# Patent

## Title
Exopeptidase catalyzed site-specific bonding of supports, labels and

## Abstract

This application is a Divisional of application Ser. No. 07/403,298 filed on Sep. 5,1989, now U.S. Pat. No. 5,134,077 which is a divisional of application Ser. no. 07/243,215 filed on Sep. 12, 1988, now U.S. Pat. No. 4,879,229 which is a continuation of application Ser. No. 06/925,538 filed on Oct. 28, 1986, now abandoned, which is a continuation of application Ser. No. 06/288, 332 filed on Jul. 30, 1981, now abandoned, each expressly incorporated by reference.

## Background
1. Field of the Invention

The present invention relates to a novel microbiological method for preparing 2,5-dike to-D-gluconic acid (hereinafter referred to as 2,5 DKG) from D-glucose. Particularly, it is concerned with a method for preparing 2,5-DKG by fermentation of new microorganisms which belong to Erwinia n. sps. or by simple contact of D-glucose with said microorganisms or their processed products, for instance, resting cells, immobilized cells, ground cells or any enzymes extracted therefrom, which may be suspended in a fluid medium or fixed on a surface of a stationary bed.

2. Description of the Prior Art

2,5 DKG is a useful intermediate in the production of L-ascorbic acid. Namely, 2,5 DKG can selectively and stereospecifically be reduced into 2- keto-L-gulonic acid, a precursor of L-ascorbic acid, in a promising new route for the latter product (See, for instance, U.S. Pat. No. 3,922,194 3,959,076, 3,963,574 and 3,998,697).

In all of the known means, the production of 2,5 DKG has been achieved by cultivating aerobic microorganism strains which belong exclusively to genera Acetobacter, Acetomonas, Gluconobacter and Pseudomonas. No instance has hitherto been reported as to its production by means of cultivation using faculatatively anaerobic strains which belong to genus Erwinia.

It is therefore a primary object of the present invention to provide a method for preparing 2,5 DKG by the use of newly isolated microorganisms which belong to genus Erwinia.

It is still another object of the present invention to provide a method for preparing 2,5 DKG in a high yield and in a high broth concentration to facilitate a commercialization of the previously mentioned promising new route.

According to the present invention, there is provided a method for preparing 2,5 DKG characterized by contacting a 2,5 DKG producing microorganism of genus Erwinia or any processed products therefrom (any product obtained by treating cells of said microorganisms) with D-glucose.

The present invention may be embodied in a variety of modes, for instance, by cultivating at least one strain of said microorganisms in an aqueous nutrient medium containing D-glucose or by simply contacting said microorganisms or their processed products, i.e., resting cells, immobilized cells, or cell free enzymes extracted therefrom, with any substrate containing D-glucose.

Throughout this specification and claims, the term "cultivating" is used to include any modes of microbiological operations wherein the microorganisms are inoculated into and incubated in a medium to effect a fermentative action therein. The term "contacting" is used to mean operations wherein the microorganisms or any processed products therefrom are combined with the substrate to effect a conversion of D-glucose into 2,5 DKG, without regard to the length of the contact, i.e., incubation.

In the process of the present invention, any microorganisms of genus Erwinia can be utilized as far as the microorganisms are capable of selectively oxidizing D-glucose and converting it into 2,5 DKG. Examples of these strains, which have been isolated and determined to belong to new species of genus Erwinia by the present inventors, are collectively listed in Table 1 below.

All of the listed strains, which have been deposited with the Fermentation Research Institute, Japan and with the American Type Culture Collection, Washington D.C., to be assigned FERM-P and ATCC numbers, respectively, are found to below to the three new species and each constitutes variant with respect to the other within the species. ______________________________________ Name given to the isolated SHS-    microorganism     FERM-P   ATCC ______________________________________ 2003    Erwinia citreus   5449     31623 2006    Erwinia punctata  5452     31626 2004    Erwinia punctata var. 5450     31624 2005    Erwinia punctata var. 5451     31625 2007    Erwinia punctata var. 5453     31627 2008    Erwinia terreus   5454     31628 2009    Erwinia terreus var. 5455     31629 2010    Erwinia terreus var. 5456     31630 2011    Erwinia terreus var. 5457     31631 ______________________________________

Details of taxonomic studies on these microorganisms will be collectively described in Table 2, below.

(Bouillon agar slants at 28.degree. C. for 24, 72 and 168 hrs.) Cells predominantly single, rods of the listed dimensions with rounded ends in common with each of the observed strains. Sometimes, the presence of deformed cells of the indicated dimensions are observed in some strains. Deformed cells: Strain: Predominant cells: Expand-  Extended cells: SHS-  (Dimensions (.mu.)) ed cells: (Dimensions (.mu.)) ______________________________________ 2003  0.8-1.2 .times. 1.0-2.9 Observed Observed, 1.6 .times. 6.0-7.0 2004  1.0-1.5 .times. 1.5-3.3 "        "       0.7-0.8 .times. 4.0-5.0 2005  1.0-1.3 .times. 1.6-2.1 "        "       0.7-0.8 .times. 2.4-3.0 2006  1.1-1.3 .times. 1.3-1.9 "        "       0.6-0.8 .times. 2.4-2.7 2007  1.0-1.2 .times. 1.7-2.3 None     None    -- 2008  0.8-0.9 .times. 1.5-1.7 "        "       -- 2009     1.0 .times. 1.2-2.1 "        "       -- 2010     1.0 .times. 1.0-2.0 "        "       -- 2011     1.0 .times. 1.3-2.0 "        "       -- ______________________________________

2. Motility and flagellation

(Bouillon agar slants at 20.degree. C. and 25.degree. C. for 18 through 24 hrs.)

Motility is confirmed by the hanging-drop method. Flagellation is confirmed under an optical microscope after being stained by Toda's method, or by means of transmission type electron microscope after being cultured in a sustained membrane culturing method. Strains SHS-2003, 2004, 2005, 2006 and 2007: Non-motile in common.

Strain SHS-2008: Motile with mono-lateral flagellum.

Strains SHS-2009, 2010 and 2011: Motile with one or two lateral flagella.

3. Spore

Not formed with each of the listed strains in common.

4. Gram staining

(Bouillon agar slants at 28.degree. C. for 24, 72 and 168 hrs.) Negative with each of the listed strains in common.

5. Acid fast

Negative in common.

1. Bouillon agar colonies at 28.degree. C. for 24, 48, 72 and 168 hrs. Initial colonies being circular, entire, smooth, translucent and butyrous in common. 2004  "         Pale    Smooth but gradually changes to beige   rough. 2005  "         Pale    Convex but gradually changes to beige   raised. 2006  "         Pale    Convex but gradually changes to beige   umbonate. 2007  "         Pale    Raised but gradually changes to beige   umbonate. 2008 Dull to    Pale    Smooth but gradually changes to glistening reddish rough and convex. Dull but grad- yellow  ually changes to glistening. 2009 Glistening Pale    Smooth but gradually changes to beige   rough and raised. 2010  "         Pale    Convex but gradually changes to beige   umbonate. 2011  "         Pale    Convex but gradually changes to reddish flat. Viscid but gradually yellow  changes to butyrous. ______________________________________

2. Bouillon agar slant at 28.degree. C. for 24-168 hours. Initial colonies being filiform and butyrous in common. Strains                         Progress of culture SHS-  Growth    Brilliancy Color (change) ______________________________________ 2003  Abundant  Glistening Pale  -- reddish yellow 2004    "       Dull to   Pale  Dull but gradually glistening beige changes to glistening. 2005  Moderate  Glistening Pale  -- beige 2006    "       Dull to   Pale  Dull but gradually glistening reddish changes to glistening. yellow 2007  Scanty    Dull      Pale  -- reddish yellow 2008  Abundant  Dull to   Pale  Dull but gradually glistening reddish changes to glistening. yellow 2009    "       Dull to   Pale  Dull but gradually glistening reddish changes to glistening. yellow 2010  Moderate  Glistening Pale  -- reddish yellow 2011    "         "       Pale  -- reddish yellow ______________________________________ 3. Buillon broth at 28.degree. C. for 24-168 hours. No odor in common. Growth on the surface of culture Ring along  Membran-  Floc-   Submerged growth Strains the tube  ous       culent       Flocculent SHS-  wall      growth    growth Turbidity sediment ______________________________________ 2003  On and    --        --    Slight Scanty after 3rd day 2004  --        On 6th day --    Slight to Initial moderate compact sediment changes to scanty flocculant sediment 2005  --          "       --    Slight Scanty 2006  --          "       --    Slight to " moderate 2007  On 3rd day On 7th day --    Slight  " 2008  On 7th day --        On 3rd "      " day 2009  --        On 3rd to On 7th "      " 6th day   day 2010  --        On 6th day --    Slight to " moderate 2011  --        On 4th day --    Slight  " ______________________________________

4. Bouillon gelatin stab at 20.degree. C. for 40 days. No liquefaction in common. Growth along the stab line.

5. Litmus milk at 28.degree. C. for 40 days. coagulation begins on about the 18th day and is completed on the 38th day. From the 18th day to the 32nd day, the upper-layer becomes pink and the lower-layer becomes grayish brown, but on the 38th day, whole layer becomes pink in uniform.

Strains SHS-2004, 2005, 2006, 2007, 2008, 2009, 2010 and 2011: No changes are observed throughout the incubation in common.

6. Potato slant at 28.degree. C. for 24-168 hours. Strains          Growth      Color ______________________________________ SHS-2003         Abundant    Pale reddish yellow 2004               "         Beige 2005             Moderate     " 2006               "         Pale beige 2007               "         Beige 2008             Abundant     " 2009               "          " 2010             Moderate     " 2011               "          " ______________________________________ results of the observation at 28.degree. C. within 14 days)

1. Nitrite: Nitrite is produced from nitrate in common.

2. Nitrate respiration: Neither growth nor gas production is observed in paraffin-sealed bouillon broth containing 1% KNO.sub.3 in common.

3. Methyl-red test: Positive in common, except strain SHS-2010 which is only very weakly positive.

4. Voges-Proskauer reaction (glucose):

5. Indole: Negative in common.

6. Hydrogen sulfide:

7. Ammonia: Not produced in common.

8. Hydrolysis of starch: Negative in common.

9. Growth on citrate media:

10. Growth with inorganic nitrogen sources: SHS-2003, 2004, 2005, 2008, 2009, 2010  and 2011: while SHS-2006 does not grow and SHS-2007 only grows very weakly. 28.degree. C. for 7 days): Negative in common. common. in common.

12. Urease: Negative in common.

13. Catalase: Positive in common.

14. Oxidase (bouillon agar slant, for 18-24 hours, containing tetramethylphenylenediamine): Negative in common.

15. Temperature relations (Bacto yeast extract 0.5%, Bacto peptone 0.5% and glucose 0.5%, pH 7): Strains     Temp. of   Optimum temp. of SHS-        growth (.degree.C.) growth (.degree.C.) ______________________________________ 2003        8.5-38.5   18-31 2004        6.5-40.0   20-34 2005        6.5-38.5   22-28 2006        6.5-38.5   21-28 2007        4.0-47.5   21-27 2008        18.0-40.0  19.5-32.5 2009        8.5-38.5   24-30 2010        6.5-40.0   20-34 2011        4.0-40.0   19.5-24 ______________________________________

16. pH relations (glycerol 1%, Bacto yeast extract 0.05%, Bacto peptone 1%, NaCl 0.5%, in 0.1 M 3,3-dimethylglutaric acid buffer, 28.degree. C. for 2-4 days) Strains                Optimum pH of SHS-        pH of growth growth ______________________________________ 2003        5.5-8.0    6.0-7.5 2004        5.5-8.5    6.0-7.5 2005        5.5-8.5    6.0-7.5 2006        5.5-8.5    6.0-7.5 2007        5.5-8.5    6.0-7.5 2008        5.5-7.5    6.0-7.5 2009        5.5-8.0    6.0-7.5 2010        6.0-8.0    6.0-7.5 2011        5.5-7.5    6.0-7.5 ______________________________________

17. Oxygen requirement: Facultatively anaerobic in common. Bacto yeast extract 0.2%, NaCl 0.5%, bromocresol purple 0.004% and agar powder 1.5%, pH adjusted at 7.0-7.2, was dispensed in test tubes of 18 mm in diameter in a depth of about 8 cm. The dispensed medium was sterilized at 121.degree. C. for 15 min. Stab inoculation was performed before complete solidification of the medium. The test tubes were then sealed with sterile liquid paraffin (5-7 ml) in 4 cm depth.) Tubes were cultured at 28.degree. C. and observed over a period of 7 days. bottom of the medium within 2-3 days and the culture begins to become slightly yellowish uniformly to show a distinct difference in color from the sealed cultured tube of the non-inoculated control with each of the listed strains. The degree of yellow color further increases with the lapse of time. 1%, Nacl 0.5% and agar powder 1.5%, or GYP agar plate, containing glycerol 0.5%, Bacto yeast extract 0.5%, Bacto peptone 0.3%, KH.sub.2 PO.sub.4 0.1%, MgSO.sub.4.7H.sub.2 O 0.02% and agar powder adjusted at 7.0-7.2), Sligh streak inoculation of a suspension of the test organism was cultured at 28.degree. C. for 48 hrs. after the oxygen was removed by two hour's reaction at 45.degree. C. in an incubator. Escherichia coli used as control.

18. O-F test (Hugh-Leifson's method): Fermentative in common. Medium containing Bacto trypton 1%, Bacto yeast extract 0.1%, bromocresol purple 0.004%, glucose (or lactose) 1% and agar powder 0.2%, pH adjusted at 7.0-7.2 was dispensed to test tubes (18 mm in diameter) in a depth of about 8 cm. Dispensed medium was sterilized at 121.degree. C. for 15 min. After the temperature of the medium became about 30.degree.-40.degree. C., duplicate inoculations were performed for each organism of the listed strains, and one tube of each pair was then sealed with sterile liquid paraffin (5-7 ml) about 4 cm in depth. Tubes were cultured at 28.degree. C. and observed over a period of 7 days. (also from lactose, in the case of SHS-2003), but no gas. A distinct growth along the stab line from the top to the bottom is observed with the sealed culture in as the non-sealed culture. Anaerobical acid formation is not so rapid as Escherichia coli used as control

19. Production of acids and gases from carbohydrates: Strains Fruc- Glu- Galac- .beta.-Methyl- Manni-           Meli-     Gly- SHS- tose cose tose glucoside Mannose Ribose tol Salicin Xylose Lactose biose Cellobiose cerol __________________________________________________________________________ 2003 +   +   +    +     +    +   +   +   +   +    +   +     + 2004 +   +   +    +     +    +   -   +   +   -    +   -     + 2005 +   +   +    +     +    +   -   +   +   -    +   +     + 2006 +   +   +    +     +    +   -   +   +   -    +   +     - 2007 +   +   +    +     +    +   -   +   +   -    +   +     - 2008 +   +   +    +     +    +   -   +   +   -    +   +     + 2009 +   +   +    +     +    -   -   +   +   -    +   +     + 2010 +   +   +    +     +    .+-. -   +   +   -    +   +     - 2011 +   +   +    +     +    -   -   +   +   -    +   +     - __________________________________________________________________________ Strains Su- Mal- Dex-       Rham- Sorbi- Raffi- Adoni- Dulci- Melezi- Arabi- .alpha.-Methyl- Inosi- SHS- crose tose trin Esuclin nose tol nose tol tol tose nose glucoside tol __________________________________________________________________________ 2003 -   -   -    -     -    -   -   -   -   -    -   -     - 2004 +   -   -    -     -    -   -   -   -   -    -   -     - 2005 +   -   -    -     -    -   .+-. -   -   -    -   -     - 2006 +   -   -    -     -    -   -   -   -   -    -   -     - 2007 +   -   -    -     -    -   .+-. -   -   -    -   -     - 2008 +   -   -    -     -    -   -   -   -   -    -   -     - 2009 +   -   -    -     -    -   -   -   -   -    -   -     - 2010 +   -   -    -     -    -   -   -   -   -    -   -     - 2011 +   -   -    -     -    -   -   -   -   -    -   -     - __________________________________________________________________________ Note: (i) Acid but no gas, produced (+) (ii) Slight acid but no gas, produced (.+-.) (iii) Neither acid nor gas, produced (-)

20. Methylene blue (bouillon broth, 18-24 hrs.): Reduced in common.

21. D-Gluconic acid: Utilized in common.

22. 2-Keto-D-gluconic acid: Utilized in common.

23. Production of reduced compound from sucrose (Bergey's manual of determinative bacteriology 8th Ed. (1974) page 335, Table 8.19, foot note f): Positive in common except for SHS-2003.

24. Decarboxylation of various amino acids: Positive with SHS-2004, 2005 2006, and 2007.

25. Lipase (Modified Starr's method, Starr, M.P., Science, 93, 333-334 (1941)): Negative in common.

26. D-gluconate oxidation (Shaw, C & Clarke, P.H., J. Gen. Microbiol., 13 155-161 (1955)): Positive in common.

27. Pectate degradation (A. M. Paton, Nature, 183, 1812-1813 (1959)): Negative in common.

28. Casein hydrolysis (D.W. Dye, N.Z. J. Sci., 11, 590-607  (1968)): Negative in common.

29. Symplasmata (Graham, D.C. & Hodgkiss, W., J. Appl. Bacteriol., 30(1), 175-189 (1967)): Negative with SHS-2003, 2004, 2005, 2007, 2008 and 2009, Positive with SHS-2006, 2010 and 2011.

30. DNase (Bacto DNase Test Agar): Negative in common.

31. Phenylalanine deaminase (Phenylalanine/Malonic acid medium, available from Nissui Pharmaceutical Co. Ltd.): positive.

34. Auxotrophy (Gray & Tatum's medium): Requires nicotinic acid or nicontinamide in common.

35. Ulilization of some organic compouns (D.W. Dye, N.Z. J. Sci., 11, 590-607 (1968), OY medium, 28.degree. C., 20 & 44 hrs, shaking): Strains Ace-   For- Fumar- Gluco-  Succi-    Galatur-       Propio- Tartar- SHS- tate Citrate mate ate nate Malate nate Benzoate onate Malonate Oxalate nate ate Lactate __________________________________________________________________________ 2003 +  +   +  +   +   +   +   -     -    -    -    -   -   + 2004 .+-. +   +  +   +   +   +   -     -    -    -    -   -   .+-. 2005 .+-. +   +  +   +   +   +   -     -    -    -    -   -   .+-. 2006 .+-. +   +  +   +   +   +   -     -    -    -    -   -   .+-. 2007 .+-. +   +  +   +   +   +   -     -    -    -    -   -   .+-. 2008 +  +   +  +   +   +   +   -     -    -    -    -   -   + 2009 +  +   +  +   +   +   +   -     -    -    -    -   -   + 2010 +  +   +  +   +   +   +   -     -    -    -    -   -   + 2011 +  +   +  +   +   +   +   -     -    -    -    -   -   + __________________________________________________________________________ Note: (i) Utilizes as a single carbon source (+) (ii) Slightly utilizes or does not utilize as a single carbon source (.+-.) (iii) Does not utilize (-)

36. Ubiquinone *Y. Yamada, K. Aida & T. Uemura, J. Gen. Appl. Microbiol., 15, 181-196 (1969)): strains SHS-2003, 2008: Ubiquinone-8 (and Ubiquinone-7) Strains SHS-2004, 2005, 2006, 2007, 2009 2010 and 2011: Ubiquinone-8.

The strains listed in Table 1 were determined by comparing the above-mentioned taxonomical properties of the respective strains in Table 2, with the descriptions in the Bergey's Manual of Determinative Bacteriology 8th Ed. 1974 (hereinafter, will be simply referred to as "Manual") to lead the conclusion which will be described below.

On the basis of the above described results of the observation that all of the listed strains are short rods of gram-negative and facultatively anaerobic, which form no spores and show a catalase activity but no oxidase activity, they are determined to belong to Family Enterobacteriaceae.

All of the listed strains are determined to belong to Genus Erwinia on the basis of the taxonomical properties, particularly on those of, the productions of acids from .beta.-methylglucoside and sucrose (except for SHS-2003 which does not produce acid from sucrose but utilizes the same as a sole carbon source) but not from adonitol, dulcitol or melezitose; no utilization of benzoate, oxalate and propionate; being incapable of hydrolysing starch; being incapable of decarboxylating glutamic acid, arginine, lysine and ortinine (except for SHS-2004, 2005, 2006, and 2007 which decarboxylate glutamic acid) and showing no urease or lipase activity.

Strain SHS-2003

Although this strain is considered to be closely related to Erwinia stewartii of group herbicola defined in the Manual on the basis of no flagellation, it still has other taxonomical properties greatly different from those of Erwinia stewartii in the following points: sole carbon source)

Furthermore, since this strain did not show a coincidence with any other known species in the genus defined in the Manual, it was concluded that it should be allocated to a new species, named as Erwinia citreus by the present inventors.

Strains SHS-2004, 2005, 2006 and 2007

Although these strains are likewise considered to be closely related to Erwinia stewartii in view of no flagellation, they still share other common taxonomical properties greatly different from those of Erwinia stewartii in the following points:

Furthermore, these strains are observed to be different from the previously described strain SHS-2003 in the points; decarboxylation of glutamic acid; being scarcely capable of utilizing lactose, acetate and lactate as sole carbon source; being unable to produce acids from mannitol and lactose; and having no activity on litmus milk.

Moreover, since these strains did not show coincidences with any other known species in the genus defined in the Manual, it was concluded that they should be allocated to a new species, named as Erwinia punctata by the present inventors.

Of these strains, the strains SHS-2004 and 2006 are common in their taxonomical properties in the points;

However, SHS-2004 differs from SHS-2006 in the points:

On the other hand, the strain SHS-2005 has the same taxonomical properties as those of SHS-2006 in the point of being scarcely capable of utilizing mannitol, acetate and lactate as their sole carbon source, but the former differs from the latter in the points:

Furthermore, the strain SHS-2007 likewise has the properties as those of SHS-2006 in the points of being scarcely capable of utilizing mannitol, acetate and lactate as their sole carbon source, but the former differs from the latter in the pints:

On the basis of the above indicated results of observation, all of the strains SHS-2004, 2005, 2006 and 2007 are likewise found to belong to said Erwinia punctata and each forms a variant with respect to the others.

Strains SHS-2008 and 2010

Although these strains are considered to be closely related to Erwinia tracheiphila or Erwinia quercina in view of their motility with mono-lateral flagellum, the taxonomical properties of them are still greatly different from those of Erwinia tracheiphila defined in the Manual, in the following points:

On the other hand, there are differences in their taxonomical properties from those of Erwinia quercina defined in the Manual in the following points: and sorbitol, and

Moreover, since these strains do not show coincidences with any other known species in the genus defined in the Manual, they were found to be suitably allocated to a new species, named as Erwinia terreus by the present inventors.

Furthermore, the strain SHS-2010 shows substantial coincidence with SHS-2008, except for certain differences observed in the following points: production by SHS-2010,

Strain SHS-2009

This strain is considered to be closely related to Erwinia tracheiphila, E. quercina and E. herbicola, var. herbicola in view of motility with mono-lateral flagellum.

However, there is remarkable differences in the taxonomical properties of this strain as compared with those of Erwinia tracheiphila defined in the Manual in the following points: glycerol and mannose.

In addition to this, there is also found remarkable differences from those of Erwinia quercina, in the following points: ribose or sorbitol,

Furthermore, there is still found remarkable difference in the properties of SHS-2010 from those of Erwinia herbicola. var. herbicola in the following points: rhamnose, ribose or sorbitol.

Although some differences are still remaining in comparing this strain with the aforedefined SHS-2008, in the points: in the other dominating properties and thus identified to be a variant of the aforedefined Erwinia terreus.

Strain SHS-2011:

This strain is considered to be closely related to Erwinia tracheiphila or Erwinia amylovora in view of its motility with a mono-lateral flagellum.

When the properties of this strain are first compared with those of Erwinia tracheiphila defined in the Manual, there is recognized remarkable differences in the following points; mannose, and

When they are compared with those of Erwinia amylovola, there is recognized remarkable differences in the following points: mannose, and

On the other hand, when this strain is compared with the SHS-2008, it is recognized to be in a coincidence with the latter in the properties except for no production of acid from ribose or glycerol, and is identified as a variant of the aforedefined Erwinia terreus.

In addition to those isolated microorganisms (wild strains), any spontaneous mutants obtained therefrom may likewise be utilized in the method of this invention with advantages as far as they are capable of producing 2,5-diketo-D-gluconic acid, and it is needless to say that any strains, obtained by artifically or inductively mutating or modifying these isolated microorganisms so that they exhibit desired properties, may likewise be utilized in embodying the present invention in a smooth way by suitably taming the strains.

The aforedefined microorganisms grow abundantly in an aqueous nutrient medium containing D-glucose, as a main carbon source, corn steep liquor as a nitrogen source and a small amount of inorganic salts. If they are aerobically cultured, they grow on media of very-high-D-glucose concentration with sufficient stability to produce 2,5-diketo-D-gluconic acid (2.5 DKG) in good yeild, as compared with the known microorganisms which have been used in producing the same product.

The concentration of D-glucose in the broth may be as high as 40 w/v % if particularly desired so, though it is usually convenient for an economical production of 2,5-diketo-D-gluconic acid to maintain this concentration to a range of 15-25 w/v % and more preferably, to approximately 20 w/v %.

The temperature of fermentation broth may be in a range of 15-35.degree. C., preferably, in that of 20.degree.-30.degree. C. and more preferably at approximately 28.degree. C. An initial pH value of the medium may be in a range of 5.5-7.5 and preferably in that of 6.0-7.0.

The pH value of the broth may be maintained at a desired range of 4.0-5.5 during the fermentation by incorporating suitable inorganic salts having a buffering action in the starting broth, or by consecutively feeding suitable bases into the broth with the progress of the fermentation. The salt can be exemplified by calcium carbonate and the base can be exemplified by sodium hydroxide.

The inoculated fermentation broth is stirred constantly with an agitator (Ca. 1,740 r.p.m.) under aeration at a ratio of about 600 N ml/min.,

The fermentation completes when the conversion of D-glucose into 2,5 DKG reaches a value which corresponds to about 90 % yield of the latter, which time is about 17-31 hours after beginning cultivation.

The accumulated 2,5 DKG or its salts may be isolated as crystals from the fermentation broth after being treated with any means such as pH adjustment, or alternatively, the broth may advantageously be utilized as such for the nutrient medium of the next stage. For instance, the broth containing 2,5 DKG may directly be utilized for the production of 2-keto-L-gluonic acid, thus the latter can be obtained in high yield in a simple procedure as compared with the conventional method.

In addition to the previously described fermentation, the present invention may be embodied by simply contacting any processed products of the microorganisms with any substrates containing D-glucose. In these cases, conditions similar to those described above in connection with the cultivation of viable cells may also be applied to the incubation of the processed products in the substrate, and the conversion starts immediately after the starting of contact.

In the following description, the present invention will be elucidated in more detail by way of examples

(1) Seed medium: D-glucose (hydrated, content 91%) 1.0 w/v %, Corn Steep Liquor (CSL)  5.0 w/v %, Potassium bihydrogen phosphate (KH.sub.2 PO.sub.4) 0.1 w/v %, Magnesium sulfate (MgSO.sub.4.7H.sub.2 O) 0.02 w/v %, and Calcium carbonate (CaCO.sub.3) 0.5 w/v % ______________________________________

is adjusted to pH 6.8-7.0 with 10 % aqueous solution of NaOH, divided into 50 ml portions and placed in sterilized 500 ml conical flasks to give the seed media.

(2) Seed culture:

Each of said media in the flasks is inoculated with one loopful of the strains listed in Table 3 below and shaken (stroke: 71 mm, 270 s.p.m.) at 28.degree. C. for about 8 hours. The culture is terminated when the optical density (O.D.) of the media becomes approximately 8 (end point.

(3) Fermentation broth:

An aqueous solution containing; D-glucose          20.0 w/v %, CSL                 3.0 w/v %, KH.sub.2 PO.sub.4   0.1 w/v % and CaCO.sub.3          6.3 w/v % ______________________________________

is adjusted to pH 6.8-7.0, divided into 455 ml portions and placed in 1 L fermentors to give the fermentation broth, into which each 45 ml of said seed media is added.

(4) Fermentation:

The fermentation is carried out under the conditions of: Temperature:       28.degree. C. Stirring:          1740 r.p.m. Aeration:          600 N ml/minute Time:              17-31 hours ______________________________________

(5) Determination:

The product is chromatographed on a filter paper carrier by ascending under the conditions specified below, and the spots are quantitatively determined by densitometry. containing anilin (0.93 g) and phthalic acid (1.66 g) followed by a color-developing treatment at 105.degree. C. for 2 minutes. Substance           Color:  Rf: ______________________________________ 2,5-diketo-D-gluconic acid Brown   0.16-0.18 2-keto-D-gluconic acid Pink    0.27-0.29 D-glucose           Brown   0.48-0.50 ______________________________________

In addition to this, a thin layer chromatography is also performed with "TLC alumisheet cellulose (tradename of E Merck A.G.) and the same developing solvent system and staining operation is described above. In this case, the determination is performed by comparison of the chromatogram with that obtained with an authentic specimen.

(6) Termination:

The fermentation is terminated at a time when the pink spot of 2-keto-D-gluconic acid disappears from said paper or thin layer chromatogram.

(7) Results: ______________________________________ Fermentation Culture 2,5-diketo-D- Seed Culture                gluconic acid. At termination At termination Conc.   Yield for Strains            Time           Time  (w/v  D-Glu- SHS-  pH    OD     (hrs.) pH  OD   (hrs.) %)    cose (%) ______________________________________ 2003  7.0   10.0   8     5.4 12.3 18    19    88 2004  6.0   8.5    8     4.9 12.0 17    19    89 2005  6.9   9.5    9     5.1 12.6 20    19    86 2006  5.9   7.5    8     4.7 12.0 21    19    89 2007  6.0   7.0    8     4.9 12.6 17    19    89 2008  5.6   3.1    10    5.3 12.4 29    17    70 2009  5.8   3.4    10    5.7 12.4 21    18    79 2010  5.4   2.5    10    5.4 12.8 31    18    75 2011  5.3   2.4    10    5.1 12.2 31    17    75 ______________________________________

(1) Medium used for obtaining microorganism cells:

An aqueous solution containing: Yeast extract (available from Daigo Eiyo Yakuhin K.K.) Yeast extract (available from Daigo Eiyo 0.1 w/v % Kauhin K.K.) KH.sub.2 PO.sub.4         0.1 w/v % NaSO.sub.4.7H.sub.2 O    0.02 w/v % and CaCO.sub.3                0.6 w/v % ______________________________________

is adjusted to pH 7.0, divided into 80 ml portions, and placed in conical flasks (500 ml).

(2) Cultures for collecting microorganism cells and for separating crude enzyme extract:

Each of said media is inoculated with the strains listed in Table 4 below in a similar manner as described in the seed culture of Example 1 and shaken (stroke, 71 mm, 270 s.p.m.) at 28.degree. C. for 16 hours.

After the shaking, the microorganism cells are collected by centrifuging the cultured media, washed twice with saline and divided into two portions.

One of said portions is suspended again in saline to give the cell suspension. The other is suspended in a 1/50 M tris-HC1 buffer (pH 7.5) wherein the cells are disruptured by ultra-sonic treatment and centrifuged to remove insoluble residue therefrom in order to obtain crude cell-free enzyme extract as its supernatant.

(3) Incubation with cell suspension:

A mixture is prepared with 1/10 M 3,3-dimethylgultaric acid buffer (pH 5.0) containing about 5 w/v % of D-glucose, wherein the cell concentration is adjusted so that the O.D. at 660 m.mu. is approximately 10. The mixture is divided into 10 ml portions, placed in test tubes of 23 mm (diameter) x 196 mm (length) and incubated at 28.degree. C. for 3 hours.

The mixture is centrifuged to give a supernatant which is analyzed by paper chromatography. The results are summarized in Table 4 below, wherein the concentrations of D-glucose in the mixture before incubation are also given. ______________________________________ (Concentrations after 3 hours shaking) D-glucose (%) before Strains incuba-         2-keto-D-gluconic 2,5-diketo-D-gluco- SHS-  tion            acid (%)   nic acid (%) ______________________________________ 2003  4.9      2.7    0.8        1.9 2004  5.0      1.9    0.7        1.5 2005  5.0      2.6    0.6        0.7 2006  5.0      1.9    0.9        1.1 2007  5.0      1.4    0.3        1.4 2008  5.0      1.3    0.8        1.2 2009  5.2      1.9    0.7        1.3 2010  5.0      2.9    0.5        0.9 2011  5.0      2.8    0.7        0.9 ______________________________________

(4) Incubation with crude enzyme extract:

To a mixture prepared in a similar manner as described in (3) above, the crude enzyme extract is added so that its concentration is 0.25 mg/ml, determined as a protein quantity (Folin's method). After being shaken as previously described, the mixture is treated with two drops of 10 w/v % trichloroacetic acid solution to remove the protein portion, and chromatographed on a filter paper. The results are shown in Table 5 below. ______________________________________ (Concentrations after 3 hours shaking) Strain                  2,5-diketo-D-gluconic acid SHS-  2-keto-D-gluconic acid (%) (%) ______________________________________ 2003  1.0               1.0 2004  0.6               0.8 2005  0.4               0.4 2006  0.5               0.5 2007  0.3               0.4 2008  0.8               0.5 2009  0.4               0.6 2010  0.3               0.3 2011  0.3               0.3 ______________________________________

## Claims

1. A method for preparing 2,5-diketo-D-gluconic acid or a salt thereof which comprises converting D-glucose into 2,5-diketo-D-gluconic acid in the presence of a strain of microorganisms consisting essentially of Erwinia citreus which is non-motile and does not effectively grow at a temperature above 39.degree. C., and produces a recoverable quantity of 2,5-diketo-D-gluconic acid or a salt thereof by cultivation in the presence of D-glucose and utilizing formate, but is incapable of liquefying gelatin and of producing any acid from esculin, rhamnose or sorbitol, respectively, or growth and isolating the 2,5-diketo-D-gluconic acid.

2. A method for preparing 2,5-diketo-D-gluconic acid or a salt thereof which comprises converting D-glucose into 2,5-diketo-D-gluconic acid in the presence of a strain of microorganisms consisting essentially of Erwinia punctata which is non-motile and does not effectively grow at a temperature above 39.degree. C., and produce a recoverable quantity of 2,5-diketo-D-gluconic acid or a salt thereof by cultivation in the presence of D-glucose, and forming reducing compound from sucrose, but is incapable of liquefying gelatin and of producing any acid from mannitol, lactose, esculin, glycerol, rhamnose or sorbitol, respectively, on growth and isolating the 2,5-diketo-D-gluconic acid.

3. A method for preparing 2,5-diketo-D-gluconic acid or a salt thereof which comprises converting D-glucose into 2,5-diketo-D-gluconic acid in the presence of a strain of microorganisms consisting essentially of Erwinia terreus which does not effectively grow at a temperature above 40.degree. C., and producing a recoverable quantity of 2,5-diketo-D-gluconic acid or a salt thereof by cultivation in the presence of D-glucose, forming reducing compound from sucrose and acetoin, and utilizing formate, but is incapable of liquefying geletin and of producing any acid from mannitol, esculin, rhamnose or sorbitol, respectively, on growth and isolating the 2,5-diketo-D-gluconic acid.

4. A method for preparing 2,5-diketo-D-gluconic acid or a salt thereof which comprises converting D-glucose into 3,5-diketo-D-gluconic acid in the presence of at least one strain of 2,5-diketo-D-gluconic acid producing microorganisms selected from the group consisting of Erwinia citreus, Erwinia punctata and Erwinia terreus which does not effectively grow at a temperature above 47.5.degree. C. and producing a recoverable quantity of 2,5-diketo-D-gluconic acid or salt thereof by cultivation in the presence of D-glucose, but is incapable of liquiefying geletin and does not produce gas or any acid from esculin, rhamnose or sorbitol, respectively, on growth and isolating the 2,5-diketo-D-gluconic acid.

5. The method of claim 26, wherein at least one strain of said microorganisms is cultivated in an aqueous nutrient medium in the presence of D-glucose to produce and accumulate 2,5-diketo-D-gluconic acid or any salts thereof in the medium during the cultivation.