Strain amycolatopsis orientalis subsp eremomycini all-russian collection of industrial microorganisms ac-807 eremomycin producent

FIELD: microbiology; medicine.

SUBSTANCE: strain Amycolatopsis orientalis subsp.eremomycini All-Russian collection of industrial microorganisms Ac-807 is eremomycin producent and can be used as source of eremomycin in medicine for treatment of severe infections.

EFFECT: increase of eremomycin output.

2 tbl, 1 ex

 

The invention relates to the medical industry and relates to a new strain actinomycete forming antibacterial antibiotic eremomycin.

Antibiotic eremomycin on chemical structure relates to polycyclic antibiotics of the vancomycin group of ristomycin, which are the reserve antibiotics for treatment of severe infections.

The structure of antibiotic eremomycin sulfate

Eremomycin effective in the treatment of diseases caused by gram-positive pathogens, including metitsillinrezistentnye Staphylococcus aureus (MRSA) and gone anaerobic Clostridium difficile is the causative agent of pseudomembranous colitis.

A known strain Amycolatopsis orientalis subsp. eremomycini PMBC-S892 - producer of antibiotic eremomycin /1/adopted as a prototype. The disadvantage of this strain is a low level of biosynthetic activity is not more than 1800 mg eremomycin 1 l fermentation liquid.

The essence of the invention lies in the fact that the new strain-producer of eremomycin by mutant transformation properties of the known strain Amycolatopsis orientalis subsp. eremomycini PMBC-S892 in the sequential three-stage impact methylnitrosourea, ultraviolet and gamma rays60With the original strain and selection of the original mutant strain increased antibiotically activity.

The new strain is deposited in culture collections under the strain VKPM-As-807.

Strain Amycolatopsis orientalis subsp. eremomycini PMBC-As-807 has increased productivity eremomycin (2300 mg per 1 l of fermentation liquid) and is characterized by the following cultural-morphological and physiological traits.

Cultural and morphological characteristics of strain Amycolatopsis orientalis PMBC-As-807

Temperature 28°C; the duration of the cultivation and study of the taxonomic characteristics (7-21) days. Spore seed material with a ten-day culture of the strain grown on glukozooksidasa agar or on the environment No. 2 Gause. Color is defined according to table Bondartsev and Prauser (table 1).

Morphological features. Hyphae of the aerial mycelium straight or winding, the disintegrating elements of different lengths; the surface of spores smooth.

Physiological and biochemical properties. Aerobe. Grows at (24-37)°C; optimum growth 28°C, gelatino thins, milk iphonesimulator interterritorial hydrolyses. On the tissue does not grow. Restores nitrates to nitrites. Melanoidin pigments are not formed during growth on provocative environment Tresner with citric acid iron.

The use of carbon sources. Well utilizes fructose, arabinose, Inositol, galactose, lures, glucose; moderately lactose, rhamnose, xylose; not utilise the quote raffinose.

Antagonistic properties. Amycolatopsis orientalis subsp. eremomycini PMBC-As-807 with the growth on agar media moderately inhibits the growth of gram-positive bacteria Staphylococcus aureus, Bacillus subfilis. Bacillus mycoides, Micrococcus luteus and has no effect on gram-negative bacteria Escherichia coli, Comamonas terrigena, Providencia stuartii.

Storing strain Amycolatopsis orientalis PMBC-As-807

Is stored in the form of culture, dried in sharethelove environment in apalach if (5-7)°C.

Antibiotic activity of strain Amucolatopsis orientalis PMBC-As-807

is determined in the culture fluid by the method of diffusion in agar (trehgogny option GFH, str) using as the test organism Bacillus subtilis 6633.

Strain Amycolatopsis orientalis PMBC-As-807 is illustrated by the following example.

Example 1. Spore material with mycelium culture Amycolatopsis orientalis PMBC-As-807 grown within (10-14) days at 28°C in the environment Lindenbaum, inoculated nutrient medium (150 ml Erlenmeyer flasks 750 ml) and grown at 28°C for 24-36 hours on a rocking chair at (220-240) rpm

The composition of the seed medium, %:

Glycerin2,0
Soy flour0,5
Potassium nitrite0,1
On the bacilli chloride 0,3
WaterWater
pH before sterilization7,0-7,2

The medium is sterilized for 30 min at a pressure of 0.6 ATM at 120°C.

Grown mycelium in the amount of (5-10)% of inoculated nutrient medium (150 ml Erlenmeyer flasks 750 ml) of the following composition,%:

Glycerin3,0
Soy flour0,5
Potassium nitrite0,1
Sodium chloride0,3
Magnesium sulfuric acid0,01
Glycine0,0025
Chalk0,2
WaterWater
pH before sterilization6,8-7,0

The fermentation is carried out at 28°C for (120-144) hours on a rocking chair at (220-240) Rev/min Eremomycin accumulated in the culture fluid of a producer. Antibiotic activity is here determined by the method of diffusion in agar with the use of test-cultures of Bacillus subtilis ATCC 6633. The content of eremomycin in the culture fluid of strain Amycolatopsis orientalis PMBC-As-807 to the end of the fermentation is 2300 mg/ml

With the aim of identifying eremomycin conduct its sorption from native solution (filtered from mycelium culture fluid) on carboxylic cation exchanger KB-2 is in salt form, elute with aqueous ammonia, and the eluate is neutralized, obezbolivaet is passed through the sorbent PTFE, an antibiotic with a sorbent displace water, concentrated in vacuo to content eremomycin (120-140) mg/ml and crystallized in the form of sulfate.

Eremomycin sulfate, obtained by biosynthesis new strain - producer Amycolatopsis orientalis PMBC-As-807, is a white crystalline powder, easily soluble in water and insoluble in organic solvents. Indicators eremomycin sulfate (physico-chemical properties, antibacterial activity and toxicity) fully complies with the indicators defined for eremomycin sulfate, protected by the RF patent №2110578 (Brutto-formula: C73H89N10C1O26×H2SO4. Mol. weight 1656 u

UV spectrum: λmax(0,01 N. HCl) nm (E%1 cm) 280 (42); λmax(0,01 N. NaOH) nm (E10,0cm) 300 (60);

IR-spectrum (KBr), νmaxcm-1: 3350, 1650,1500, 1200-1000; [α]20D=-100°(1,0, H2O).

The yield of the target product is cleaned is about (97% purity) of crystalline drug eremomycin sulfate content in the native solution is (60,0-65,0)%.

Thus, created a new mutant strain of Amycolatopsis orientalis PMBC-As-807, which differ from the known character of the growth diagnostic agar media, degree of assimilation of carbon sources (Inositol, galactose) and by the ability of antibioticaccutane, which is 78% larger than the activity of the strain prototype Amycolatopsis orientalis PMBC-S892 (table 2).

Antibiotic activity of a new strain of Amycolatopsis orientalis PMBC-As-807 is 2300 µg/ml Using a new strain of Amycolatopsis orientalis PMBC-As-807 as producer for the biosynthesis of eremomycin allows to increase the yield of the target product. The output eremomycin is 1.2 grams per 1 liter native solution.

Sources of information

Patent RU 2110578 C1 from 18.06.97

Table 1.
Cultural and morphological characteristics of strain Amycolatopsis orientalis PMBC-As-807
Agar mediumThe growth of the air mycelium*The color of the myceliumSoluble pigment
AirSubstrate
No. 1 GauseNo ColorlessNo
Oatmeal+ -White (D-1)ColorlessNo
Of čapekNoWhite (D-1)ColorlessYellow
Glucosamina+ -From white (D-1) to yellowish (W-1)Yellow (G-6)Yellow (G-6)
Lindenbaum (glycerol-nitrate)+ +White (D-1),ColorlessYellow (G-6)
GlucosinolateNoWhite (D-1)Colorlessno
No. 2 Gause+ -
Tresner (peptone and citric acid iron) Melanoidin pigments are missing
*/++moderate;+, weak.

Strain Amycolatopsis orientalis PMBC-As-807 - producer of antibiotic eremomycin.



 

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