The present invention relates to a process for producing glucose-1-phosphate making use of microorganisms.

Glucose-1-phosphate (hereafter abbreviated as "G-1-P") is useful as a substrate for synthesis of drugs and saccharides.

G-1-P is mainly obtained by phosphorolysis of starch or dextrin with maltodextrin phospholylase (MDPase), and a process making use of MDPase derived from potato (Japanese Patent Publication No. 95492/1994) and the so-called enzymatic process using MDPase derived from microorganisms have been reported to date.

As examples of the enzymatic process using MDPase derived from microorganisms, have been reported a process using an enzyme derived from Escherichia coli (Weinhäusel et al., Enzyme Microb. Technol. 17, 140-146 (1995) and a process using an enzyme derived from Corynebacterium callunae (Nidetzki et al., J. Carbohydrate Chem., 14, 1017-1028 (1995)). More recently, processes for producing G-1-P making use of heat-stable MDPase derived from medium thermophilic bacteria and high thermophilic bacteria such as Bacillus stearothermophilus (Japanese Patent Application Laid-Open No. 14580/1998) and Thermus caldophilus (Shin et al., J. Industrial Microbiol., 24, 89-93 (2000)) have been reported.

However, such enzymatic processes using the enzyme itself require complicated steps such as a step of extracting an enzyme from plants or bacteria and preparation of an immobilized enzyme, and it has hence been desirable to develop a simpler process for producing G-1-P.

It is an object of the present invention to provide a process for producing G-1-P by which a mass of G-1-P can be provided without using complicated steps.

The present inventors have carried out various investigations as to bacteria producing a mass of G-1-P in a medium by culture. As a result, it has been found that when bacteria of the genus Corynebacterium are cultured under conditions of the presence of a saccharide and a high concentration of phosphoric acid or a derivative thereof, a high concentration of G-1-P can be produced directly in a medium to produce a mass of G-1-P.

According to the present invention, there is thus provided a process for producing glucose-1-phosphate, comprising the steps of culturing bacteria of the genus Corynebacterium in a medium containing a saccharide and at least 1 mM of phosphoric acid or metaphosphoric acid, tripolyphosphoric acid, polyphoshoric acid, diphosphoric acid, polymetaphosphoric acid, phosphates or salt thereof, and collecting glucose-1-phosphate produced and accumulated in the medium.

The bacteria of the genus Corynebacterium used in the present invention are not particularly limited , only so far as they belong to the genus Corynebacterium and produce G-1-P in a medium in the presence of a saccharide and a fixed concentration of phosphoric acid or a derivative or salt thereof, and examples thereof include Corynebacterium callunae, Corynebacterium glutamicum, Corynebacterium vitaeruminis and Corynebacterium pilosum. Among these, Corynebacterium callunae, Corynebacterium glutamicum and Corynebacterium vitaeruminis are preferred, with Corynebacterium callunae IFO 15359, Corynebacterium glutamicum JCM 1321 and Corynebacterium vitaeruminis JCM 1323 strains being particularly preferred.

Preferable examples of the saccharide added to the medium include monosaccharides, disaccharides, oligosaccharides and polysaccharides containing glucose as a constitutive saccharide, and α-1,4-glucane-containing saccharides, for example, starch, amylose, dextrin, maltose, maltooligosaccharides, amylopectin and glycogen are mentioned as more preferable examples. Among these, cheap starch, dextrin and maltooligosaccharides are particularly preferred.

Such saccharides may be used either singly or in any combination thereof.

Examples of the derivative or salt of phosphoric acid to be added to the medium include metaphosphoric acid, tripolyphosphoric acid, polyphosphoric acid, diphosphoric acid, polymetaphosphoric acid, phosphates and salts of these derivatives. As the salts, are preferred sodium and potassium salts. Examples of particularly preferred phosphates include monopotassium phosphate, dipotassium phosphate, monosodium phosphate and disodium phosphate. In the present invention, it is preferable to use a mixture of phosphoric acid or a derivative thereof and a salt thereof, or a mixture of a few kinds of phosphates or salts of the derivatives.

The concentration of phosphoric acid, or the derivative or salt thereof in the medium must be at least 1 mM from the viewpoint of the effect, and it is desirable that the concentration be in a range of preferably 1 mM to 1 M, more preferably 5 mM to 500 mM, particularly preferably 100 mM to 500 mM.

The medium used in the present invention is not particularly limited, only so far as bacteria of the genus Corynebacterium can grow therein, and a medium containing a carbon source, a nitrogen source, metal minerals, vitamins, etc. in addition to the above-described saccharide and phosphoric acid or the derivative or salt thereof may be used.

Examples of other carbon sources than the saccharide include organic acid salts such as acetates.

Examples of the nitrogen source include ammonia, inorganic and organic ammonium salts such as ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium carbonate, ammonium phosphate and ammonium acetate, and nitrogen-containing organic substances such as urea, peptone, meat extracts, yeast extracts and hydrolyzates of casein, and amino acids such as glycine, glutamic acid, alanine and methionine.

Examples of the metal minerals include sodium chloride, ferrous sulfate, magnesium sulfate, manganese sulfate, zinc sulfate and calcium carbonate. These metal minerals may be used either singly or in any combination thereof as needed.

The culture is conducted by suitably adjusting pH and temperature so as to obtain conditions under which the microorganisms can sufficiently grow. However, it is generally preferred that the culture be conducted for 12 to 96 hours at a pH of 5 to 8 and a temperature of 25 to 40°C.

As a culturing method, a resting-bacterial reaction and an immobilized-bacterial reaction may also be used in addition to shaking culture and culture by a fermentor.

The collection of G-1-P produced and accumulated in the medium may be conducted by, for example, separating and removing the bacteria used and combining centrifugation, ultrafiltration, ion exchanging, reverse osmosis, electrodialysis, salting out, crystallization etc. with one another in accordance with a publicly known method.

According to the process of the present invention, a high concentration of G-1-P can be thus produced directly in the medium, and so a mass of G-1-P can be produced without conducting any complicated steps compared with the enzymatic process using an enzyme. As demonstrated in Examples, which will be described subsequently, such an effect is scarcely recognized in the MDPase derived from bacteria of the genus Bacillus (Japanese Patent Application Laid-Open No. 14580/1998) in which the MDPase is contained in the bacteria like the bacteria of the genus Corynebacterium , and the specific activity thereof is 4.2 U/mg and comparable with MDPase derived from the bacteria of the genus Corynebacterium the specific activity of which is 5.3 U/mg (J. Carbohydrate Chem., 14, 1017-1028 (1995)), and is a characteristic effect of the bacteria of the genus Corynebacterium .

The determination of G-1-P was conducted in accordance with the Weinhausle's method (Enzyme Microb. Technol., 17, 140-146 (1995)) with the method modified.

More specifically, an enzymatic reaction solution (100 µL, containing 100 mM Tris-acetate buffer (pH 6.8), 2 mM EDTA, 10 mM magnesium sulfate, 2 mM NAD, 10 µM glucose-1,6-diphosphate, 1.2 U/mL phosphoglucomutase (derived from rabbit muscle, product of Roche Diagnostic Co.) and 1.2 U/mL glucose-6-phosphate dehydrogenase (derived from Leuconostoc mesenteroides, product of Roche Diagnostic Co.)) was added to a sample (100 µL) suitably diluted on a 96-well microplate, and the mixture was incubated at 37°C for 30 minutes to measure an absorbance at 340 nm.

One platinum loop of Corynebacterium callunae IFO 15359, Bacillus subtilis IFO 3037, Bacillus subtilis IFO 1372 or Bacillus licheniformis JGM 2505 was inoculated in a medium (containing 3.5% soluble starch, 3.0% Lablemco powder (product of OXOID Co.), 0.05% magnesium sulfate, 0.04% monopotassium phosphate and 0.1% disodium phosphate) to conduct a shaking culture overnight at 30°C as species culture.

Each (1%) of the species bacteria cultured above was inoculated on a medium prepared by adding a potassium phosphate buffer (pH 7.0) to 3.5% soluble starch, 3.0% Lablemco powder (product of OXOID Co.), 0.05% magnesium sulfate so as to give a phosphate concentration of 10 mM, 20 mM or 40 mM to conduct culture at 30°C for 5 davs as principal culture. The concentration of G-1-P in the supernatant thus obtained was determined. The results are shown in Table 1. Productivity of G-1-P (g/L) Concentration of phosphate 10 mM 20 mM 40 mM C. callunae IFO 15359 0.15 0.41 0.58 B. subtilis IFO 3037 <0.01 <0.05 <0.01 B. subtilis IFO 1372 0.03 <0.01 <0.01 B. licheniformis JGM 2505 0.07 0.01 0.01

One platinum loop of Corynebacterium callunae IFO 15359, Corynebacterium glutamicum JCM 1321, Corynebacterium vitaeruminis JCM 1323, Bacillus subtilis IFO 3037, Bacillus subtilis IFO 1372 or Bacillus licheniformis JGM 2505 was inoculated into a medium (containing 3.5% soluble starch, 3.0% Lablemco powder (product of OXOID Co.), 0.05% magnesium sulfate, 0.04% monopotassium phosphate and 0.1% disodium phosphate) to conduct a shaking culture overnight at 30°C as culture.

The bacteria cultured above were respectively collected, and each of them was inoculated on a medium prepared by adding a potassium phosphate buffer (pH 7.0) to 0.67% yeast nitrogen base (product of Difco Co.) and 10% dextrin (derived from potato, product of SIGMA Co.) so as to give a phosphate concentration of 100 mM, 200 mM, 400 mM or 500 mM in such a manner that OD_{600 nm} is 10, thereby conducting culture at 30°C for 5 days as principal culture. The concentration of G-1-P in the supernatant thus obtained was determined. The results are shown in Table 2. Productivity of G-1-P (g/L) Concentration of phosphate 100 mM 200 mM 400 mM 500 mM C. callunae IFO 15359 2.1 5.1 12.7 12.7 C. glutamicum JCM 1321 - - 11.5 - C. vitaeruminis JCM 1323 - - 13.0 - B. subtilis IFO 3037 0.01 0.02 - 0.04 B. subtilis IFO 1372 0.02 0.03 - 0.02 B. licheniformis JGM 2505 0.01 0.01 - 0.01

As a result of the above, it was possible to produce G-1-P with good efficiency depending on concentration by using bacteria of the genus Corynebacterium and adjusting the concentration of the phosphate.

According to the process of the present invention, as described above, a mass of G-1-P can be provided without conducting complicated steps such as a step of extracting an enzyme from plants or bacteria and preparation of an immobilized enzyme, which are requiredfor the enzymatic process.