 strain, 12-4 strain and 22-3 strain were added to L-sorbose, 2-
When cultured in a medium containing keto-L-gulonic acid or 5-keto-D-gluconic acid, a significant amount of an unknown sugar oxidation product (sometimes referred to as SOP-] 1) is accumulated in the culture solution. I found a thing. As a result of various studies on the chemical structure of the substance isolated from the culture solution, it was revealed that this substance was 2-keto-L-glaric acid.

Furthermore, the reactivity of 2-keto-L-gularic acid was investigated, and it was found that L-grosaccharoascorbic acid can be easily obtained by treating this compound with an acid.

Furthermore, 2-keto-L-glaric acid is acetalized or ketalized to give 2-keto-L in one step.
It has been found that 2,3-O-acetals or ketals of glural acid are obtained in high yield.

As a result of intensive studies on the above points, the present invention has been completed.

That is, in the present invention, (1) 2-keto-L-glaric acid or a salt thereof, (2) L-sorbose, 2-keto-L-gulonic acid or 5-keto-D-gluconic acid are converted into 2-keto- Bacteria belonging to the genus Pseudogluconone Bacter, which have the ability to oxidize to L-gularic acid, were treated with L-sorbose, 2-keto-L
A method for producing 2-keto-L-glaric acid, which comprises contacting with gulonic acid or 5-keto-D-gluconic acid to generate and accumulate 2-keto-L-gularic acid, and collecting the resulting product. 3) A method for producing L-grosaccharoascorbic acid, which comprises treating 2-keto-L-glaric acid with an acid, and (4) acetalizing or ketalizing 2-keto-L-glaric acid. A method for producing a 2,3-O-acetal or ketal of 2-keto-L-glaric acid, which comprises:
It is.

First, the method for producing 2-keto-L-glaric acid by the fermentation method will be described.

Speaking of strains used, K591s strain is 1 in Wakayama prefecture.
The 2-5 strain, 12-15 strain, 12-4 strain and 22-3 strain are bacteria isolated from soil data of Shiga prefecture. These 5
Each strain is a fungus of its own.

The various degradative properties of the five soil-separating bacteria used in the present invention were analyzed by the Bergey's Manual of Determinating Bacteriology.
8th Edition (1984) and Bergey's Manual of Systematic Bacteriol
When compared with ogy Volume 1 (1984), 5 strains, namely K
The 591s strain, 12-5 strain, 12-15 strain, 12-4 strain, and 22-3 strain are Gram-negative, polar flagella-motile rods, and are aerobic and oxidase-positive, so Pseudomonas (Pseudomonas)
monas) genus bacteria is thought to be. Require growth factors, the total amount of DNA guanine and cytosine 67 ± 1
Mol%, quinone type has 10 isoprene units
Since it is a ubiquinone of Section IV of this genus
It is similar to Pseudomonas diminuat and Pseudomonas vesicularis belonging to RNA group IV.
However, the production of acetic acid from ethanol, though weak, and the production of dihydroxyacetone from glycerol are different from the properties of Pseudomonas.

These properties make Gluconobacte
r) It is that of a genus. However, these strains are also positive for an oxidase test, cannot grow at pH 4.5, can grow well in a yeast extract-containing broth medium or peptone-yeast extract medium that does not contain sugar (power), and can guanine DNA. The total amount of and cytosine is 67 ± 1 mol%
And other properties are different from those of Gluconobacter species.

Therefore, these 5 strains, namely, K591s strain, 12-5 strain, 12-1
The 5 strains, 12-4 strains and 22-3 strains could not be found to belong to any known genus and could not be regarded as new strains of a new genus. Therefore, K591s strain, 12-5 strain, 12-15 strain, 12-4 strain and 22
5 strains of -3 strains are Pseudogluconobacter sacchroketogene
s) was named. Here, the auxotrophy of these 5 strains is mentioned. K591s strain, 12-5 strain and 12-15 strain are Co
It has an unusual property that requires A for growth. These three
The strain's CoA requirement cannot be replaced by pantothenic acid. On the other hand, strains 12-4 and 22-3 can grow in the presence of CoA and pantothenic acid.

Hereinafter, these Pseudogluconobacter saccharoketgenes may be referred to as oxidizing bacteria.

The strains used in the present invention are not limited to the above-mentioned 5 strains, for example, the 5 strains are irradiated with ultraviolet rays or X-rays, or N-
A mutant strain obtained by treatment with a mutagenizing agent such as methyl-N'-nitro-N-nitrosoguanidine (nitrosoguanidine), methylmethanesulfonic acid, and nitrodiene mustard is also advantageously used. Examples thereof include TH14-86 strain derived from Pseudogluconobacter saccharoketgenes K591s strain by nitrosoguanidine treatment, and further SOP-809 strain derived therefrom. Both mutants are 2-keto-L- from L-sorbose.
The parent strain K591, except that the ability to produce glamic acid is enhanced.
It showed the same taxonomic properties as the s strain.

Pseudogluconobacter Saccharoketgenes above
K591s strain, 12-5 strain and TH14-86 strain were
On September 19th, Pseudogluconobacter saccharoketgenes 12-15, 12-4 and 22-3 were
On December 16, 1985, the deposit numbers were respectively IFO 1446
4, IFO14465, IFO 14466, IFO 14482, IFO14483 and IFO
14484 was deposited at the Fermentation Research Institute.
See also Pseudogluconobacter Saccharoketgenes K5
91s strain, 12-5 strain and TH14-86 strain were pseudogluconobacter saccharoketgenes 12 on October 7, 1985.
-15 strains, 12-4 strains and 22-3 strains were deposited with the deposit number FERM P-8481, FERM P-8481, FERM on December 20, 1985 at the Institute for Microbial Technology (FRI), Ministry of International Trade and Industry. P-84
79, FERM P-8577, FERM P-8576 and FERM P-85
Each of the deposits was converted into a deposit under the Budabest Treaty and stored at the same research institute with the deposit number shown below.

K591s strain: FERM BP-1130 12-5 strain: FERM BP-1129 TH14-86 strain: FERM BP-1128 12-15 strain: FERM BP-1132 12-4 strain: FERM BP-1131 22-3 strain: FERM BP -1133 Also Pseudogluconobacter Saccharoketgenes
The SOP-809 share was changed to IFO 14608 on May 12, 1987,
Deposited to the Fermentation Research Institute, on June 10, 1987, Ministry of International Trade and Industry, Industrial Technology Institute, Microbial Industrial Technology Research Institute (FR
It has been deposited with I) under deposit number FERM P-9407.

In the present method for producing 2-keto-L-glaric acid, when the starting material L-sorbose, 2-keto-L-gulonic acid or 5-keto-D-gluconic acid was added to the medium, the whole amount used was initially cultured. To the medium, or the total amount may be added to the culture medium in several divided portions or continuously. L-sorbose, 2-keto-L-gulonic acid or 5 in the culture solution
The concentration of -keto-D-gluconic acid is 2 to 20% (W / V), preferably 2 to 10% (W / V) with respect to the medium.

In the present invention, Pseudogluconobacter bacteria are
-Sorbose, 2-keto-L-gulonic acid or 5-keto-
When culturing in a liquid medium containing D-gluconic acid to allow 2-keto-L-glaric acid to grow and accumulate in the culture medium,
Rather than culturing the oxidizing bacterium Pseudogluconobacter genus alone, mixing a different type of bacterium with the oxidizing bacterium 2
-The amount of accumulated keto-L-glaric acid is significantly increased.

Examples of the bacteria to be mixed include bacteria belonging to the genus Bacillus, Pseudomonas, Proteus, Citrobacter, Enterobacter, Erwinia, Xanthomonas, Flavobacterium, and the like, and specific examples are shown below. Examples include bacteria.

Bacillus sereus IFO 3131 Bacillus licheniformi
s) IFO 12201 Bacillus megaterium IFO
12108 Bacillus pumilus IFO 12090 Bacillus amylolique fancy
oliquefaciens) IFO 3022 Bacillus subtilis IFO 137
19 Bacillus circulans IFO
3967 Pseudomonas trifolii I
FO 12056 Pseudomonas malto
philia) IFO 12692 Proteus inconstan
s) IFO 12930 Citrobacter freundi
i) IFO 13544 Enterobacter cloaca
e) IFO 3320 Erwinia herbicola IFO
12686 Xanthomonas pisi IFO 1355
6 Xanthomonas citri IFO
3835 Flavobacterium meningocepticum (Flavobacte
rium meningosepticum) IFO 12535 Micrococcus varians
IFO 3765 Escherichia coli IFO 3366 One of these fungi in an appropriate medium at 20-40 ° C
A culture solution obtained by culturing for 4 days from the day can be used as a seed culture solution of a mixed bacterium. The transplant amount is usually 1/10 of that of oxidizing bacteria (Pseudogluconobacter sp.)
1/1000 is preferable. When the mixed bacterium is mixed and cultured with the oxidizing bacterium in such a transplantation amount, the growth of the oxidizing bacterium is promoted.
Sorbose, 2-keto-L-gulonic acid or 5-keto-D
-Gluconic acid is oxidized to 2-keto-L-glaric acid in a shorter time. The bacteria used as the mixed bacteria are L-sorbose, 2-keto-L-gulonic acid or 5-keto-D-gluconic acid, which are the raw materials of the present invention, and 2-keto-L-glaric acid, which is a product. It is desirable that the fungus has no or only a slight chemical activity. Other culture conditions are the same as those of the oxidizing bacteria alone culture.

The medium used for culturing the oxidizing bacteria may be liquid or solid as long as it contains a nutrient source that can be used by the strain, but it is preferable to use a liquid medium for obtaining a large amount. For the medium, a carbon source, a nitrogen source, inorganic salts, organic acid salts and micronutrients which are usually used for culturing microorganisms are used.

L-sorbose is used as it is as a carbon source, but other auxiliary carbon sources such as glucose,
Glycerin, saccharose, lactose, maltose, and sugar sugar can be used.

As a nitrogen source, for example, various ammonium salts (ammonium sulfate, ammonium nitrate, ammonium salt, phosphorus ammonium), corn steep liquor (hereinafter
(Sometimes referred to as CSL), peptone, meat extract, yeast extract, dry yeast, soybean flour, cottonseed meal, and urea-containing inorganic or organic nitrogen-containing substances.

As inorganic salts, salts of potassium, sodium, calcium, magnesium, iron, manganese, cobalt, zinc, copper and phosphoric acid are used.

As a micronutrient, Co which is an essential growth factor of the above-mentioned bacteria
A, pantothenic acid, biotin, thiamine, and riboflavin, as well as flavin mononucleotide (hereinafter referred to as FMN) that has a promoting effect on growth and 2-keto-L-glaric acid production.
, Other vitamins, L-cysteine, L-glutamic acid, sodium thiosulfate, etc. are added as compounds or natural products are appropriately added.

The culture may be performed by static culture, shaking culture, aeration-agitation culture, or the like, but so-called deep aeration-agitation culture is preferable for mass production.

The culture conditions will of course vary depending on the type of strain, the composition of the medium, etc., and may be selected according to each case so that the desired product can be produced most efficiently. For example,
The culture temperature is preferably 25-35 ° C, and the pH of the medium is 5-
About 9 is desirable.

By culturing under the above conditions for 20 to 170 hours, 2
-Keto-L-glaric acid accumulates at the highest concentration. In this case, since the pH generally decreases with the formation of the target substance, an appropriate basic substance such as caustic soda, caustic potash, or ammonia is added to always keep the microorganism 2-keto-L.
It may be maintained at the pH most suitable for the production of gularic acid, or an appropriate buffer may be added to the medium to maintain the optimum pH.

In addition, a sterilized culture of a bacterium different from the above oxidizing bacteria can be effectively used as a medium component. Bacteria that can be used include, for example, bacteria belonging to the genus Bacillus, the genus Pseudomonas, the genus Citrobacter, the genus Escherichia, and the genus Erwinia, and specific examples thereof include the bacteria shown below.

Bacillus cereus IFO 3131 Bacillus subtijis IFO 30
23 Bacillus pumilus IFO 12089 Bacillus megateriumu IFO
12108 Bacillus amylolique fancy
oliquefaciens) IFO 3022 Pseudomonas trifolii I
FO 12056 Citrobacter freun
dii) IFO 12681 Esherichia coli IFO 3546 Erwinia herbicola IFO
12686 Add these bacteria to the medium in which they can grow for 20-40
Cultivate at 2 ℃ for 2 to 4 days, sterilize the resulting culture solution, add this to the medium of the present oxidizing bacteria at a rate of 0.5 to 5.0% (V / V),
It can also promote the growth of oxidizing bacteria.

In this way, 2-keto-L- that grew and accumulated in the culture medium
Glaric acid can be separated and purified by a means known per se utilizing its properties.

2-Keto-L-gularic acid may be separated as a free acid, for example, as a salt of sodium, potassium, calcium, ammonium or the like.

As a method of separation, for example, if necessary, after filtration from the culture solution, after removing the bacterial cells by centrifugation, the solution as it is, or treated with activated carbon, concentrated, and the precipitated crystals are collected by filtration, The method of recrystallizing and extracting the desired product, the solvent extraction method, the chromatography method, the salting-out method and the like can be used alone or in an appropriate combination, or can be used repeatedly.

When 2-keto-L-glaric acid is obtained in a free form, it may be converted into a salt of sodium, potassium, calcium, ammonium or the like by an appropriate method, and when it is obtained as a salt, This may be changed to a free form or another salt by an appropriate method.

It is clear from the following explanation that the object of the present invention is 2-keto-L-glaric acid.

The elemental analysis values of the sugar oxidation product (SOP-1) obtained in Example 4 are as follows.

Elemental analysis value (%): C ₆ H ₆ O ₈ Ca · 5H ₂ O theoretical value (%): C; 21.43, H; 4.89, (Ca; 11.92) Measurement value (%): C; 21.34, H; 4.83, (Ca; 11.5) (Ca is the value measured by the atomic absorption method) The NMR spectrum is as follows. ¹³ C-NMR (D ₂ O + 10% DCl) δ76.1 (d), 76.7 (d), 7
8.4 (d), 79.7 (d), 81.4 (d), 82.9 (d), 101.7
(S), 106.4 (s), 171.8 (s), 172.5 (s), 173.1
(S), 173.4 (s) Although a complicated chart can be obtained from the above ¹³ C-NMR spectrum, 12 peaks observed after careful examination were 6 pairs of pairs of 2 peaks each. I understood. From this result, this substance is not an oxidation product in which the carbon-carbon bond of L-sorbose is cleaved, but has a similar structure.
It suggests that it is a 6-carbon sugar consisting of isomers of species.

Based on this ¹³ C-NMR spectrum and elemental analysis value, this substance was found to have two carboxyl groups (C-1,6 position), three hydroxyl-substituted carbons (C-3,4,5 position) and one anomeric It is presumed to have carbon (C-2 position).

From the above-mentioned analytical data of the starting material L-sorbose and 2-keto-L-gulonic acid and the product, from L-sorbose to the hydroxymethyl group at the C-1 position and the carboxyl group at the hydroxymethyl group at the C-6 position. It is presumed that oxidation occurs, and from 2-keto-L-gulonic acid, oxidation of the hydroxymethyl group at the C-6 position to a carboxyl group occurs. In addition, in the ¹³ C-NMR spectrum, a peak around 200 ppm derived from a free carbonyl group (C-2 position) was not observed. Therefore, it does not exist as a linear structure (1) under the measurement conditions and is represented by the following formula. Anomeric isomers such as (1a)
It was presumed to exist as a mixture of (1b) and (1b).

Since it is difficult to determine the structure of the dicalcium salt (1c) as it is, the structure was determined by inducing a known compound by the method shown in the formula below.

That is, the sugar oxidation product [(1c), SOP-1: later 2-keto-
It was confirmed by the following examination that it was a L-dicalcic acid dicalcium salt. Was reacted with sulfuric acid in an acetone solvent to give a free carboxylic acid, and then heated under reflux under dehydration conditions to obtain an isopropylidene compound (2). Further, (1c) was reacted with sulfuric acid in water to form a free carboxylic acid, which was then heated and subjected to a lactonization reaction to obtain a compound [(3), which was later identified as L-grosaccharoascorbic acid]. It was

On the other hand, the compound (2 ′) is a known compound [USP, No. 2,428,4
38; USP, No.2,483,251; Carbohydrate Research, 60 , 251
~ 258 (1978)], the inventors improved the literature method to synthesize (2 ') by the following method.

That is, 2,3: 4, which is an intermediate in the industrial production method of vitamin C,
6-di-O-isopropylidene-2-keto-L-gulonic acid [(4), 2,3: 4,6-di-O-isopropylidene-α-L
-Xylo-2-hexulosonic acid] is partially hydrolyzed to give 2,3-O-isopropylidene-2-keto-L-gulonic acid [(5), 2,3-O-isopropylidene-α-L-xy
lo-2-hexulosonic acid] and then compound (5)
Was subjected to catalytic air oxidation reaction in the presence of a Pt catalyst to obtain a compound (2 ') in which only the 6-position hydroxymethyl group was selectively oxidized to a carboxyl group. The compound (2 ') synthesized by this method and the compound (2) derived from the compound (1c) have good melting points, and IR, ¹ H-NMR and ¹³ C
-The various spectra such as NMR were completely in agreement.

From this, it can be determined that the structure of the acetal form of the compound (1c) is (2), and from this fact, the structure of the sugar oxidation product (SOP-1) is 2-keto-L- Dicalcium glalarate pentahydrate (Calcium
L-xylo-2-hexulosarate).

Furthermore, from the compound (2 ′), a standard L-grosaccharoascorbic acid (3 ′) was prepared according to the method described in the above literature.
Was synthesized. The compound (3 ′) is the compound (1c) described above.
When the compound (3) derived from the above was compared and examined, the melting point and various spectra were completely in agreement, which confirmed the structure of the sugar oxidation product (1c) obtained above.

Next, the method for producing L-grosaccharoascorbic acid from 2-keto-L-glaric acid will be described in detail.

The 2-keto-L-glaric acid used as a raw material compound in the production method of the present invention may be a free acid or a salt. Examples thereof include alkali metal salts (lithium, sodium, potassium salts, etc.), alkaline earth metal salts (magnesium, calcium, barium salts) or ammonium salts.

The acid used in the method for producing L-grosaccharoascorbic acid of the present invention is not particularly limited, but examples thereof include hydrogen fluoride, hydrogen chloride, hydrogen bromide, hydrogen iodide, phosphoric acid, sulfuric acid, fluorosulfuric acid, and persulfate. Mineral acids such as chloric acid, trifluoromethanesulphonic acid, methanesulphonic acid, paratoluenesulphonic acid, acetic acid, monochloroacetic acid, trichloroacetic acid, trifluoroacetic acid and organic acids such as H ⁺ type ion exchange resins can be mentioned by themselves. Alternatively, it may be dissolved or suspended in water as needed.

The acid used may be a single acid or a mixture of two or more of the above-mentioned acids.

The amount of acid used is not particularly limited, but it is usually 0.1
A large excess amount of about 2 times to about 100 times the molar amount is used,
More preferably, it is in the range of about 0.2 times to about 50 times mol.

When using salts of 2-keto-L-glaric acid as a starting material, a theoretical amount or a slight excess of an acid for neutralizing these salts is added to neutralize, and the salt produced as a by-product at this time is subjected to a filtration method and ion exchange. The salt may be previously removed from the reaction system by a known technique such as a membrane method, an ion exchange resin method, an extraction method with a suitable solvent, column chromatography or electrodialysis method.

Water is usually used as the reaction solvent used in the production method of the present invention, but any solvent that does not inhibit the reaction can be used. For example, benzene, toluene, xylene, chlorobenzene, dichloromethane, chloroform, carbon tetrachloride, 1,1-dichloroethane, 1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,1-trichloroethane, trichloroethylene, tetrachloroethylene, Examples include nitromethane, nitroethane, t-butanol, ether, tetrahydrofuran, dioxane, ethylene glycol dimethyl ether, diethylene glycol dimethyl ether, acetone, methyl ethylketone, cyclopentanone, cyclohexane, acetonitrile, propionitrile, and benzonitrile. 2 of these solvents
It is also possible to carry out the reaction in a mixed solvent composed of one or more species.

When an organic solvent is used as the reaction solvent, the reaction rate may be improved by adding a surfactant to the reaction system.

Examples of the surfactant include nonionic surfactants such as 18-crown-6, dibenzo-18-crown-6, [2,2,2] -cryptate and polyoxyethylene alkylallyl ether, such as tetrapropylammonium. Chloride, benzyltrimethylammonium bromide
Phenyltriethylammonium chloride, cetyltrimethylammonium chloride, stearylmethylammonium bromide, 1-phenacylpyridinium chloride,
Various ammonium salts such as stearyl propylene diamine dihydrochloride, and cationic surfactants such as phosphonium salts such as benzyltriphenylphosphonium chloride and vinyltriphenylphosphonium bromide, such as higher fatty acids and anions such as alkylallyl sulfonate. Examples thereof include ionic surfactants, and these may be used alone or in combination of two or more kinds. The amount of surfactant added is about 0.05 to 5% by weight, preferably 0.1 to 3% by weight, based on the substrate.

The reaction temperature is not particularly limited. Usually about 0 ℃ to about 150
The temperature is in the range of about 0 ° C, preferably about 10 ° C to about
It is in the range of 100 ° C.

The reaction time varies depending on the starting material, the type of salt, the type of acid, the amount used, and the reaction conditions, but it is usually about 10
Minutes to about 24 hours, preferably about 20 minutes to 12 hours.

The isolation of the L-grosaccharoascorbic acid thus produced from the reaction system is slightly different depending on whether the raw material used is a free acid or a salt thereof. That is, when the starting material is 2-keto-L-glaric acid and the salt by-produced by the neutralization reaction with an acid during the reaction has not been removed in advance, it is necessary to remove the salt. For the desalting operation, the reaction solution as it is or after treatment such as removal of the solvent, filtration, ion exchange membrane method, ion exchange resin method, extraction method with an appropriate solvent, column chromatography, or electrodialysis method is used alone. Alternatively, it is performed according to an appropriate combination.

Even if the starting material used is a salt of 2-keto-L-glaric acid, it is desalted during the reaction, or 2-keto-L-glaric acid.
In the case of glural acid, L-grosaccharoascorbic acid can be easily prepared by known means such as extraction, column chromatography and recrystallization from the residue obtained by distilling off the solvent and low-boiling substance from the reaction product by a conventional method. Can be isolated and purified.

When isolated as a salt of L-grosaccharoascorbic acid, L-grosaccharoascorbic acid obtained by the above-mentioned method and a suitable base such as an alkali metal oxide, an alkali metal hydroxide or an alkali metal carbonate. Reaction with salts or bicarbonates, alkaline earth metal oxides, alkaline earth metal hydroxides or alkaline earth carbonates or suitable amines, or substitution with suitable alkali metal or alkaline earth metal or ammonium It can be exchanged by contact with the cation exchange resin. Moreover, the reaction product is not isolated once as L-grosaccharoascorbic acid, and the reaction product is treated with a suitable base such as an alkali metal oxide, an alkali metal hydroxide, an alkali metal carbonate or bicarbonate, an alkaline earth metal oxide. , Neutralized with alkaline earth metal hydroxides or carbonates or suitable amines, or contacted with a cation exchange resin substituted with a suitable alkali metal or alkaline earth metal or suitable ammonium By directly converting the salt of L-grosaccharoascorbic acid to the desired salt, the desired L-grosaccharoascorbic acid salt is isolated by a known means such as solvent distillation and recrystallization or reprecipitation. , May be purified.

Finally, mention is made of the acetal compounds and ketals of 2-keto-L-glaric acid.

As the 2,3-O-acetal or ketal of 2-keto-L-glaric acid of the present invention, the constituent component of the acetal or ketal has the general formula (In the formula, R ₁ and R ₂ are the same or different, and hydrogen, alkyl, an optionally substituted phenyl group, and R ₁ and R ₂ may combine to form a cycloalkyl group. Shown).

Examples of the alkyl group represented by R ₁ and R ₂ include an alkyl group having 1 to 5 carbon atoms, and specific examples include:
For example, methyl, ethyl, propyl, i-propyl, butyl, i-butyl, sec-butyl, t-butyl, amyl, i-amyl and the like can be mentioned.

As the substituent of the optionally substituted phenyl group,
Examples thereof include halogen such as chlorine, hydroxyl group, alkoxy having 1 to 3 carbon atoms, alkyl having 1 to 3 carbon atoms, amino group, nitro group, cyano group, carboxyl group, ester group, carbamoyl group and trifluoromethyl group. .

When R ₁ and R ₂ are taken together to form a cycloalkyl group, the carbon number thereof is preferably in the range of 2 to 6,
Specific examples include ethylene, methylene, tetramethylene, pentamethylene and hexamethylene.

In this method, 2-keto-used as a starting compound
L-Glaric acid may be the free acid itself, or may be a method of reacting an acid from the corresponding salt in the reaction system to purify the free acid.

Next, the acetalization or ketalization in the present production method is carried out by reacting 2-keto-L-glaric acid with an aldehyde, a ketone or a reactive derivative thereof in the presence of an appropriate acetalization or ketalization catalyst. Be seen.

The above aldehyde or ketone has the general formula R ₁ COR ₂
(R ₁ and R ₂ have the same meanings as described above) are used.

Specific examples of the aldehyde include formaldehyde, acetaldehyde, propionaldehyde, n-butyl aldehyde, i-butyraldehyde, benzaldehyde, and halogen such as chlorine, hydroxyl group, alkoxy having 1 to 3 carbons, and 1 to 3 carbons. Alkyl, amino, nitro, carboxyl, ester, carbamoyl,
Examples thereof include a benzaldehyde derivative which may have a substituent such as a trifluoromethyl group. Specific examples of the ketone include, for example, acetone, methyl ethyl ketone, diethyl ketone, di-n-propyl ketone, di-i.
-Propyl ketone, di-n-butyl ketone, di-i-butyl ketone, methyl n-propyl ketone, methyl n
-Butyl ketone, methyl i-butyl ketone. Amyl
Examples thereof include ethyl ketone, diamyl ketone, amyl methyl ketone, i-amyl methyl ketone, cyclobutanone, cyclopentanone, cyclohexanone and cycloheptanone.

On the other hand, as a reactive derivative of aldehyde or ketone, (In the formula, R ₁ and R ₂ have the same meanings as described above, and R ₃ and R ₄
Are the same or different and have 1 to 5 carbon atoms, or
R ₃ and R ₄ may be combined to form a cycloalkyl group having 2 to 6 carbon atoms). Specific examples of R ₃ and R ₄ include, for example, methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, sec-butyl, t-butyl, amyl, i-amyl and
R ₃ and R ₄ together form ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene and the like.

In the production method of the present invention, the amount of the aldehyde or ketone used is about 2 to 10 times the stoichiometric amount based on the starting 2-keto-L-glaric acid or its salt, but it is usually a reaction reagent / solvent. It is advantageous to use a large excess as.

The amount of the reactive derivative of acetal or ketal used is about 1.5 to 10 times the stoichiometric amount with respect to the starting material, 2-keto-L-glaric acid or a salt thereof. It is advantageous to use a mixture of reactive derivatives. In this case, it is preferable that the reactive derivative of acetal or ketal is kept to a necessary minimum and the aldehyde or ketone is used in a 5-fold molar to large excess.

As the acetalization catalyst or ketalization catalyst in the production method of the present invention, a known acid catalyst can be used, and specific examples thereof include the acid catalysts shown below.

Mineral acids include, for example, hydrogen chloride, hydrogen bromide, hydrogen iodide, hydrogen fluoride, perchloric acid, sulfuric acid, fluorosulfuric acid, phosphoric acid, boric acid, and the like, and organic acids include, for example, paratoluenesulfonic acid, Examples thereof include benzene sulfonic acid, naphthalene sulfonic acid, methane sulfonic acid, trifluoromethane sulfonic acid, trifluoroacetic acid, trichloroacetic acid and H ⁺ type ion exchange resins. Examples of Lewis acids include boron trifluoride, boron trifluoride, and boron trifluoride. Ether complex, boron trichloride, boron tribromide, boron triiodide,
Antimony pentoxide, antimony pentafluoride, ferric chloride,
Cupric chloride, cupric bromide, zinc chloride, stannous chloride, stannic chloride, stannous bromide, stannic bromide, aluminum chloride,
Aluminum bromide, aluminum iodide, titanium tetrachloride, phosphorus trioxide, phosphorus pentachloride, phosphorus oxychloride, iodine, iodine monochloride, iodine monobromide, iodine trichloride, phosphorus iodide and the like can be mentioned. These may be used by themselves or, if necessary, dissolved or suspended in water or a solvent. Further, the acid to be used may be a single acid or a mixture of two or more of the above-mentioned acids may be used.

The amount of the catalyst used is, for example, 2-keto used as a raw material.
It is appropriately selected depending on whether L-glaric acid is a free form or a salt thereof. For example, 2-
In the case of keto-L-glaric acid, it is about 0.001-10 with respect to the substrate.
% By weight, preferably in the range of about 0.01 to 5% by weight.

When the starting material is a salt of 2-keto-L-glaric acid, the amount of 2-keto-L-glaric acid that is once free in the reaction system can be increased by adding an organic acid or mineral acid in a slight excess over the theoretical amount required for neutralization.
Keto-L-glaric acid is added, and a necessary amount of an acetalization or ketalization catalyst is added as a catalyst for the acetalization or ketalization reaction. Therefore, the amount of the acid used is in the range of the theoretical neutralization amount to 120% by weight of the substrate.

As the reaction solvent used in the production method of the present invention, any solvent can be used as long as it does not inhibit the reaction. For example, acetonitrile, propionitrile, nitromethane, niloethane, nitrobenzene, dichloromethane, chloroform, carbon tetrachloride, 1,1 -Dichloroethyne, 1,2-dichloroethane, ethyl bromide, pentane, cyclopentane, hexane, cyclohexane, heptane, benzene, toluene, xylene, dimethylformamide, dimethyl sulfoxide, etc., and also use the above-mentioned ketone as a solvent. You can also do 2 of these solvents
It is also possible to carry out the reaction in a mixed solvent composed of one or more species.
In addition, a small amount of water may be added at the start of the reaction in order to increase the solubility of the raw material compound and the catalyst in the solvent.

Since this reaction is an equilibrium reaction and the yield obtained by removing the water produced in the reaction is generally better, the reaction may be carried out while removing water from the reaction system by a known method. In this case, known methods include distilling off water or using a desiccant. When distilling water off, it is common to use the azeotrope with a solvent and water.The water that is obtained by cooling the azeotropic vapor is separated and removed, and the remaining solvent is returned to the reactor. Alternatively, the azeotropic vapor may be removed from the reaction system and the same amount of dry solvent may be newly added to the reaction system. As a method of using a desiccant, a liquid obtained by directly or once cooling an azeotropic vapor is dried with a desiccant represented by anhydrous calcium sulfate, molecular sieves, alumina, etc., and then returned to the reactor. Good.

The reaction temperature is usually about 0 ° C to 150 ° C, preferably about 10 ° C to 100 ° C. Further, the reaction may be carried out under reduced pressure in order to adjust the azeotropic point of aldehyde or ketone which is a solvent or a reaction reagent, or a reactive derivative thereof and water.

The reaction time varies depending on the type of 2-keto-L-glaric acid or its salt, the type of reaction reagent, the type and amount of acid, the reaction conditions, etc., but is usually about 30 minutes to 24 hours, and preferably It takes about 1 to 12 hours.

The 2-keto-L-glaric acid 2,3-
O-acetal or 2,3-O-ketal is isolated from the reaction system by the following method. In this isolation method, 2
-When a salt of keto-L-glaric acid is used as a raw material,
Since a salt is by-produced in the reaction system by the neutralization reaction with an acid, there are known methods such as a filtration method, an ion exchange membrane method, an ion exchange resin method, an extraction method with a suitable solvent, a column chromatography or an electrodialysis method. The technique removes salt from the reaction.
On the other hand, when free 2-keto-L-glaric acid is used as the raw material, the desalting operation described above is unnecessary. If a salt-free reaction product is obtained, the solvent and low-boiling-point reaction reagent are distilled off from the reaction product by a conventional method, and the resulting residue is subjected to known means such as concentration, extraction, column chromatography, and recrystallization. Thus, the desired 2,3-O-acetal or 2,3-O-ketal of 2-keto-L-glaric acid can be easily isolated and purified.

EXAMPLES The present invention will be described in more detail below with reference to Reference Examples and Examples. The% of the fermentation medium indicates weight / volume% unless otherwise specified.

Reference Example 1 50.0 g (0.171 mol) of 2,3; 4,6-di-O-isopropylidene-2-keto-L-gulonic acid monohydrate was dissolved in 300 ml of distilled water at 90 ° C. Stir for 15 minutes. After the reaction,
Water and acetone were distilled off under reduced pressure, and the total amount was concentrated to about 100 ml. This concentrated solution was extracted with ethyl acetate (20
The extract was washed with water and dried over anhydrous sodium sulfate. After treatment with about 3 g of activated carbon, the solvent was distilled off to give 2,3-O-isopropylidene-2 as a colorless paste.
23.8 g of -keto-L-gulonic acid was obtained. Yield 60%.

Elemental analysis value (%) Calculated value as C ₉ H ₁₄ O ₇ C 46.16; H 6.02 actual value C−46.61; H 6.12 IR (liq.film) cm ⁻¹ 3650 to 3100,1740 ¹ H-NMR (DMSO− d ₆ ) δ 1.31 (s, 3H), 1.42 (s, 3H),
3.50 (m, 2H), 3.96 ~ 4.40 (m, 2H), 4.68 (m, 1H), 6.0 ~
13.5 (br, 3H) ¹³ C-NMR (DMSO-d ₆ ) δ 25.9 (q) 27.0 (q), 58.8
(T), 73.3 (d), 83.4 (d), 87.8 (d), 109.8
(S), 112.2 (s), 168.3 (s) Reference Example 2 5.86 g of 2,3-O-isopropylidene-2-keto-L-
Gulonic acid was dissolved in 70 ml of distilled water, and 2.10 g of sodium hydrogen carbonate was added thereto to neutralize it. To this solution, 5.86 g of 5% Pt / C was added, heated to 80 ° C., and 0.7 to 0.9 L of air was passed every minute. At this time, the pH of the reaction solution was maintained at about 7.0 using a pH controller (addition of NaHCO ₃ water). The starting compound was consumed in about 4 hours, so the catalyst was filtered off and the filtrate was concentrated. After the concentration, about 50 ml of acetone was added and the insoluble matter was collected by filtration to obtain 6.3 g of powdery 2,3-O-isopropylidene-2-keto-L-glaric acid disodium salt.

This powder was dissolved in 160 ml of distilled water, 30 ml of cation exchange resin IR-120B (H ⁺ type, manufactured by Rohm and Haas Co.) was added, and the mixture was stirred at room temperature for 10 minutes. The resin was filtered off, the filtrate was extracted with ethyl acetate (200 ml × 2 times), washed with water and dried over anhydrous sodium sulfate. After treatment with activated carbon, the mixture was concentrated under reduced pressure and then n-hexane was added to obtain 0.88 g of 2,3-O-isopropylidene-2-keto-L-glaric acid as primary crystals.

Further, 0.88 g of secondary crystals and 0.63 g of tertiary crystals were obtained from the mother liquor. Yield 39%.

Melting point 198-202 ℃ (decomposition) [cf. literature value 202-204 ℃ (decomposition): Carbohydrate Research, 60,251-258 (1978)] IR (KBr) cm ^-1 3500,3300-2800,1730 Elemental analysis value (% ) Calculated as C ₉ H ₁₂ O ₈ C 43.56; H 4.87 Found C 43.54; H 4.93 ¹ H-NMR (DMSO-d ₆ ) δ 1.31 (s, 3H), 1.44 (s, 3H),
4.25 (d, 1H), 4.70 (d + s, 1H) ¹³ C-NMR (D ₂ O) δ 25.8 (q), 26.9 (q), 75.6
(D), 82.7 (d), 87.8 (d), 110.8 (s), 116.0
(S), 169.8 (s), 171.5 (s) Reference Example 3 Document [USP, NO, 2,428,438 (1947); USP, No.2,483,251
(1949)] and conducted the experiment. 2,3-O-
10 ml of concentrated hydrochloric acid was added to 2.80 g of 2,3-O-isopropylidene-2-keto-L-glaric acid synthesized by the catalytic air oxidation method of isopropylidene-2-keto-L-gulonic acid.
Stirred at ~ 80 ° C for 20 minutes. After adding 20 ml of acetonitrile to the reaction solution and treating with activated carbon, the reaction product was concentrated under reduced pressure and left overnight. The precipitated crystals were collected by filtration to obtain 0.80 g of colorless crystals of L-grosaccharoascobic acid. Yield 37%.

Melting point 215 to 218 ° C (decomposition) [cf. literature value 206 to 210 ° C (decomposition) ^{* 1} ; 212 to 215 ° C (decomposition) ^{* 2} ] ^{* 1} : USP, NO.2,48
3,251 ^{* 2} : Carbohydrate Research, 60 , 251-258 (197
8) Elemental analysis value (%) Calculated value as C ₆ H ₆ O ₇ C 37.91; H 3.18 Measured value C 37.80; H 3.22 IR (KBr) cm ^-1 3560,3500,3400 to 2700,1770,1750,1700
sh, 1680,1665 ¹ H-NMR (DMSO-d ₆ ) δ 4.31 (d, 1H), 4.95 (d, 1H), c
a.6.12.8 (br, 4H) ¹³ C-NMR (D ₂ O) δ 68.6 (d), 77.8 (d), 119.2
(S), 154.3 (s), 173.1 (s), 174.5 (s) Reference Example 4 1.0 kg of 2-keto-L-dicalcium dicalcium salt-5
Hydrate (purity determined by high-performance liquid mattography)
98.8%) in 3 L of acetone and 300 ml with stirring
Concentrated sulfuric acid was added dropwise and reacted for 4 hours. Filter off insolubles,
It was washed with about 500 ml of acetone and combined with the filtrate. After concentrating the filtrate under reduced pressure, 0.3 L of concentrated hydrochloric acid was added to the residue at 50 ° C.
Reacted for 40 minutes. The reaction product was concentrated to dryness under reduced pressure and 300 ml.
The colored substance and the like were washed with acetonitrile. To the residue, 4 L of acetone was added and dissolved by heating, then about 5 L of benzene was added and the mixture was allowed to stand in a cold place to obtain 320 g of L-grosaccharoascorbic acid primary crystals.

The mother liquor was concentrated and then recrystallized from acetone-benzene to obtain 132 g of secondary crystals. Yield 80.9%.

Elemental analysis value (%) Calculated value as C ₆ H ₆ O ₇ C 37.91; H 3.18 Measured value C 37.87; H 3.15 Example 1 of Pseudogluconobacter saccharoketgenes K591s strain grown on the slope medium in Table 1 Inoculate the test tube (16 x 160 mm) containing 5 ml of complete medium with 1 bacterial ears of cell and
Shaking culture was performed for a day. 1 ml of this culture solution was transferred to a test tube containing 5 ml of the same medium, and cultured with shaking for 4 hours. Obtained culture solution
Aseptically centrifuge 5 ml at 5 ° C for 15 minutes (12,000 rpm) to collect the bacteria, and then 10 ml of tris-maleic acid buffer solution (pH 6.5,
It was suspended in 0.05M) and centrifuged again. Washed cells obtained by repeating this twice were suspended in 5 ml of the above buffer containing 1 mg / ml of nitrosoguanidine, and shaken at 30 ° C. for 2 hours to be treated with a mutagen. The treated solution was centrifuged at 5 ° C. for 15 minutes to collect the cells, and the cells were washed twice with 10 ml of tris-maleic acid buffer solution to obtain nitrosoguanidine-treated cells. 0.85%
It was diluted appropriately with the saline solution of, and seeded on a plate (diameter 9 cm) containing 15 ml of complete medium (solid type) and cultured at 28 ° C. for 5 days to form colonies. When the generated colonies were counted and compared with the untreated ones, the killing rate by this nitrosoguanidine treatment was 90.4%. Further, the colonies on the complete medium plate were replicated on the minimal medium plate shown in Table 3, and after culturing at 28 ° C for 3 days, the appearance frequency of the auxotrophic mutant strain was about 6.6%.

Approximately 2 cm of colonies on the complete medium treated with the mutagen as described above were transferred to a new complete medium plate with 12 strains per plate.
Streaks were transplanted to the length.

After culturing at 28 ° C for 2 days, the grown platinum cells 1 platinum loop L-sorbose 7.0% (separate sterilization), dry yeast 1.0%, peptone 1.
It was transplanted to a test tube containing 3 ml of a medium (pH 6.5) consisting of 0%, ferrous chloride 0.1%, and CaCO ₃ 3.0%, and cultured at 30 ° C. for 4 days with shaking. Under this condition, 2 times that of the parent strain K591s
-TH14-86 strain capable of producing keto-L-gulone (IFO 1
4466, FERM BP-1128) was selected as a strain with enhanced L-sorbose oxidation ability.

The TH14-86 strain and the parent K591s strain were mixed with the slant medium shown in Table 1
After growing for 3 days at 0 ° C, 1 platinum loop of each bacterial cell was used for 3% L-sorbose, 2% CSL, 0.3% dry yeast, 0.2% peptone, Fe
_{SO 4 · 7H 2 O 0.1%} , Na 2 S 2 O 3 · 5H 2 O 0.05%, 0.3% ammonium sulfate,
The cells were inoculated into a test tube containing 3 ml of a medium containing CaCO ₃ 4% (pH 6.6), and cultured at 30 ° C. for 5 days with shaking.

The obtained culture solution was diluted 3 times with 0.3N sulfuric acid and then centrifuged (12,000 rpm) to obtain a diluted supernatant. 1 μl of this supernatant was spotted on a cellulose plate (Merck) and developed with a solvent of phenol: water: formic acid (75: 25: 5) at room temperature for 3 hours. The plate was air-dried and then developed with a silver nitrate reagent.
The black-brown spot around the Rf value of 2.1 (2-keto-L-glaric acid) shown by the TH14-86 strain was much larger than that of the parent K591s strain.

Table 1 Slope medium (g / L) D-sorbitol 25 peptone 10 Yeast extract 10 CaCO ₃ 2 agar 20 pH 7.0 Table 2 Complete medium (g / L) D-sorbitol 25 peptone 10 Yeast extract 10 pH 6.5 (In solid medium, add 20 g of agar) (g / L) Table 3 Minimal medium (g / L) Sucrose 5 K ₂ HPO ₄ 3 KH ₂ PO ₄ 1 (NH ₄ ) ₂ SO ₄ 1 NaCl 1 MgSO ₄・ 7H ₂ O 0.1 MnCl ₂ · nH ₂ O 0.002 L-sodium glutamate 0.1 L-Cysteine 0.1 CoA 0.002 FMN 0.002 Thiamine 0.002 Biotin 0.001 pH 7.0 (20 g of agar in solid medium is added) Example 2 Pseudo-obtained in Example 1 Gluconobacter saccharoketgenes TH14-86 strain was treated with nitroguanidine in the same manner as in Example 1 and grown on a complete medium plate. Each fungus body 1 platinum loop 3% L-sorbose, CSL 2
%, Dried yeast 0.5%, 0.2% Lou _{acid, FeSO 4 · 7H 2 O 0} · 1
%, Na ₂ S ₂ O ₃ .5H ₂ O 0. 05%, CaCO ₃ 4% (pH 6.6) in a test tube containing 3 ml of the medium, and shake culture was carried out at 30 ° C. for 3 days.

The resulting culture was treated as in Example 1 and subjected to thin layer chromatography. Colored with silver nitrate, Rf value 2.
SOP- that gives the largest black-brown spot near 1
It was selected as strain 809 (IFO 14608).

Example 3 The Pseudogluconobacter saccharoketgenes SOP-809 strain obtained in Example 2 was grown in the slant medium shown in Table 1 at 30 ° C. for 3 days. This bacterial body 1 white bacterium ear glucose 2.0%,
A 200 ml Erlenmeyer flask (steam sterilized at 120 ° C. for 20 minutes) containing 20 ml of a seed medium consisting of peptone 1.0%, dry yeast 1.0% and CaCO ₃ 2.0% was inoculated. The cells were cultured at 30 ° C for 2 days with shaking (200 rpm). 20 ml of the obtained culture solution was transferred to a flask containing 200 ml of the same seed medium as above, and cultured under the same conditions to obtain a seed culture solution of SOP-809 strain.

On the other hand, a 200 ml Erlenmeyer flask containing 20 ml of the seed medium was inoculated with 1 platinum loop of Bacillus megaterium IFO12108 strain grown on a slant medium for 2 days at 28 ° C.
After shaking culture for a day, a mixed culture of seed cultures was obtained.

L-sorbose 6.0% (separate sterilization), CSL 2.0%, dry yeast 1.0%, Na ₂ S ₂ O ₃ / 5H ₂ O 0.05%, FeSO ₄ / 7H ₂ O 0.1%, ammonium sulfate 0.35, FMN 1mg / L, ₃ L of fermentation medium consisting of thiamin 1 mg / L, biotin 0.5 mg / L, ACTCOL (manufactured by Takeda Pharmaceutical Co., Ltd.) 0.02%, CaCO ₃ 5.0%, was steam sterilized at 120 ° C for 30 minutes and pre-sterilized 5 L fermentation tank I put it in. This fermentor is as described above
300 ml of the seed culture liquid of SOP-809 strain and 4 ml of the seed culture liquid of the mixed bacteria were transplanted, and cultured for 70 hours under the conditions of 30 ° C., aeration 2.0 L / min, and stirring 800 rpm. The culture broth thus obtained had 48.0 mg / ml.
Accumulated 2-keto-L-glaric acid.

Example 4 To 1 L of the fermentation broth obtained in Example 3 was added about 35 ml of hydrochloric acid to adjust the pH.
Adjusted to 2.5. This was centrifuged (7,000 rpm, 10 min) and the precipitate was collected. It was washed with 750 ml of water and separated by hydrochloric acid again to obtain a washed precipitate. After suspending this in an appropriate amount of water, about 35 ml of hydrochloric acid was added to adjust the pH of the suspension to 1.2. Then, 8606 ml of supernatant was obtained by centrifugation. Ten
After adding g of activated carbon and stirring for 3 hours, the activated carbon was removed by centrifugation to obtain 810 ml of a supernatant liquid. While stirring, 28% ammonia water was added dropwise to the supernatant to adjust the pH to 2.5, and the mixture was allowed to stand overnight at 8 ° C. The resulting crystals were collected by filtration and dried over phosphorus pentoxide for 2 days under reduced pressure to give 2-keto-L-dicalcium dicalcium salt (57.6 g) as colorless columnar crystals.
The properties of the obtained 2-keto-L-glaric acid are as follows.

(1) Elemental analysis: C ₆ H ₆ O ₈ Ca · 5H ₂ O, theoretical value (%): C; 21.43, H; 4.89, (Ca; 11.92) Measurement value (%): C; 21.34, H; 4.83, (Ca; 11.5) (Ca is the value measured by atomic absorption method) (2) Molecular weight: 208.12 (free acid) (3) Melting point: 148-150 ℃ (decomposition, dicalcium pentahydrate) (4) Ratio Illuminance: [α] _D ²⁵ = + 5.1 ° (c = 1.0% in
(0.2N HCl, dicalcium pentahydrate) (5) Ultraviolet absorption spectrum: no endothermic absorption (6) IR (KBr) cm ^-1 3375, 3330 to 2600 (br), 1660, 16
10,1580,1450,1420,1380,1330,1315,1295,1280,1265,12
20,1160,1115,1080,1065,1040,1015,1000,980,880,860,
840,780,750 (7) ¹³ C-NMR (D ₂ O + 10% DCl) δ 76.1 (d), 76.
7 (d), 78.4 (d), 79.7 (d), 81.4 (d), 82.9
(D), 101.7 (s), 106.4 (s), 171.8 (s), 172.5
(S), 173.1 (s), 173.4 (s) (8) Solubility in solvent: water-soluble, easily soluble in hydrous alcohol. Calcium salt is sparingly soluble in water and only acid is easily soluble.

(9) Color reaction: Silver nitrate reagent gives a blackish brown color, aniline phthalate gives a yellow color, and O-phenylenediamine gives an orange color to a purple color. Ninhydrin, naphthoresorcin, and Pauli reagent are negative.

(10) Basic, acidic, neutral: acidic substance (11) Color of substance: colorless (12) High-performance liquid chromatography retention time ^* : 6.66 minutes ^* Note High-performance liquid chromatography measurement conditions HPLC: Hitachi 655A System Column: SCR101H (sulfonated polystyrene gel), 300 ×
7.9 mm (Shimadzu) Flow rate: 0.8 ml / min (Pressure: 50 kg / cm ² ) Moving layer: Dilute sulfuric acid (pH 2.1) Detector: UV (214 nm) and differential refractometer (13) Thin layer chromatography Rf Value ^** : 0.21 ^** Note Thin layer chromatography conditions Solvent: Phenol: Water: Formic acid = 75: 25: 5 Thin layer: Cellulose (Merck) Time: 3 hours Temperature: Room temperature Color: Silver nitrate reagent, o-phenylene Diamine reagent (14) Moisture content: Water content measured by thermobalance 24.3
%showed that. This value is C ₆ H ₆ O ₈ Ca
The calculated water content of ₂ O was almost the same as 26.8%. Furthermore, the water of crystallization of this product easily fluctuates depending on the drying conditions.
It dried to give 3.5 hydrate, dried at room temperature for 24 hours to give trihydrate, and dried at 60 ° C. for 6 hours to give dihydrate. The elemental analysis values are shown below.

When dried at 40 ℃ for 6 hours Elemental analysis value (%) Calculated as C ₆ H ₆ O ₈ Ca ・ 3.5H ₂ O C 23.30; H 4.24 Measured value C 23.20; H 4.18 Elemental analysis when dried at room temperature for 24 hours Value (%) Calculated as C ₆ H ₆ O ₈ Ca ・ 3H ₂ O C 24.00; H 4.03 Actual value C 24.05; H 4.05 When dried at 60 ° C for 6 hours Elemental analysis value (%) C ₆ H ₆ O ₈ Calculated value as Ca · 2H ₂ O C 25.54; H 3.57 measured value C 25.81; H 3.43 Example 5 10 ml of Bacillus megaterium (IFO12108 strain) cells grown on the slant medium of Table 1 for 2 days at 28 ° C. The suspension was suspended in sterilized water, the whole amount was inoculated into a Sakaguchi flask containing 500 ml of the seed medium of Example 3, and cultured at 28 ° C. for 2 days with reciprocal shaking (85 spm) to give a Bacillus megaterium seed culture solution.

Sucrose 4.0%, cottonseed meal 4.0%, K ₂ HPO ₄ 0.65%, KH ₂ PO ₄ 0.55%,
0.05% ammonium _{sulfate, NaCl0.05%, MgSO 4 · 7H} 2 O0.05%, and consisting of 0.05% calcium pantothenate medium (pH 7.0) 30L
Was placed in a 50 L fermentor and sterilized with steam at 125 ° C. for 20 minutes. 1 L of Bacillus megaterium seed culture was transplanted to this fermentor and cultured for 4 days under the conditions of stirring 200 rpm, aeration 24 L / min, internal pressure 1.0 Kg / cm ² , 28 ° C. The obtained culture solution was sterilized by steam at 120 ° C. for 20 minutes and stored in a cold place, and used as a sterilized culture of Bacillus megaterium (sometimes referred to as megabroth) as one component of the fermentation medium.

Glucose 2.0%, Megabroth 35 (V / V), CSL1.0%,
Peptone 0.1%, yeast extract 0.5%, ammonium sulfate 0.3%, CaCO ₃ 2.0
The seed medium (pH 6.5) (25 ml) was dispensed into a 200 ml Erlenmeyer flask, and steam sterilized at 120 ° C. for 20 minutes. Pseudogluconobacter saccharoketgenes K591s strain (FERM, which was grown in a slant medium of Table 1 at 30 ° C for 3 days in this flask, was used.
BP-1130, IFO 14464), TH14-86 strain (FERM BP-1128, IFO
14466), 12-5 strain (FERM BP-1129, IFO 14465), 12-1
5 strains (FERM BP-1132, IFO 14482), 12-4 strains (FERM BP-
1131, IFO 14483), and 22-3 strain (FERMBP-1133, IFO 144
Each of the flasks was inoculated with 1 platinum loop of the bacterial cell of 84),
Culture was carried out at 30 ° C. for 3 days with shaking (200 rpm) to obtain each seed culture solution. Each seed culture 1.25ml obtained, L- sorbose 5.0%, CSL2.0%, Megaburosu _{2.0%, Na 2 S 2 O} 3 .5H
_{2 O0.05%, FeSO 4 · 7H} 2 O0.1%, 0.5% ammonium sulfate, CaCO ₃ 4.0%,
Was inoculated into a 200 ml Erlenmeyer flask containing 25 ml of a fermentation medium (pH 6.5) consisting of and cultured with shaking at 30 ° C. The fermented liquor thus obtained contained 2-keto-L-glaric acid in the amounts shown in Table 4 when quantified by high performance liquid chromatography.

Example 6 Pseudogluconobacter saccharoketgenes SOP
The -809 strain was cultured in the same manner as in Example 5 to obtain a seed culture solution. 1.25 ml of this seed culture solution is added to 2-keto-L-gulonic acid 5.0
% (Another sterilization), CSL1.0%, dry yeast 0.5%, ammonium sulfate 0.5%, Na
_{2 S 2 O 3 · 5H 2} O0.05%, implanted in an Erlenmeyer flask containing the fermentation medium (pH 6.5) 25 ml consisting of _{FeSO 4 · 7H 2 O0.1%,} 3 at 30 ° C.
Culture was carried out with shaking (200 rpm) for a day. The fermented liquid obtained contains 3
7.2 mg / ml of 2-keto-L-glaric acid had formed. Also, replace 2-keto-L-gulonic acid with L-sorbose,
Fermentation broth similarly cultivated was 54.5 mg / ml 2-keto-L.
-Contains glural acid.

Example 7 Pseudogluconobacter Saccharoketgenes K591
S strains, TH14-86 strains, 12-4 strains, 12-5 strains, 12-15 strains, 22-3 strains and SOP-809 strains were cultured in the same manner as in Example 3 to obtain respective seed culture solutions. Obtained.

CSL4.0% dry yeast 1.0%, 1.0% ammonium sulfate, FeSO ₄ .7H ₂ O0.3
_{%, Na 2 S 2 O 3} .5H 2 O 0.1%, CaCO 3 4.0% Medium 10ml consisting (pH 6.5) was dispensed sterilized 200ml conical flask. To this flask, 10 ml of 5% sodium 5-keto-D-gluconate solution was filtered and sterilized, and 1 ml of each of the above-mentioned seed cultures was further transplanted and shake-cultured at 30 ° C. for 3 days. 2-Keto-L-glaric acid in the obtained culture solution was quantified by high performance liquid chromatography, and the results are shown in Table 5.

Example 8 1.0 g of dicalcium 2-keto-L-glaric acid pentahydrate was suspended in 30 ml of distilled water, and about 10 ml of cation exchange resin IR-120B (H ⁺ type) was added thereto. The mixture was stirred at room temperature for 20 minutes. The cation exchange resin was filtered off, and 0.1 g of activated carbon was added to the obtained filtrate for decolorization, and then the filtrate was freeze-dried to give 0.79 g of a colorless transparent syrup-like 2-keto-L. −
Glaric acid was obtained. Yield 97.3%.

Elemental analysis value (%) Calculated value as C ₆ H ₈ O ₈ · 1.3H ₂ O C 31.12; H 4.61 actual value C 31.31; H 4.63 IR (liq.film) cm ^-1 3650 to 2700,1725 (br), 1620,140
0,1220,1060,840,800,700 ¹ H-NMR (DMSO-d ₆ ) δ 3.9 to 4.3 (m, 2H), 4.4 to 4.7
(M, 1H), 5.2-8 (br, 5H) ¹³ C-NMR (DMSO-d ₆ ) δ 75.8 (d), 76.7 (d), 77.9
(D), 78.1 (d), 79.3 (d), 81.4 (d), 101.9
(S), 105.5 (s), 170, 5 (s), 171.2 (s), 171.7
(S), 171.9 (s) Example 9 33.63 g of 2-keto-L-dicalcic acid dicalcim salt pentahydrate was suspended in 100 ml of distilled water, and 9.1 g (0.9 molar equivalent) of concentrated sulfuric acid was added thereto. Was added dropwise with stirring. After the completion of dropping, the mixture was stirred for 2 hours. The precipitate formed was filtered off, washed with a little water and combined with the filtrate. 180 ml of 1N NaOH was added dropwise to this filtrate. After completion of the dropping, this solution was added dropwise to 1.5 L of methanol while stirring, and the precipitate was collected by filtration and dried under reduced pressure.
17.8 g of disodium 2-keto-L-glaric acid dihydrate was obtained. 75% yield.

Melting point ca. 160 ℃ (decomposition) IR (KBr) cm ^-1 3600 to 2700,1620 (br) Elemental analysis value (%) Calculated as C ₆ H ₆ O ₈ Na ₂ · 1.5H ₂ O C 25.82; H 3.25 Found C 25.59; H 2.92 Example 10 336.3 g of 2-keto-L-dicalcium dicalcium salt.5
The hydrate was suspended in 1 L of pure water, and 91 g (0.9 molar equivalent) was added to it.
Concentrated sulfuric acid was added dropwise with stirring. After the completion of dropping, the mixture was stirred for 2 hours. The precipitate formed was filtered off, washed with a little water and combined with the filtrate. Take 1/6 volume of the filtrate and add 150 ml to this filtrate.
1N KOH was added dropwise while cooling, and then this solution was added dropwise to 2 L of cooled methanol. The precipitate is collected by filtration, 200m
After being washed with 1 l of methanol, it was dried under reduced pressure to obtain 33.7 g of dipotassium 2-keto-L-glaric acid monohydrate. yield
74.3%.

Melting point ca. 150 ℃ (decomposition) IR (KBr) cm ^-1 3600 to 2700,1620 (br) Elemental analysis value (%) Calculated value as C ₆ H ₆ O ₈ K ₂ · H ₂ O C 23.84; H 2.67 actual measurement Value C 23.60; H 2.61 Example 11 49.0 g of pasty 2-keto-L-glaric acid prepared according to the method described in Example 8 (2-keto-L-glaral as determined by high performance liquid chromatography). 8 as acid
4.9% content, 0.20 mol), add 20 ml of concentrated hydrochloric acid and
Stirred for minutes. The reaction was concentrated to dryness under reduced pressure to a residue.
15 ml of distilled water was added, and this solution was passed through a column filled with about 100 ml of chromatographic white eagle charcoal (manufactured by Takeda Pharmaceutical Co., Ltd.) using distilled water. This column was eluted with distilled water, the eluate was concentrated under reduced pressure, a small amount of ethyl acetate was added to the residue, the insoluble matter was filtered off, and dried to give 32.6 g of crude L-grosaccharoascorbic acid. Was obtained (purity 97.8%,
Yield 83.7%). This was recrystallized from an acetone-benzene mixed solution to obtain L-grosaccharoascorbic acid.

Melting point 215 to 218 ° C (decomposition) Elemental analysis value (%) Calculated value as C ₆ H ₆ O ₇ C 37.91; H 3.18 measured value C 37.80; H 3.22 MS (m / e) 190 (M ⁺ ) Specific irradiance [ α] _D ²² = + 76.1 ° (c = 1.035% in H
₂ O), [α] _D ²² = + 76.0 ° (c = 1.07% in MeOH) ¹ H-NMR (DMSO-d ₆ ) and ¹³ C-NMR (D ₂ O) were measured. It was completely the same as L-grosaccharoascorbic acid synthesized separately in. In addition, the Rf value, RT value, melting point, and IR (KBr) spectrum of TLC and HPLC, respectively, were also in perfect agreement. From the above experimental facts, the product obtained by acid-treating the pentahydrate of 2-keto-L-dicalcium dicalcium salt obtained by the oxidative fermentation of L-sorbose is L
-Grossaccharoascorbic acid was confirmed.

Example 12 341 g of crude 2-keto-L-dicalcium dicalcium salt.5
Hydrate [determined by high performance liquid chromatography
Keto -L- Gral dicalcium salts _{(C 6 H 6 O 8 Ca} · 5H 2 O)
98.8% 1.0 mol] was suspended in 1000 ml of distilled water, 152 g of 97% sulfuric acid (1.50 mol) was gradually added dropwise while stirring at room temperature, and the mixture was stirred at room temperature for 3 hours after the completion of the addition. The insoluble matter was filtered off, washed with about 500 ml of distilled water and combined with the filtrate. The filtrate was concentrated under reduced pressure to about 200 ml and cooled. The insoluble matter was filtered off, the mixture was again heated in a water bath having a bath temperature of 50 to 55 ° C. for 6 hours, and then concentrated under reduced pressure. About 100 ml of distilled water was added to the concentrate, the insoluble matter was filtered off, and this solution was passed through a column filled with 200 ml of a special white egret charcoal for chromatography (manufactured by Takeda Yakuhin Kogyo) using distilled water to remove distilled water. Elute with. The eluate was concentrated under reduced pressure, and the precipitated crystals were collected by filtration and dried to obtain 120 g of primary crystals of L-grossaccharoascorbic acid. The filtrate was decolorized with activated carbon powder, concentrated under reduced pressure, and the precipitation result was collected by filtration and dried to obtain secondary crystals. This filtrate was similarly decolorized, concentrated, filtered and dried to obtain a tertiary crystal. The second and third crystals thus obtained were recrystallized from water to obtain 39.8 g of L-grosaccharoascorbic acid. Total yield 84%.

Example 13 10.0 g of L-grosaccharoascorbic acid was added to 30 m of distilled water.
Dissolve in l, cation-ion exchange resin IR-120B (Na ⁺ type) 15
After passing through a column filled with 0 ml, the effective fraction was concentrated under reduced pressure, and the precipitated colorless needle crystals were collected by filtration to obtain 3.8 g of L-grosaccharoascorbic acid monosodium salt.

Yield 31.4%.

Melting point ca. 192 ° C (decomposition) Elemental analysis value (%) Calculated value as C ₆ H ₅ O ₇ Na · 1H ₂ O C 31.32; H 3.07 Measured value C 31.23; H 3.01 Example 14 10.0 g of L-grossac 30m of distilled water from roascorbic acid
It was dissolved in 1 and passed through a column packed with 150 ml of cation exchange resin IR-120B (K ⁺ type), and the effective fraction was concentrated under reduced pressure.
The precipitated white crystals were collected by filtration to obtain 7.6 g of L-grosaccharoascorbic acid monopotassium salt. Yield 60.9%.

Melting point ca. 250 ℃ (decomposition) IR (KBr) cm ^-1 3500 to 3050 (br), 1755, 1660 Elemental analysis value (%) Calculated as C ₆ H ₅ O ₇・ 0.5H ₂ O C 30.38; H 2.55 Measured value C 30.67; H 2.29 Example 15 To 200 ml of acetone was added 30 g of 2-keto-L-dicalcium glutarate pentahydrate, and 10 ml of concentrated sulfuric acid 8.77 g was added with stirring under ice cooling.
It was added dropwise over a period of minutes. The precipitated calcium sulfate was filtered off, 30 mg of 57% hydrogen iodide was added, and the mixture was heated under reflux for 20 hours while drying the solvent with Molecular Sieve 3A in Soxhlet. After completion of the reaction, sodium methylate 9.
72 g was added to the reaction solution, and the precipitated disodium salt of 2,3-O-isopropylidene-2-keto-L-glaric acid was collected by filtration. Dissolve this in 500 ml of distilled water and use ion exchange resin IR
280 ml of -120B (H ⁺ type) was added and stirred for 2 minutes. After removing the resin and treating with activated carbon, water was distilled off to solidify. This solid was dissolved in 600 ml of acetone-ethyl acetate (1: 1), dried over sodium sulfate, and then concentrated. The product obtained is
Crystallized from a mixture of ethyl acetate-hexane, 13.30 g
To give 2,3-O-isopropylidene-2-keto-L-glaric acid. Yield 60.1%.

Melting point 198 to 202 ° C Elemental analysis value (%) Calculated value as C ₉ H ₁₂ O ₈ C 43.56; H 4.87 measured value C 43.59; H 4.84 IR (KBr), ¹ H-NMR (DMSO-d ₆ ) and ¹³ C -NMR (D ₂ O)
Was measured, and it was completely the same as 2,3-O-isopropylidene-2-keto-L-glaric acid separately synthesized in Reference Example 2. In addition, the Rf value, RT value, and melting point of TLC and HPLC, respectively, were also in perfect agreement. From the above experimental facts, the product obtained by acid treatment of 2-keto-L-diglyceric acid dicalcium salt pentahydrate obtained in the oxidative fermentation of L-sorbose in the presence of acetone was 2,3. It was confirmed to be -O-isopropylidene-2-keto-L-glaric acid.

Example 16 A mixed solution of 3.36 g of dicalcium 2-keto-L-dicalcium glutamate pentahydrate and 50 ml of acetonitrile was cooled to 0 ° C., 0.98 g of concentrated sulfuric acid was added with stirring, and the mixture was further stirred for 30 minutes. .
The produced insoluble material was filtered off, and the filtrate was concentrated under reduced pressure to give a paste-like substance. To this residue, 30 ml of acetonitrile and 10 ml of cyclohexanone were added, and the mixture was heated and refluxed for 3 hours while drying the reflux solvent with Molecular Sieve 3A in Soxhlet. After completion of the reaction, 100 ml of methanol was added to the paste-like residue obtained by concentrating under reduced pressure, and about 20 mmol of sodium methylate was added thereto at 0 ° C. The reaction product was concentrated to dryness under reduced pressure, n-hexane was added to the residue, the insoluble material was collected by filtration, and washed with hexane.
The obtained powder was dissolved in 50 ml of distilled water, and this was passed through a column filled with 100 ml of cation exchange resin IR-120B (H ⁺ type). The effective fraction obtained by elution with distilled water was freeze-dried to obtain 2.1 g of powder. This product was subjected to silica gel chromatography (solvent: ethyl acetate-chloroform), and 1.66 g of
2,3-O-Cyclohexylidene-2-keto-L-glaric acid was obtained. Yield 56.2%.

Melting point 148 to 152 ° C (recrystallized from dichloromethane-n-hexane) Elemental analysis value (%) Calculated value as C ₁₂ H ₁₆ O ₈ · 0.4H ₂ O C 48.78; H 5.73 Measured value C 48.83; H 5.71 IR (KBr ) Cm ^-1 3600 to 2800, 1750 ¹ H-NMR (DMSO-d ₆ ) δ 1.1 to 2.2 (br, 10H), 4.25 (d,
1H, J = 3HZ), 4.66 (m, 2H), 6.8 to 9.3 (br, 3H) Example 17 1.93 g of 5-keto-L-dicalcium dicalcium salt 5
The hydrate (C ₆ H ₆ O ₈ Ca.5H ₂ O, 0.0057 mol) was suspended in 10 ml of distilled water, about 10 ml of ion exchange resin IR-120B (H ⁺ ) was added, and the mixture was stirred for 20 minutes. The resin was filtered off, washed with about 10 ml of distilled water and combined with the filtrate. The filtrate was freeze-dried to obtain a paste-like crude 2-keto-L-glaric acid. Add 2 ml benzaldehyde, 50 ml acetonitrile and 30 mg 57% hydrogen iodide and add molecular sieve 3A in Soxhlet.
The solvent was heated to reflux for 4 hours while being dried. Analysis of reactants by high-performance fluid malography [Analysis conditions-column: Amix HPX-87H (Bio-rad), mobile phase: 0.008NH ₂
SO ₄ , flow rate: 0.6 ml / min, detector: SE-31 (Showa Denko KK)]
It was found that 2,3-O-benzylidene isomers were produced in a ratio of about 60:40. The reaction mixture was concentrated under reduced pressure, and 50 ml of ethyl acetate and 50 ml of distilled water were added to the paste-like residue to separate the layers. Sodium sulfate (anhydrous) was added to the ethyl acetate layer and dried. This was filtered and the filtrate was concentrated under reduced pressure. 10 ml of distilled water was added to the residue to dissolve it, and column chromatography was performed using Sephadex G-10 with distilled water as a developing solvent. The target fraction was distilled off under reduced pressure, 30 ml of ethyl acetate was added to the residue to dissolve it, and 0.1 g of activated carbon Shirasagi A was added.
(Takeda Pharmaceutical Co., Ltd.) was added to decolorize, and 30 ml of n was added to the filtrate.
Recrystallization from the addition of -hexane gave 0.44 g of 2,3-O-benzylitene-2-keto-L-glaric acid. Yield 26
%.

Melting point 175 to 176 ° C (decomposition) Elemental analysis value (%) Calculated value as C ₁₃ H ₁₂ O ₈ C 52.71; H 4.08 Measured value C 52.85; H 4.15 IR (KBr) cm ^-1 3500 to 3000,1760,1720 ¹ _{H-NMR (DMSO-d 6} ) δ 4.3 (d, 1H), 4.72 (s, 1H), 4.
8 (d, 1H), 5.93 (s, 1H), 7.43 (s, 5H) Effect of the invention L- belongs to the genus Pseudogluconobacter of the present invention.
Sorbose, 2-keto-L-gulonic acid or 5-keto-D
-By using a microorganism having the ability to oxidize gluconic acid to 2-keto-L-glaric acid, a novel compound 2-
Keto-L-glaric acid can be efficiently produced.
Further, by using this acid as a raw material, 2,3-O-acetal and ketal of L-grosaccharoascorbic acid and 2-keto-L-glaric acid can be efficiently produced.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP 62-228091 (JP, A) JP 62-228092 (JP, A) JP 62-228287 (JP, A) JP 62- 228288 (JP, A) JP-A-63-91088 (JP, A)

Claims (4)

(57) [Claims] 1. 2-Keto-L-glaric acid or a salt thereof. 2. A bacterium belonging to the genus Pseudoglucobacter having the ability to oxidize L-sorbose, 2-keto-L-gulonic acid or 5-keto-D-gluconic acid to 2-keto-L-glaric acid. A 2-keto characterized by being brought into contact with L-sorbose, 2-keto-L-gulonic acid or 5-keto-D-gluconic acid to produce and accumulate 2-keto-L-glaric acid, which is then collected. -A method for producing L-glaric acid. 3. A method for producing L-grosaccharoascorbic acid, which comprises treating 2-keto-L-glaric acid with an acid. 4. A method for producing a 2,3-O-acetal or ketal of 2-keto-L-glaric acid, which comprises acetalizing or ketalizing 2-keto-L-glaric acid.