Process for producing 2-keto-L-gulonic acid

An improved process for producing 2-keto-L-gulonic acid which comprises culturing a microorganism belonging to the genus Pseudogluconobacter which has an ability to oxidize L-sorbose to 2-keto-L-gulonic acid in a culture medium supplemented with a rare earth element in the presence of L-sorbose.

FIELD OF THE INVENTION 
The present invention relates to a fermentation process for producing 
2-keto-L-gulonic acid which is useful as an intermediate for synthesizing 
L-ascorbic acid. 
BACKGROUND OF THE INVENTION 
2-Keto-L-gulonic acid which is useful as an intermediate for synthesizing 
L-ascorbic acid has been produced by the industrially established 
so-called Reichstein method [see, Helvetica Chimica Acta, 17, 311 (1934)]. 
However, this method involves many steps and requires a large amount of 
solvent and, therefore, is insufficient for industrial technology of 
today. 
On the other hand, instead of Reichstein method, several methods mainly 
employing microorganisms have been proposed. For example, a method which 
comprises subjecting D-glucose to microbiological oxidation to produce 
5-keto-D-gluconic acid, reducing it chemically or microbiologically to 
obtain L-idonic acid and then oxidizing the resultant microbiologically to 
obtain 2-keto-L-gulonic acid [see, U.S. Pat. No. 2,421,611]; and a method 
which comprises oxidizing D-glucose microbiologically to obtain 
2,5-diketo-D-gluconic acid, reducing it microbiologically or chemically to 
obtain 2-keto-L-gulonic acid [see, Japanese Patent Publication Nos. 
39-14493, 53-25033, 56-15877 and 59-35920]have been investigated. 
However, chemical reduction steps employed in these methods, i.e., the 
reduction of 5-keto-D-gluconic acid to idonic acid in the former method 
and the reduction of 2,5-diketo-D-gluconic acid to 2-keto-L-gulonic acid 
in the latter method are accompanied with problems in stereospecificity 
and they produce D-gluconic acid and 2-keto-D-gluconic acid as 
by-products, respectively, which results in decrease in yield. Further, 
when the above reduction is carried out microbiologically, excessive 
glucide should be supplied to the microorganisms as a reduction energy 
source, which also results in lowering of yield. In this respect, when 
L-sorbose is used as a starting material, 2-keto-L-gulonic acid can be 
produced only by an oxidation step. 
In fact, several trials utilizing this advantage have been made by using 
bacteria belonging to the genera Gluconobacter, Pseudomonas, Serratia, 
Achromobacter and Alcaligenes [see, Biotechnology and Bioengineering, 14, 
799 (1972); Japanese Patent Publication No. 41-159 and No. 41160; U.S. 
Pat. No. 3,043,749; USSR Patent No. 526,660; Japanese Patent Publication 
No. 49-39838; Acta Microbiological Sinica, 20, 246 (1980) and 21, 185 
(1981); Japanese Patent Laid Open Publication No. 62-48389]. 
However, the disclosed strains give insufficient yield and therefore, they 
are insufficient for industrial use. 
Recently, there has been reported a method for producing 2-keto-L-gulonic 
acid from D-glucose by using one bacterial strain obtained by introducing 
2,5-diketo-D-gluconic acid reductase gene of a microorganism belonging to 
Corynebacterium into a microorganism belonging to Erwinia according to DNA 
recombination technique [see, Science, 230, 144 (1985)]. However, this 
method is also insufficient for utilizing industrial use from the 
viewpoint of the amount of 2-keto-L-gulonic acid produced. 
OBJECTS OF THE INVENTION 
Under these circumstances, the present inventors have studied intensively 
to obtain an industrially advantageous method for producing 
2-keto-L-gulonic acid. As the result, we have already found that bacteria 
isolated from soil and designated as Pseudogluconobacter saccharoketogenes 
can produce a considerable amount of 2-keto-L-gulonic acid from L-sorbose 
(see, European Patent Published Application No. 221,707). Then, during the 
study on the improvement of that method, we have unexpectedly found that 
the fermentation time is shortened and that the production yield of 
2-keto-L-gulonic acid from L-sorbose is remarkably improved by culturing 
the bacteria in a culture medium supplemented with a rare earth element. 
It has not been found that a rare earth element can exhibit such a 
fermentation-promoting effect. We have studied intensively on this 
phenomenon and, as the result, have attained the present invention. 
The main object of the present invention is to provide an improved process 
for producing 2-keto-L-gulonic acid from L-sorbose. 
This object as well as other objects and advantages of the present 
invention will become apparent to those skilled in the art from the 
following description. 
SUMMARY OF THE INVENTION 
According to the present invention, there is provided an improved process 
for producing 2-keto-L-gulonic acid which comprises culturing a 
microorganism belonging to the genus Pseudogluconobacter which has an 
ability to oxidize L-sorbose to 2-keto-L-gulonic acid in a culture medium 
supplemented with a rare earth element in the presence of L-sorbose. 
By culturing a microorganism belonging to the genus Pseudogluconobacter 
having an ability to oxidize L-sorbose to 2-keto-L-gulonic acid in the 
presence of a rare earth element, 2-keto-L-gulonic acid which is an 
important starting material for synthesis of L-ascorbic acid can be 
produced efficiently. 
DETAILED DESCRIPTION OF THE INVENTION 
The microorganism of Pseudogluconobacter used in the present invention 
includes, for example, the following strains described in European Patent 
Published Application No. 221,707: 
Pseudogluconobacter saccharoketogenes K591s: FERM BP-1130, IFO 14464; 
Pseudogluconobacter saccharoketogenes 12-5: FERM BP-1129, IFO 14465; 
Pseudogluconobacter saccharoketogenes TH14-86: FERM BP-1128, IFO 14466; 
Pseudogluconobacter saccharoketogenes 12-15: FERM BP-1132, IFO 14482; 
Pseudogluconobacter saccharoketogenes 12-4: FERM BP-1131, IFO 14483; 
Pseudogluconobacter saccharoketogenes 22-3: FERM BP-1133, IFO 14484. 
Hereinafter, these Pseudogluconobacter saccharoketogenes bacteria may be 
referred to as oxidation bacteria. 
The rare earth element used in the present invention includes, for example, 
scandium (Sc), yttrium (Y), lanthanum (La), cerium (Ce), praseodymium 
(Pr), neodymium (Nd), samarium (Sm), europium (Eu), gadolinium (Gd), 
terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), 
ytterbium (Yb) and lutetium (Lu). These rare earth elements can be 
supplemented in the form of metallic powder or slug. Or, they can be used 
in the form of compounds such as their chlorides, carbonates, sulfates, 
nitrates, oxides and oxalates. They can be used alone or in combination of 
two or more rare earth elements, for example, cerium carbonate and 
lanthanum chloride can be used simultaneously. Further, a crude product 
obtained during isolation and purification steps of the respective 
elements can also be used. 
The amount of the rare earth element supplemented to the culture medium can 
be selected from such a range that it does not inhibit growth of the 
microorganism used. Generally, the effective amount ranges from 0.000001 
to 0.1% (W/V), preferably, from 0.0001 to 0.05% (W/V). 
As a manner for supplementing the element to the culture medium, it can be 
previously supplemented to the culture medium, or it can be supplemented 
intermittently or continuously during culture. 
In the process of the present invention, when the starting material, i.e., 
L-sorbose is added to the culture medium, the total amount thereof can be 
added to the culture medium at the beginning of culture, or it can be 
added in several portions or continuously to the liquid culture. The 
concentration of L-sorbose in the culture medium can be 2 to 40% (W/V), 
preferably 5 to 30% (W/V) based on the culture medium. 
In the culture medium used for culture of the above oxidation bacteria, 
nutrient sources which can be utilized by the bacterial strains, that is, 
carbon sources, nitrogen sources, inorganic salts, organic salts and trace 
nutrients which can be utilized by the strains can be used. 
As carbon sources, L-sorbose can be used as it is. In addition, as 
supplementary carbon sources, for example, glucose, fructose, glycerin, 
sucrose, lactose, maltose, molasses and the like can be used. 
Nitrogen sources include, for example, various kinds of ammonium salts 
(e.g., ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium 
phosphate), inorganic or organic compounds containing nitrogen such as 
corn steep liquor (hereinafter may be referred to as CSL), peptone, meat 
extract, yeast extract, dried yeast, soybean flour, cottonseed meal, urea 
and the like. 
As inorganic salts, in addition to the above rare earth elements, salts of 
potassium, sodium, calcium, magnesium, iron, manganese, cobalt, zinc, 
copper and phosphoric acid can be used. 
As trace nutrients needless to say, CoA, pantothenic acid, biotin, thiamine 
and riboflavin which are essential growth factors of the above bacteria 
can be added. In addition, flavin mononucleotide (hereinafter may be 
referred to as FMN) which exhibits promotion activities for growth and 
production of 2-keto-L-gulonic acid, other vitamins, L-cysteine, 
L-glutamic acid and sodium thiosulfate and the like, as compounds or 
native products containing them, can be appropriately added. 
These components of the culture medium can be previously added to the 
culture medium at once. Or, a part or all of them can be added 
intermittently or continuously to the liquid culture. 
As the means for culturing, there can be employed stationary culture, 
shaking culture or agitating culture or the like. However, for mass 
production, so-called submerged culture is preferred. 
Of course, the culture conditions vary depending on the particular kind of 
strain, the particular composition of the culture medium and the like and, 
briefly, they can be selected for each particular case so that the 
objective product can be produced with the highest efficiency. For 
example, the culture temperature is preferably 25.degree. to 35.degree. C. 
and pH of the culture medium is desirably about 5 to 9. 
Upon culturing for 10 to 120 hours under the above conditions, 
2-keto-L-gulonic acid can be accumulated at the highest concentration. In 
this case, since pH generally lowers as the objective product accumulates, 
a suitable basic material, for example, sodium hydroxide, potassium 
hydroxide or ammonia can be added to always maintain the optimal pH level 
for microbiological production of 2-keto-L-gulonic acid. Or, a suitable 
buffer can be added to the culture medium to maintain the optimal pH. 
In the present invention, when the microorganism belonging to the genus 
Pseudogluconobacter is cultured in the presence of a rare earth element in 
a liquid medium containing L-sorbose to produce and accumulate 
2-keto-L-gulonic acid in the culture medium, the amount of accumulated 
2-keto-L-gulonic acid can be remarkably increased by mixing the above 
oxidation bacteria with another microorganism in comparison with using the 
oxidation bacteria, i.e., the microorganisms belonging to 
Pseudogluconobacter alone. 
The microorganisms to be mixed include, for example, bacteria belonging to 
the genera Bacillus, Pseudomonas, Proteus, Citrobacter, Enterobacter, 
Erwinia, Xanthomonas, Flavobacterium, Micrococcus, Escherichia and the 
like. More particularly, the following bacteria are included: 
Bacillus cereus IFO 3131; 
Bacillus licheniformis IFO 12201; 
Bacillus megaterium IFO 12108; 
Bacillus pumilus IFO 12090; 
Bacillus amyloliquefaciens IFO 3022; 
Bacillus subtilis IFO 13719; 
Bacillus circulans IFO 3967; 
Pseudomonas trifolii IFO 12056; 
Pseudomonas maltophilia IFO 12692; 
Proteus inconstans IFO 12930; 
Citrobacter freundii IFO 13544; 
Enterobacter cloacae IFO 3320; 
Erwinia herbicola IFO 12686; 
Xanthomonas pisi IFO 13556; 
Xanthomonas citri IFO 3835; 
Flavobacterium meningosepticum IFO 12535; 
Micrococcus varians IFO 3765; 
Escherichia coli IFO 3366. 
A liquid culture obtained by culturing any of these bacteria in a suitable 
medium at 20 to 40.degree. C. for 1 to 4 days can be used as a seed 
culture of the microorganism to be mixed. In general, the amount to be 
inoculated is desirably 1/10 to 1/1000 of that of the oxidation bacteria 
(Pseudogluconobacter). When mixed culture is carried out by mixing the 
microorganism to be mixed with the oxidation bacteria in such an amount to 
be inoculated, the growth of the oxidation bacteria can be promoted and 
thereby L-sorbose a higher concentration can be oxidized to 
2-keto-L-gulonic acid within a shorter period of time in comparison with 
culture using the oxidation bacteria alone. The bacteria to be used as the 
microorganism to be mixed desirably have no or weak assimilation property 
with L-sorbose which is the starting material of the present invention, or 
2-keto-L-gulonic acid which is the objective product of the present 
invention. Other culture conditions are the same as those using the 
oxidation bacteria alone. In addition, the sterilized culture of certain 
kinds of bacteria other than the above oxidation bacteria can be 
effectively utilized as an ingredient of the culture medium. Bacteria 
which can be utilized include, for example, those of the genera Bacillus, 
Pseudomonas, Citrobacter, Escherichia and Erwinia. More particularly, the 
following bacteria are included: 
Bacillus cereus IFO 3131; 
Bacillus subtilis IFO 3023; 
Bacillus pumilus IFO 12089; 
Bacillus megaterium IFO 12108; 
Bacillus amyloliquefaciens IFO 3022; 
Pseudomonas trifolii IFO 2056; 
Citrobacter freundii IFO 12681; 
Escherichia coli IFO 3546; 
Erwinia herbicola IFO 12686. 
These bacteria can be cultured in a medium in which they can grow at 20 to 
40.degree. C. for 2 to 4 days. The resultant culture can be sterilized and 
added to the culture medium of the present oxidation bacteria in an amount 
of 0.5 to 5.0% (V/V) to promote the growth of the oxidation bacteria. 
2-Keto-L-gulonic acid thus produced and accumulated in the culture medium 
can be isolated and purified by known means utilizing its properties. 
2-Keto-L-gulonic acid can be isolated as a free acid. Or, it can be 
isolated, for example, as a salt with sodium, potassium, calcium or 
ammonium. 
As a method for isolation, there can be employed, for example, a method 
wherein bacterial cells are removed from the culture medium by filtration 
or centrifugation as needed, subsequently, the solution is concentrated 
without or after treatment with activated carbon, and separated crystals 
are collected by filtration and recrystallized to obtain the objective 
product; solvent extraction; chromatography; salting out and the like. 
These methods can be employed alone, in appropriate combination thereof, 
or in repetition. 
When 2-keto-L-gulonic acid is obtained in the free form, it can be 
converted into, for example, a salt of sodium, potassium, calcium, 
ammonium or the like and, when it is obtained as a salt, it can be 
converted into the free form or other salts by an appropriate method. 
2-Keto-L-gulonic acid produced in the culture medium was determined by high 
performance liquid chromatography under the following conditions. 
The conditions of measurement of high performance liquid chromatography: 
HPLC: 655A system (manufactured by Hitachi Seisakusho, Japan) 
Column: SCR 101H (sulfonated polystyrene gel), 300.times.7.9 mm 
(manufactured by Simadzu Seisakusho, Japan) 
Flow rate: 0.8 ml/min. (pressure: 50 kg/cm.sup.2) 
Mobile phase: diluted sulfuric acid (pH 2.1) 
Detector: UV (214 nm) and differential refractometer. 
Retention time: 7.20 min. for 2-keto-L-gulonic acid and 8.33 min. for 
L-sorbose. 
The following examples further illustrate the present invention in detail 
but are not to be construed to limit the scope thereof. All percentages 
used for the culture media are % (W/V) unless otherwise described. The 
yields for the cultures in the examples are expressed by mole conversion 
yield of the amount of 2-keto-L-gulonic acid produced based on that of 
L-sorbose used.