BG98319A - Strain streptomyces tenebraruis 2444 producent of tobramycin - Google Patents

Abstract

The strain Streptomyces tenebrarius 2444 is used in the pharmaceutical industry as producent of the tobramycin antibiotic. It is registered in NBIMCC under No. 2342. It was produced by mutagenesis and selection of strain Streptomyces tenebrarius 5519 which include protoplasting, regeneration, treatment with ultraviolet light with 253.7 nm wavelength and survivability 0.01-1% and natural selection. The strain has a 6-time increased productivity of Tobramycin (factor 5') and low productivity of Apramycin and Carbomoylcanamycin B, which enables the desired product of Tobramycin to be made at considerably lowered material costs.

Description

The invention relates to a strain of Streptomyces tenebrarius 2444, a producer of tovramyin used in the Pharmaceutical Industry.

S. tenebrarius sells a neuramycin antiviotic complex consisting of 11 amino-clitol components with a known chemical stator. Three of them are synthesized in substantially higher amounts - Factor 2Sapramyin) j Factor 4 (carvamoylkanamycin B) and ·· ···· · · · ···· · · · · · ·

Factor 5 'Scarbamoyltobramycin) and the others are

MINORIS 2,3,6)

Factor (apramycin) is used in veterinary medicine lysis after carvamoyl grape has been removed. Factor 5 'is converted to Factor 6 (tovramycin) under the same conditions caused by

Gram positive and Gram negative microorganisms and

And others. <3,5)

Strains producing different components or ratios of components in this are known

You are known to be predominantly producing Factor 5 '(. 8,10 is also that under deep conditions strain us 1575 produces

ie the production of carmamoylturamicin is 1/7 of the total amount of products 1).

It is known that the strain S.tenebrarius

4: 5'in a ratio of 0.5: 0.1: 1.3 in the longitudinal conditions

FermentationC11)

Disadvantages of the above

strains are the predominant προ of dakia of Factors 2 and 4 in the calcuric fluid, which makes them for industrial production due to the low target product - Factor 5 ', which leads to significant costs for obtaining ma

It is an object of the invention to produce a strain of Streptomyces tenebrarius that has an altered ratio of production

Factors, increased production of Factor 5 ', decreased production of

Factors and 4 and low cost of getting ma

S. tenebrarius 2444 strain was obtained by mutagenesis and selection

The task is solved by creating a strain of Streptomyces tenebrarius 2444, a predominant producer of tovramycin, registered with NBPMC under No. 2342.

· · · · · · · · · · · · · • a · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · , taught by the parent S.tenebrarius

1575 (11).

Breeding there S.tenebrarius 2444 includes protoplasts

wound and regeneration, UV light treatment with a wavelength of 253.7 nm and a survival rate of 0.01-17., natural opening.

The International Systematics Project (7) methods were used to characterize the strains and the S.tenebrarius 2444 mutant obtained.

Air color (VI>, co-myeloma (CM) and medium pigmentation are reported on a Bondartsev scale <9).

Taul. 1. Characterization of the starting strains and the mutant obtained.

strain S, tenebrarius S.tenebrarius S.tenebrarius

WEDNESDAY 1575 5519 2444

3 4

VM is weighed down sluggishly VM is weighed down sluggishly VM is weighed down sluggishly ISP - 2 / d3 /; cm-pink te- (d3); CM-dark cre (d3); CM-dark yeast to the left had / about /. Colonies creamy / about /. Svilen malts rice / v5-g2 /. Svilen with unpriced prices height and sporulation agar height and sporulation. Not annoying Does not bother you- Turns pink- soluble with medium- we create in the middle purple soluble in and pigment. pigment. medium pigment / g3 /

ISP - 3 oat arab

BH-weighted sluggish to shamoua / d3-t5 /; CM is colorless to creamy /vB/. It is glorious

BM-weighted sluggish (d3); CM-vesicle Good growth. Does not form soluble

BM-weighted sluggish to dark cream (d3-ob); CM-soluble tan. Does not form a medium-to-medium pigment. pigment / L3 /.

soluble medium pigment.

ISP - 4 starch salts

VM shamoua (r5); CM ~ cream / wb /. Good growth, ovate SGYURU laia-Does not form

VM-weighted sluggish VM-weighted sluggish (d3); CM-yellow brown to cream (d3-eb); /d4/. Good height, CM-creamy to yellowish sporulation at the end of brown / wb-d4 /.

Ag soluble in medium and pigment.

Not colonies are soluble in

Good growth, ovate sporulation.Not the focal pigment.

is a soluble medium pigment

ISP - 5 glycerin asparagines

BM weighted sluggish (d3); CM-apricot yellow to dark brown / d2 ~ vb / .Good growth »Forms pink-liVM-weighted sluggish BM-weighted white / d3 /; CM-dark brown / d3 /; CM-dark brown to shamua / bb-g5 / . up to shamoua / wb-d5 Very good growth. Do Not Grow Solution Not soluble Soluble soluble aral lava Soluble medium pigment (W3).

Rome in the middle pigment.

in the middle pigment «···

ISP - 7 TIR03IN0V agar

VM is expanded / ι / δ / jCM-pink purple / gЗ /. Very good growth. Turns pink

VM ~ pale / d1 /; CM pale brown / v7-dZ / Very good growth.

Does not form solubilized white to cream (d3-eb); CM dark brown to bamboo (db-d5). Medium-soluble pigment (midwife).

Rome in the middle pigment.

fast growth. Does not cause pigment in the middle.

mineral environment of Gazse 1

Colonies with a diameter of 5-mm with a convex center, radial with oval collisions. Forms a pink-purple pigment soluble in agar »

Colonies 2-4mm in diameter, FLAT with point-like unpolished center, concave »Does not form soluble pigment in agar.

Colonies 4-mm in diameter with concave center and abundant sporulation - Does not form soluble pigment in agar

The number of spores in the sporophilic hyphae is over 10. The spore hyphae are spirals with multiple turns.

With regard to the absorption of monosaccharides, there was no difference in their absorption for the three strains. There are differences in their resistance to different antibiotics.

Table 2. Resistance of Streptomyces tenebrarius strains to various antibiotics

bw / 1m

Km

Tm

See

Um

Pen NeF Hlf Reef

TC

1575

I

4 "

551'9

4 »

44444

4K control; 1H-apramycin; Km-kanamyin? Tm ~ gobRamycin; Cm ~ streptomycin; Hm-gentamicin; Pen-penicillin; DEF-cephalosporin; HLF-chloramPhenicol; Rif-Rifamian? Tz-tetracycline? + -stable; - resistant.

Investigations were made on a Gazse 1 agar medium, such as Fr.

antibiotics were added to the medium at a temperature below 50 s.

· · · · · · · · · · · · · · · · · · · · · · · · · · · ·

The important thing here is that the resulting mutant Streptomyces tenebrarius 2444 is resistant to the major components of the neuramiin complex to a much higher degree than the strains

S.tenebrarius 1575 and S.tenebrarius 5519.

In terms of pH, temperature and other requirements, the strains did not show significant differences.

The fermentation is carried out in two stages using the following nutrient media:

Inoculation medium: glycerol 27. ·, maize extract 27. ·, ammonium chloride 0.2%; sodium chloride 0.3%; calcium carbonate 0.5%; tap water; pH 7.0.

Fermentation medium: soybean meal 3%; ammonium chloride 0.6%; magnesium sulfate 1.1%; calcium carbonate 0.8%; glucose 2%; soybean oil 0.6%; tap water; pH7.0.

Tov.Z. Productivity of Streptomyces tenebrarius strains.

Strain

S.tenelrarius 1575

S.tenelrarius 5519

Produced Factor Ratio 2: 4: 5 '

4: 2: 1

0.5: 0.1: 1.3

2: 1: 6

The advantages of the strain according to the invention are expressed in the changed ratio of Het production of the main Factors in the neuramycin complex - increase of Factor 5 'formation in the medium, reduced production of Factors 2 and 4 at low cost for its production »

The invention is illustrated by the following examples:

Example 1 »A strain of Streptomyces tenebrarius 2444 was obtained from strain s.tenebrarius 5519, which was protoplastic by the method of ·· ····

Ferenzy (1). A vegetative culture with an optical density of 600 ng, 4 - 0,8, of the transit Development Phase, cultured three times on glycine containing medium 4 - 67, is used.

The myelin is centrifuged and washed, then treated with o

with lysoiium 0.57. for 1 hour at 37 s. The resulting protoplasts were washed and sieved on a regeneration medium. The grown colonies were reassembled in a Gause 1 medium and checked for productivity. The resulting colonies of heterocariosis type were treated with

ultraviolet light with a wavelength of 253.7 nm with a survival rate of 0.01-0.1 X of the treated suspension. An aqueous spore suspension having a density of 1 - 1.5.10 is applied to the lavage. Two milliliters of the suspension was placed in 50 mm diameter glass petri dishes

agitation, at a distance of 20 cm from the source for 30 - 60 sec.

After irradiation, serial dilutions are made and seeding on an agar medium Gauze 1 «The colonies are replanted on tubes and tested for Fermentation productivity.

EXAMPLE 2 To a 50 ml inoculum medium, a piece of agar with a surface area of about 1 cm from a previously sporulated culture was placed in 250 ml flasks and cultured in the dark for 37 h with 18 hours on a rotary shaker. transfer ίο by volume percent of inoculum developed and fermentation continued under the same conditions for 120 hours- After removal of the micelles 10 microliters Filtrate was terminated on a Merk 5553 plate for thin layer chromatography developing in ethylmethyl ketone: ethanol: ammonia 1: 1 ratio: 1 1. After drying the plaque and containing samples and standards in appropriate concentrations, the latter bioavtograFIRa on the surface of an agar medium containing peptone O 17 "yeast extract 0.37; sodium phosphate 0.27; agar 27; pH 7.8 - 8.0 which is contaminated with 17 of a standardized suspension of Bacillus subtilts ATCC 6633 at 530 · · · · · · · · · · · · · · · · · · · ·

20 20% pass for pass. The chromatography plates were deposited on the surface for a minimum and thermostatized for 16-18 hours at 37 sec.

After staining with a solution of methylene blue, prints are taken - According to the relevant standard, the ratio of

Factors 2: 4: 5 'which is 2: 1: 6

Specific performance analysis is done by high performance liquid chromatography C 4)

Example 3

A previously sporulated tube of S.teneb rarius 2444 was seeded on a Gause 1 medium with a 200-liter Erlenmeyer flask with 200 ml of inoculum medium of the following composition; ammonium chloride 0.2%; sodium chloride 0.3%;

glycerin 2%;

calcium min

Sterilization Fr.

with rotary shaker 18 carbonate 0.5%; tap water, o at 120 C. The culture was incubated

PH 7,0

For 50 media.

Cultivation is a liter Fermenter, charged with the same o

C; 100 revolutions of the stirrer;

performed at 37

INFLOW

air volume

1: 1; pressure 0,

5atm. in about an hour- Ten percent of the seed so developed is fed to 50 soybean meal 37.

; ammonium chloride

0, 67;

magnesium sulfate 1.1%; calcium carbonate

0.8%; glucose 3%;

tap water; pH 7, about.

lead: 37 C;

The fermentation process is carried out with the following

SC

0.5 atm. for

100 revolutions of the stirrer; air 1: 1;

pressure for about 120 hours.

The antibiotic content of the culture fluid was determined by thin layer chromatography followed by bioautography with the B.subtilis ATCC 6633 test microbe described in Example 2 »

The ratio of components in the culture fluid is as follows: Factor

Factor 4: Factor 5 '= ·· ····

Literature '3 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

1. Ferenzy, L., I.Ott, G.Ambrus, and T. Lang (1988). Process for the improvement of antibiotic production by in vivo genetic recombination, US Pat. 4 729 951.

z !. k.och, K. F., F. A. Davies, J. A. Rhoades. (1973) «Nebramycuns Separation of the complexand identification of factors 4,5 and 5 '. J.Antibiot. 26, (12) 745-751.

3. Koch K.F. , J. A. Roades. (1970). Structure of nebramycin factor 6, a

new aminoglycoside antibiotic, Antimicrob.Agents Chemother., 309-313.

4. Manova S., Dimitrova A., Tzoneva D., (1988). Determination of tobra- mucin, apramycin and kanamycin in broth fermentation using HPLC analysis, Abstracts of Vll-th Nat.Conf, on Antibiotics, Razgrad, 7.

5. New H. C. (1976). Tobramycin, an overviev, J.Inf. Diseases, 134 Suppl. S3-SI9.

6. O'Conor S., L. k .. T. Law, N.D. Jones, M-0. Chaney ”(1976). Apramycin, an unique aminocyclitol antobiotic, J. Org.Chem., 41, 2087-2092.

7. Shirking, E.B., and D.Gottlieb. (1966). Methods for characterizati-

on Streptomyces species, Int.J.Syst.bacteriol. 16 (3), 313-340.

8. Stark, M.W., N.G.Knox, R.Wilgus, and R.DuBus (1976). The nebramycin fermentations Culture and fermentation development, in Develop.Ind. Microbiol., Vol.17, L. A.Underkofler (ed.), American Inst.of Biol.

Science, Washington D.C., 61-77.

9. Bondartsev, Zl. C. (1954). Scale of Flowers, L., Ed. / 14 USSR, p. 27.

ίο. Sinyagina 0. P. et al. (1980). Obtaining nutrients of Ramiaiin-forming tauruses into the culture of the producer of the aninoglycoside complex of antibiotics Streptomyces cremeus var.tobramycini var. nov.2242,

ZlHTHBHOTHKM 25 (12), 891-894.

11. P Bulgaria Certificate No47276.

Claims (1)

Strain of Streptomyces tenebrarius 2444, registered with NBPMCC under No. 2342, percent of tovraminin.