JPS604720B2 - Fermentation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to Streptomyces clavarigelus (Streptomyces clavarigelus).
The present invention relates to the improvement of fermentation of a strain of Eptomyces clavuli.

In the specification of German Patent Application No. 2604697 of the present inventors, the present inventors have disclosed that Streptomyces
The formula (1
) and its salts have been described.

Clavalanic acid can be named based on "clavam".

That is, the name is J. Amer. Chem. So
c. (1962) No. 84, p. 3400, “Cepham” is used in the naming of cephalosporin compounds.
A name is given to the core heterocycle of formula A in the same manner as the word .

That is, the compound of formula (1) is (arc, station, Z)-2-(
It is named hydroxyethylidene) clapam 3-carboxylic acid. This compound is described in German Patent Application No. 2517
This is also stated in the specification of Publication No. 316. Clavalanic acid and its salts have been found to exhibit antibacterial activity against a wide range of Gram-positive and Gram-negative microorganisms, and furthermore they have been shown to inhibit the 3-lactamase enzyme produced by Gram-positive and Gram-negative microorganisms. It has also been discovered that

The German Patent Application No. 2,517,316 suggests a relatively wide scope for fermentation and specifically describes fermentation at a pH of 7.0 or higher for other antibiotics. British Patent No. 1315 describing the fermentation of Streptomyces clavarigelus to produce
No. 177 describes a pH range from 6.7 to 7.5 or higher.

However, the present inventors have shown that when fermentation is carried out under conditions where the pH is strictly controlled in the range of 6.3 to 6.7, the amount of clavalanic acid produced in the cone of Streptomyces clavarigelus can be reduced. It was discovered that the scales were raised to an unexpected degree. Accordingly, the present invention provides a method for producing a Streptomyces strain comprising culturing a Streptomyces clavarigelus strain in a nutrient medium therefor, maintaining the pH of the medium in the range of 6.3 to 6.7 during most of the fermentation period. The present invention provides a method for fermenting Ses clavarigelus to produce clavalanic acid.

A pH of about 6.5 is optimal. Although substantial benefits derive from controlling the pH within the stated limits during most of the fermentation, it is generally preferred that such control be effected throughout the fermentation period.

However, during the early growth phase, such as the first three field hours, it may not be necessary to control the pH within the above limits. However, in general the pH of the medium should never be outside the range of 6-8 for any significant period of time. The present inventors 6.3-6.
It has been found that the use of a pH range of 7.7 is very advantageous, so that at pH 6.5, for example, two or more times more clavalanic acid can be produced than that produced at pH 6.0 or 7.0.

In an experiment conducted by the present inventors, fermentation was carried out at pH 6.5.
The yield of clavalanic acid when carried out at pH 6.7
Or it was almost three times as much as the result with 7.0.
The inventors have discovered that it is most preferable to automatically control the pH so that the fermentation takes place. This includes a measured amount of an aqueous mineral or carboxylic acid (e.g. hydrochloric acid, sulfuric acid, citric acid or acetic acid) and/or a base (e.g. gaseous ammonia or an aqueous sodium or potassium hydroxide or carbonate or ammonium hydroxide). ) is added automatically in response to pH changes during the dovetail process. It can be carried out using a spot control device. p
Aqueous solutions of acids or bases used for H adjustment are preferred.
contains 5% to 15% of said acid or base. Except for very strict pH control, Streptomyces
The acid extraction method for the production of clavalanic acid from S. clavarigelus can be carried out by conventional means, namely by culturing Streptomyces clavarigelus in the presence of assimilable carbon, nitrogen and mineral salt sources.

Cultivation is preferably carried out in deep culture under aerobic conditions. Assimilable carbon, nitrogen and mineral sources can be provided by single and/or multiple nutrient sources.

Carbon sources generally include glucose, starch, glycerin, maltose, sucrose, molasses, carboxylic acids, dextrin, and lactose. Nitrogen sources generally include soybean flour, corn steep liquor, distiller's solubles, yeast extract, cottonseed flour, veptone, casein, and amino acid mixtures. Urea and other amides may also be used. Nutrient mineral salts that can be incorporated into the culture medium include commonly used salts such as sodium, potassium, ammonium, iron, calcium, magnesium, zinc, nickel, cobalt, manganese, phosphoric acid, sulfuric acid, chloride and carbonic acid. Examples include salts that can generate ions.

An antifoam agent is generally present for excessive foam control and can be added intermittently as needed. Streff.

Tomyces clavarigelus is generally cultured at 20oC.
-37q0 is preferably carried out at a temperature of 25° to 30°, and this is desirably carried out with stirring and aeration, for example by shaking or otherwise. The growth medium can first be inoculated with a small amount of a spore suspension of the microorganism.

However, in order to avoid growth retardation, a vegetatively grown inoculum solution of the microorganism is generated by inoculating a small amount of beach land with the spore form of the microorganism, and the resulting vegetatively grown inoculum solution is It can be transferred to a broccoli culture medium. Or more preferably, it is brought to seed stage where further growth takes place before being transferred to the main sour lotus medium. Microorganisms are stref.

It is a strain of Tomyses clavarigelus. The inventors have found that NRRL3583 and selected strains and mutants thereof are particularly satisfactory strains for clavalanic acid production. The form of the NRRL3589 bead is described in British Patent No. 1,315,177. As used herein, the expression "variant" refers to the effect of an external agent, which may be naturally occurring or applied either intentionally or otherwise. It includes all resulting mutant strains. The mutant strain is “Radiation
and RadioiSotopeS br lndship trialMiCm. rganiSmS “[Fro
CeedingS. f theS posl skin
, Vienna 1973, page 241 (l
ntemationaIAtomicEnergyAu
thoriV) 1. It can be produced by a variety of methods, including those outlined by Adler. These methods include: i) photosensitizers such as 8-methoxypsoralen,
Nitrous oxide, hydroxylamine, pyrimidine, base analogs such as 5-furomouracil, acridine, alkylating agents such as ethyl methanesulfonate or mustard gas, hydrogen peroxide, phenol, formaldehyde, e.g. and ii) genetic techniques such as recombination, artificial antigen conversion, conversion, lysogenization, lysogenic conversion and selection techniques for naturally occurring mutants.

As used herein, "selected strains" refer to
selected from a parent strain cultivated to produce a strain that has one or more properties that differ quantitatively or qualitatively from that of the parent strain (e.g., resistance to substances produced during fermentation) It refers to the microbial strain derived from the colony.

Such a selected strain can, of course, be a naturally occurring variant of the parent microorganism, but sometimes it is not. Therefore, in a preferred fermentation example, slopes of Streptomyces clavarigelus NRRL 3585 or mutants or selected strains thereof are used to produce assimilable carbon sources such as sucrose or glycerol, assimilable nitrogen sources such as tributone or assimilable carbon and nitrogen. Complex mixtures such as distiller's solubles and media containing yeast extracts and nutrient mineral sources can be pre-inoculated.

This medium can be grown for up to 3 days at 250 to 30 qo with drying. The inoculum thus produced can then be used to inoculate (up to about 10%) a nutrient medium containing similar assimilable carbon, nitrogen and mineral sources. .

Preferably, the lotus gills are cooked in batches at 25° to 300°.
The process is carried out at a pH of 6.5 under lighting and aeration for 3 to 10 days. The pH is controlled by automatic addition of dilute mineral acids and bases as described above. Clavalanic acid produced during fermentation can be detected using the cup agar plate assay system described by Lees and Toutill [AM1ystl955, 8].
0 (947) p. 95-123 and Anal t l
95580 (952) p. 531-535] for Acinetobacter species.

The isolation of clavalanic acid, in particular in the form of its lithium salt, is described in detail in our above-mentioned German Patent Application Publication No. 2003-100002.

Generally, the clavalanic acid is preferably isolated from the nutrient medium as the lithium salt in one or more steps. If desired, this is converted into a salt other than clavalanic acid or a lithium salt by ion exchange. Clarify Yoki Yomo broth and apply it to a charcoal column to adsorb clavulanic acid and/or its salts, which is then eluted with an aqueous solvent such as aqueous acetone, and the eluate is adsorbed onto a basic ion exchange resin. can be done. The resin adsorbate can then be eluted with an aqueous solution of a lithium salt, such as lithium chloride, and the eluate concentrated to precipitate lithium clavulanate in a particularly high purity state. The present invention will now be described more specifically in the following examples, which should not be considered as limiting the invention.

Example 1 Growth of 'a' Inoculum (Inoculum) Sterile distilled water (10w') was added to 14 day old malt/yeast extract agar slants of Streptomyces clavarigelus NRRL3585 and a suspension was prepared.

A portion of this suspension (1.5 [) was used to steam sterilize medium 1 in 2 Tough Lawrence flasks containing the following ingredients:
Inoculated at 50'. Yu/Yuu Sugou 20 Day Taylor's Solubles 15 Yeast Extract
5K2HP04
0.2 tryptone
5 Glycerin 10 Water
So I'm going to take a nap.

This flask was heated at 2 o'clock for 4 hours at 2" amplitude at 2"
Culture was carried out on a rotary shaker at 20 revolutions/min.

(b) Sour-filled inoculum A large number of inoculum solutions thus grown were used to inoculate each of the 5-inoculated fermenters having 4-4 steam-sterilized medium containing the following ingredients: (3.7
5%) Yu/So Day Taylor's Solubles 5.2 Hydrolyzed Casein 5.2 Soybean Flour
21 Soluble starch
47 glucose
7.8 Ferrous sulfate (7 days 20)
0.1 Antifoaming agent (polyglycol) 0.5'/Filling water Total amount 1 amount The fermentation liquid was aerated at 2800°C (0.73 volume/volume/min) and stirred (750 revolutions/min, (2 x 3 inch diameter blades) and were cultured for 9 hours.

Adjust the pH of the drug using an ammonia solution (0.88
An automatic device was used in which ammonia solution (10% V/V) or hydrochloric acid solution (concentrated hydrochloric acid 5% V/V) was added, controlled as described from 32 hours after inoculation. Culture solution pH
'Well, it was measured using a steam sterilizable glass electrode. This electrode is coupled to a pH monitor (model 539, Pether) and control system that regulates the supply of acid or alkali to the culture vessel by a Watson-Marlow pump (Watson-Marlow). Ta. The clavalanic acid content of the fermentation medium is
Using the cup plate agar assay method, the maximum titer of clavalanic acid was determined against Acinetobacter species on samples taken between the hairs.

The following results were discovered. Example 2 In a second experiment carried out as in Example 1, the Sardine Keywort solution was adjusted to pH 6.5 starting at various times after inoculation.
was controlled by.

Here again, the clavulanic acid content was determined on samples taken during the acid rum. pH control starting point of 6.5
Similar results were obtained when acetic acid solution (10% V/V of glacial acetic acid) was used instead of hydrochloric acid solution when pH control at 6.5 was started from 0 hours. Obtained.

Example 3 A third experiment was carried out under conditions as in Example 1, except that the thawed casein was removed from the medium and Nawahai was
m revolutions/min (2 x 3.5 inch straight blades) and the fermentation temperature was 30°C.

The fermentation pH was controlled at 6.5 as described above from the start of the fermentation, but it was stopped at various times after inoculation. Clavalanic acid content was determined on samples taken during fermentation. Stopping point of shame control〃Evening/I Clavalanic acid

Claims (1)

[Claims] 1. Cultivating a Streptomyces clavarigelus strain in a nutrient medium therefor, maintaining the pH of the medium within the range of 6.3 to 6.7 during the majority of the fermentation time. A method for fermentation of Streptomyces clavarigelus for the production of clavalanic acid, comprising: 2. Adjust the pH of the medium to 6.3-6 throughout the fermentation period.
．． 7. The method according to item 1 above. 3. The method according to any one of items 1 to 2 above, wherein the pH is automatically controlled. 4 a measured amount of aqueous mineral acid or carboxylic acid and/or
Or the method according to item 3 above, wherein the base is automatically added during the fermentation process in response to pH changes. 5. The method according to any one of items 1 to 4 above, wherein the culture is performed in deep culture under aerobic conditions. 6. A method according to any preceding clause, wherein the nutrient medium includes assimilable carbon sources, nitrogen sources and mineral salts provided by single and/or complex nutrient sources. 7. The method according to item 6 above, wherein glucose, starch, glycerin, maltose, sucrose, molasses, dextrin and/or lactose are included as carbon sources. 8. Items 1 to 7 above, in which soybean flour, corn steep liquor, Daytailers Solubles, yeast extract, cottonseed flour, peptone, casein, amino acid mixtures and/or urea and other amides are used as the nitrogen source. Any method described. 9. Said 1 to 1, wherein a salt capable of producing sodium, potassium, ammonium, iron, calcium, magnesium, zinc, nickel, cobalt, manganese, phosphoric acid, sulfuric acid, chloride and/or carbonate ions is used as the nutritional mineral salt. The method described in any of Item 8. 10. The method according to any one of items 1 to 9 above, wherein the culturing is carried out at a temperature of 20°C to 37°C. 11. The method according to any one of items 1 to 10 above, wherein the nutrient medium is stirred during fermentation. 12 Streptomyces clavarigelus strain is NR
12. The method according to any one of items 1 to 11 above, which is RL3585 or a mutant strain thereof. 13. After fermentation, the clavalanic acid is isolated from the nutrient medium in one or more steps as a lithium salt and optionally converted by ion exchange to clavalanic acid or a salt other than the lithium salt, as described in paragraphs 1 to 12 above. Any method described.