CS239293B1 - Microorganism stem pseudomonas sp.ccm 3635 - Google Patents

Abstract

New strain of Pseudomonas sp. CCM 3635, producing intracellular a hydrolase that catalyzes deacylation 7-beta- (4-carboxybutamamido) -cephalosporanO ' acid and its derivatives to form the corresponding 7-amino compounds, important raw materials for semi-synthetic production cephalosporin antibiotics.

Description

The invention relates to a microorganism strain of Pseudomonas sp. CCM 3635, producing intracellular hydrolase, useful for the enzymatic preparation of 7-aminocephemic compounds, in particular for the preparation of 7-aminocephalosporanoic acid (hereinafter referred to as 7-ACK), an important starting material for obtaining semisynthetic cephalosporin antibiotics.

It is known that the 7-aminocephem compounds of the general formula

Η ₂ Ν-ρΥ>

CH ₂ X

COOH in which X represents a hydrogen atom, a -OH, -OCOCH3 group or a nucleophilic group IIz® obtained from their 7-acylamino derivatives of the formula II

R = CO — HN ch ₂ x

COOH / 11 / wherein R denotes - / CH _2/3 -COOH, - / _CH2 / ₃ -CO-COOH / CH / ₃ --CHNH ₂ -COOH or -CH ₂ CGH ₃ and X mean the same as in formula I, by the action of specific enzymes that hydrolyze the amide bond.

These enzymes are produced by suitable microorganisms, as discussed below. Either isolated enzymes or whole cells of certain strains of microorganisms selected with respect to the starting substrate may be used for the hydrolysis.

For example, the use of cells with pecinilinacylase activity is disclosed for the hydrolysis of N-phenyl-acetamido-desacetoxvoephalosporin C, a compound of formula II wherein X is hydrogen and R-CH ₂ CgHg in the preparation of 3-desacetoxy-7-aminocephalosporanoic acid. čs. Certificate No. 218 023 /.

Furthermore, it is known that direct hydrolysis of a compound of formula 11 in which X is -OCOCH ₃ and R is - (CH ₂ ) ₃ -CHNH ₃ -COOH, ie. cephalosporin C is difficult. Procedures using cells of the microorganisms Aspergillus, Penicillium, Alternaria, Giberella, Glomerella et al., Japanese Pat. No. 77,143,289, British Pat. No. 1,566,830) with yields of 5% 7-ACK and 3-deacetyl-7-amino-naphthalosporanoic acid.

The procedure using Pseudomonas sp. with a yield of 7-ACK of about 18% / Japanese Pat. No. 78 94,093].

For the conversion of cephalosporin C to 7-ACK, a two-step process so far seems to be the most convenient, which involves the preparation of cephalosporin c derivatives, i.e. 7-acylamino compounds of formula II, wherein R is - / CH ₂ / ₃ -COOH or - / CH _2/3 -CO-COOH and its hydrolysis using either strain Comamonas / Japanese Pat. Documents Nos 75 101, 584, 78 59, 095 and 81 55,

298) or strains of Bacillus and Arthrobaoter (Japanese Pat. Nos. 77 128, 293 and 78 86, 094].

The 7-acylaminocephem compounds of formula I can be obtained from cephalosporin either chemically / Japanese pat. Nos. 77 77, 078 and 78 114, 891), or enzymatically by treatment with D-amino acid oxidase or activated cells of microbial producers of this enzyme, such as Trigonopsis variabills, Fusarium sp., Aspergillus flavus, Candida et al. No. 1,272,769, Japanese Pat. Nos. 77 44, 695, 76 88, 694, 77 128, 295, 77 125, 696 and 80 23, 966].

The known methods of obtaining the 7-acylamino compounds of the formula II utilize strains of microorganisms which synthesize said hydrolases in the presence of an inducer or, moreover, synthesize undesirable degradation enzymes.

These disadvantages are eliminated by a new strain of microorganism identified as Pseudomonas sp., Which has constitutive synthesis hydrolase aoylamlnooefalosporánové acid and its derivatives, i.e. compounds of formula II in which R represents a group --COOH or - / CH _2/3 -CO-COOH .

The novel strain of the present invention was obtained by isolation from topsoil by accumulation in a culture medium containing 3-methyl-7-beta- [4-carboxybutanamido] -ceph-3-em-4-carboxylic acid sodium salt as a carbon source, mutagenesis and selection of a monocolonium isolate that does not require an inducer-glutaronic acid hydrolase synthesis.

The new tribe was stored in Cs. Collection of Microorganisms / University of E. E. Purkyně, Brno, tř. Defenders of peace 10, under the name Pseudomonas sp. CCM 3635.

The strain characteristics are listed in the following overview:

A. Morphology:

1. Size // οχλ /, cell shape:

2. Mobility:

3. Gram-staining:

rods 0.5 to 0.9 times 1.0 to 1.5 individual, ev. negative polar flagella in clusters

B. Observation of cultures:

I. Broth-agar plates:

2. Culture on sloping broth-agar

3. Bujon-agar puncture:

4. Gelatin puncture:

5. Liquid broth:

colonies round, convex, with rough, wrinkled surface, with slightly wrinkled margin, white to yellowish, slightly opalescent good growth, rough and wrinkled surface, white to slightly yellowed, slightly opalescent, agar without color changes surface growth surface growth, no fluid growth in turbidity, formation of sediment without blank

C. Growth conditions:

it grows well at 25 to 34 ° C and pH 6.5 to 8.5 aerobic

D. Physiological characteristics:

1. Indole production:

2. Production of hydrogen sulphide:

3. Formation of acetylmethylcarbinol:

4. Methyl red test:

5. Nitrate reduction:

6. Denitrification

7. Catalase:

8. Urease:

9. Oxidase:

10. Arginine dihydrolase:

11. gelatine:

12. Starch:

13. Ox-ferm test:

14. Fermentaoe of sugars:

negative negative positive negative negative negative positive negative positive negative

15. Carbon utilization:

no liquids no changes hydrolysis glucose, fructose, mannose, galactose, xylose, sucrose, maltose, lactose, mannitol acetate, glutarate, pyruvate neutralized with ethanol, propionate, citrate and glyoerol neutralized

16. Beta-lactamase: negative

The amount of hydrolase formed in the cells was monitored based on the rate of hydrolysis of the salts of the 7-acylamino-cep compounds by the spectrophotometric method (Balasingham et al., Biochim). Biophys. Acta 276, 250, 1972) or by thin layer chromatography.

Using Pseudomonas sp. CCM 3635 verified the hydrolysis of 3-acetoxymethyl-7beta- (4-carboxybutanamido) cef-3-em-4-carboxylic acid (hereinafter glutaryl-7-ACK), 3-acetoxymethyl-7beta- (5-carboxy-5-oxopentamido) (-ceph-3-em-4-carboxylic acid, 3-methyl-7beta- (carboxybutanaraido) -ceph-3-em-4-carboxylic acid (hereinafter glutaryl-7-ADCK)),

N- [7beta- [4-carboxybutanamido] -ceph-3-em-3-ylmethyl] pyridinium-4-carboxylic acid.

Cell specific activity was determined based on the rate of hydrolysis of glutaryl-7-ADCK (at a concentration of 5 mg / ml 0.1m phosphate buffer pH 7.0 and 37 ° C). For 7-ACK chromatography using a thin layer of cellulose and of n-propanol / water 7: 3 / with ninhydrin detection / R _f 0.5 /. The identity of 7-ACK was verified by high pressure liquid chromatography.

Pseudomonas sp. CCM 3635 according to the invention can be advantageously used for the preparation of 7-aminocephem compounds. Due to the constitutive hydrolase synthesis, it is not necessary to add an enzyme synthesis inducer to the soil.

Another advantage of this strain is that it does not exhibit undesirable beta-lactamase activity. Incubation of cells at a concentration of 15 mg dry matter / ml in 1% glutaryl-7-ACK solution (4 hours at pH 7.0 and 37 ° C) did not open the beta-lactam ring, as measured by UV-light absorption at 255 nm and comparison with control measurements of substrate incubated under the same cell-free conditions. '

Possible uses of Pseudomonas sp. CCM 3635 for the preparation of 7-ACK are documented in the Examples.

Glutaryl-7-ACK was prepared from cephalosporin C by the D-amino acid oxidase enzyme Trigonopsis varlabills CBS 4095 or CBS 1040 in the presence of sodium azide to inhibit catalase, under aeration.

Trigonopsis variabilis cells with D-amino acid oxidase activity, prepared by two-stage aerobic fermentation in complex or synthetic culture broths with the addition of 0.3% D, L methionine as an inducer, permeabilized with suitable permeabilizing agents (e.g. toluene or wetting agents) may be used.

The strain Pseudomonas sp, CCM 3635 is useful for the hydrolysis of glutaryl-7-ACK prepared chemically or isolated from the reaction mixture after deamination of oephalosporin C by Trigonopsis variabilis cells.

Separate the cells by centrifugation, acidification with hydrochloric acid, extraction with ethyl acetate and evaporation of the dried extract in vacuo.

The strain according to the invention is also useful for the hydrolysis of glutaryl-7-ACK produced by D-amino acid oxidase of Trigonopsis variabilis cells from cephalosporin C in the presence of sodium azide, directly in the reaction mixture after cell separation, without isolation.

Finally, it is useful for the hydrolysis of glutaryl-7-ACK produced by deamination of cephalosporin C in crude eluates from Trigonopsis variabilis cell purification.

Further details of the novel strain according to the invention and its applicability are given in the following examples which illustrate the invention but do not limit it in any way.

He did

Pseudomonas sp. CCM 3635 was obtained from ornioe by the following procedure:

g ornioe was suspended in physiological saline, and the filtrate was inoculated with the following composition (in wt / wt): 0.2% potassium dihydrogen phosphate, 0.05% magnesium sulfate, 0.02% manganese chloride crystals, 0.003% sulfate ferrous crystals, 0.05% yeast extract and 1.5% glutaryl-7-ADCK (pH 7.2, filter sterilization).

The grown culture was further passaged on soil of the same composition and then sown on solid soil with Masopepton agar. The obtained monocolonium isolates were verified for deacylating activity after cultivation in the presence of an inducer, i.e. glutaric acid, in the soil of the following composition: 5% acidic protein hydrolyzate (commercial product), 0.1% corn extract,

0.5% yeast extract, 0.1% glutaric acid (pH 7.2).

The selected isolate which, after cultivation in a broth of the same composition, under the conditions of Example 2, exhibited a specific activity of 1.2-10 / mol 7-ADCK / min / mg dry cell mass was further mutagenized.

Cells after growth on complex broth were separated, washed with 0.1 M phosphate 7 buffer (pH 7.0) and the suspension containing about 10 cells and 150 µg of N-methyl-N-nitro-N-nitrosoguanidine / ml buffer was stirred 60 min. at room temperature.

Then, the suspension was sown on solid medium with masopepton agar and a selection of monocolonium isolate was performed which showed a constitutive hydrolase synthesis when grown in the aforementioned broth with an acidic protein hydrolyzate without glutaric acid.

The selected monoclonal isolate exhibited hydrolase activity of 2.5 / mol 7-ADCK / min / mg dry cell mass under the culture conditions shown in Example 2, i.e. about 100% increase in activity without the need for an inducer.

Example 2

In 50 ml Erlenmayer flasks, 50 ml of nutrient broth of the following composition was added: 1% peptone, 0.4% beef extract, 0.2% yeast extract, 0.5% sodium chloride. After sterilization and seeding with Pseudomonas sp. CCM 3635, obtained according to Example 1, was incubated on a rotary shaker at 28-29 ° C for 48 h.

Then, 5 ml of the grown culture was used to incubate 100 ml of broth in 500 ml flasks. This broth contained 5% acid protein hydrolyzate, 0.1% corn extract and 0.5% yeast extract (pH 7.2, sterilized at 120 ° C for 20 min).

After incubation for 45 h on a rotary shaker at 28-29 ° C, the grown cells were collected by centrifugation (10,000 rpm, 10 min), washed with an equal volume of 0.1 M phosphate buffer pH 7.0 and, after centrifugation, were suspended in the same buffer at a concentration of 20 mg dry matter / ml.

ml of the prepared cell suspension was mixed with 50 ml of 0.1 M phosphate buffer containing 1.2 g glutaryl-7-ACK (substrate content 88%), whose pH was adjusted to 7.0 with 1% sodium hydroxide solution. .

The reaction was run at 37 ° C for 4 h. After removal of cells by centrifugation, 7-ACK was detected at a concentration corresponding to 90% conversion. The 7-ACK was isolated after concentration tenfold by precipitation with 2 N hydrochloric acid to pH 3.2 and separation of the precipitate after standing overnight at 5 ° C.

Yield 647 mg 7-ACK with an active compound content of 83%, total yield of pure substance 72.2 i.

Example 3

In 500 ml flasks, 100 ml of culture medium was placed: 2% glucose,

0.4% potassium dihydrogen phosphate, 0.1% crystalline magnesium sulfate, 0.05% calcium chloride, 0.01% boric acid, 0.004% ammonium molybdate, 0.04% manganese sulfate,

0.004% crystalline zinc sulfate, 0.045% copper sulfate, 0.025% ferrous sulfate crystals, 2. 10 ~ ⁵ % blotin, 1. 10 ^-3 % thiamine, 0.3% D, L-methionine (glucose and vitamins sterilized by filtration, soil pH 6.0).

After inoculation with Trigonopsis variabilis strain CBS 4095, it was cultured on a rotary shaker / 240 rpm / 72 h at 29 ° C. Then, 2 ml of the grown culture was used to inoculate 100 ml of broth of the above composition and incubated under the same conditions for 72 h.

Cells were centrifuged, permeabilized and washed twice with a 5-fold volume of buffer. Permeabilization was carried out for 90 min at 29 ° C in a suspension containing about 30 mg dry cell mass / ml of 0.1 M phosphate buffer pH 8.1 and 10% toluene.

g of cephalosporin C potassium salt was dissolved in 0.1 M phosphate buffer pH 8.1 to a volume of 100 ml, 0.025% sodium azide and Trigonopsis variabilis cells prepared as described above were added at a concentration of 2 mg dry matter / ml.

The reaction mixture was aerated at 33 ° C for 2 h. The degree of conversion of cephalosporin C to glutaryl-7-ACK was 77-90%.

To the obtained glutaryl-7-ACK solution, Pseudomonas sp. CCM 3635, obtained according to Example 2, at a concentration of 10 mg dry matter / ml and the mixture was incubated with stirring at 37 ° C for 4 h.

Cells were then removed by centrifugation (10,000 rpm, 10 min) and 7-ACK was determined in the supernatant at a concentration corresponding to 80% conversion of glutaryl-7-ACK.

Example 4

Pseudomonas sp. CCM 3635 were prepared as described in Example 2 and mixed with a solution containing glutaryl-7-ACK (6 mg of active ingredient / ml), obtained by treatment with Trigonopsis variabilis cells in the presence of 0.1% sodium azide on cephalosporin c in rich eluates from its purification. The conversion to 7-ACK was 80%.

Claims (1)

SUBJECT OF THE INVENTION Pseudomonas sp. CCM 3635, producing intracellular hydrolase, which catalyzes the deacylation of 7-beta- (4-carboxybutanamido) -cephalosporanoic acid and its derivatives of formula II R-CO-Hn CH, X / 11 /, COOH in which R represents - (C 10 _O ) _o -COOH or - (CH 2) -CO-COOH, and X represents a hydrogen atom, £. i and J are -OH, -OCOCH3 or a nucleophilic group contained in aqueous solutions to give the corresponding 7-aminocephene compounds of formula I COOH in which X denotes the same as in formula II.