SU1390242A1 - Method of producing nourseotricin - Google Patents

Abstract

This invention relates to biotechnology and is related to the enzymatic production of nourzheothricin, an antibiotic belonging to the group of streptotricins. The purpose of the invention is to reduce the consumption of the stimulator while maintaining the output of the antibiotic. The essence of the method is that the strain Str. Noursei IMET JA3890 is absorbed in a complete nutrient medium containing an antibiotic stimulant — sodium azide, brencatehine, amital, zinc sulfate or 6-alanine. In this case, the stimulator can be introduced both before the inoculation of the nutrient medium and during the cultivation process until the exponential growth phase of the producer is completed. The fermentation process is carried out under aerobic conditions at 25-30 ° C for 3-10 days, and the target product is isolated from the filtrate of the culture fluid. 4 tab. with S (L

Description

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I The invention relates to the field of biotechnology and is related to the enzymatic production of noorzeotricin, an antibiotic belonging to the group of streptothricins.

Isolated by Bradler and Trumm (1963–1965) from the culture of the variant Streptomyces noursei ATCC 11455 anti-: biotic-noorzeotricin in vitro is active against gram-positive and gram-negative bacteria, as well as against microbacteria, moreover, -; lt both antiviral and teniacidal action. With regard to the chemical structure, Nourzheotricin is mainly a mixture of streptothricin F and D (Grefe et al., 1974).

The streptomycete strain, which forms the antibiotic Nourzeothricin, called otreptorayces noursei IMET LA 13890b, is stored in the collection of strains of the Central Institute of Microbiology and Experimental Therapy AN-GDR.

mainly 26-28 ° C. Deep cultivation to obtain vegetative sowing material lasts 24-72 hours, in particular 48 hours, and the main crop grows 72-144 hours.

The antibiotic is separated from the culture filtrate separated from the mycelium by adsorption to the corresponding cation exchangers, for example, Vofatite CP-300, and subsequent elution with dilute acids (Bradler and Trum, 1963; Grefe et al. 1977).

On a well-known nutrient substrate (Bradler and Trum, 1985), the strain of Streptomyces noursei IMET LA 3890 b forms only insignificant amounts of nerurothiotricin (below 100 µg / ml) by the addition of aminoaryl carboxylic acids, in particular 5-10 mM o-aminobenzoic acid, at the beginning of cultivation cultures are known

According to limestone methods (Brady-25 conditions to increase the yield

Lehr and Trom, 1963/1965; Boker and Trom, 1966; Trefe et al., 1974) is cultivated under aerobic conditions. Dp this lyophilized on

 In soil, the spore material of this strain of streptomycetes is subcultured on suitable agar media. After 6–10 days incubation at 28–30 ° C, the sporulating mycelial lawn grown in such a way is used to inoculate sterile liquid nutrient media consisting of carbon sources and

 nitrogen, as well as inorganic salts.

A: Different types of starch can be used as carbon sources. Q. Secondary exchange is rotated due to

vegetable flours, for example, potato starch, glucose and / or glycerin. In particular, soybean flour, various amino acids and / or ammonium salts are used as nitrogen sources. At the beginning of fermentation, the acidity of the medium most favorable for antibiotic formation is in the range of pH 6.0-6.7.

Deep cultivation of the microorganism Streptomyces noursei IMET lA 3890 b can be carried out in wide-necked flasks or round flat-bottomed flasks of various contents on a rocking chair, as well as in fermenters aerated with sterile air with a corrosion-resistant material mixing device with 25 fermentation 5-10 times (B-5 times) (5-10 times) (B). and Trom, 1966; Trom and Boker, 1965; Grefe et al., 1974).

o-Aminobenzoic acid and / or 0 other a min oarylcarboxylic acids (Grefe et al., 1974, 1977, 1978, 1979) cause a specific suppression of the formation of cytochrome a-type (cytochromoxidases), indirectly inhibiting the transfer of amino acids from the nutrient medium in the mycelium and oxidative deamination of amino acids in the cells of the nerzeotricinogen. Due to this

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with cellular abundance of nitrogen catabolites and, in addition, it is possible that the amino acids that serve as precursors of noorzeotricin are largely consumed by the antibiotic forcing during its growth phase. Thus, these amino acids are better available for secondary exchange during the formation of noorzeotricin, since predominantly inorganic nitrogen sources (ammonium nitrogen) are used to form biomass.

Enzymatic production of nourcingotricin using o-aminobenzoic acid significantly increases the yield of the antibiotic. However, the necessary use of large quantities of aminoarylcarbono

This is adversely affected by the cost-effectiveness of the method due to the cost of such additional substances. Thermal sterilization and, accordingly, chemical or radiation sterilization of solutions of salts of aqueous amino acrylcarboxylic acids cannot be carried out because of the decomposition of aminoaryl carboxylic acids to form TOK conversion products, for the necessary disinfection of such solutions remains only sterile filtration. Last, however, is relatively time consuming. The presence of amino-arylcarboxylic acids in the effluent of the chromatographic columns makes biological treatment of wastewater difficult.

The purpose of the invention is to reduce the consumption of the stimulator while maintaining the output of the antibiotic.

The method is carried out as follows.

The objective of the invention is the creation of a method that provides industrial production of Nourzeotricin without the use of aminoaryl carboxylic acids.

Supplements of the respiratory chain inhibitors, such as sodium azide or other alkaline azides, brancatechine, and amital (ethylisoamylbarbituric acid), and / or amino acid transfer inhibitors in a living cell, such as water-soluble zinc or beta-alanine salts, are added to the main culture in seeding time, have a similar facilitating effect on the biosynthesis of nerzeotricin, which does not change the biological properties of the fermentation product. The concentrations of one of these substances necessary for this lie in the range of 0.025–0.25 mmol, generally about 0.15 mm (sodium azide) or 0, 5–1.75 mmol, mostly 0.50, to 0.75 mmol (zinc sulphate of yl-iuZ-alanine) or 0.05-2.0 mmol, generally about 0.4, to 1.5 mm (brencatechine or amine).

For the method using the following fermentation medium containing,%: Wednesday In-30 - glucose 2.9; soybean flour, defatted 1.3, sodium chloride 0.5; calcium carbonate 0.3; tap water to obtain 100 ml, pH 6.5 (before sterilization) BO-31 medium - corn starch 3.0; glucose 0,3; soy flour is defatted

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0.7; ammonium nitrate 0.5; crystalline magnesium sulfate 0.2; sodium chloride 0.5; calcium carbonate 0.3; tap water to obtain 100 MP, pH 6.0 (before sterilization), medium W-32 — potato starch 3.2; glucose 2,9; Soya flour defatted 1,4; crystalline ammonium nitrate magnesium sulfate 0,2; sodium chloride 0.1; calcium carbonate 0.6; tap water to obtain 100 ml, pH 6.0 (before sterilization).

These media used for the producer culture provide a much higher yield of nourzeotricin as compared with the known fermentation medium.

In the presence of sodium azide and, accordingly, other alkaline azides, brencatechol, amytal, zinc sulfate and / or beta alanine, added to the nutrient medium alone or in a mixture at the time of sowing the culture, the neuroteotricin formation increases by another 2-10 times.

Naurzeotricin can be isolated from the culture filtrate by adsorption using activated carbon and cation exchangers (strongly-and weakly acidic). The preferred method is based on alkali-based adsorption of weakly acidic cation-exchange salts. Elution of the adsorbates is carried out with aqueous or alcoholic solutions of mineral acids. From the mineral-acid eluates, a pure salt of ecotricin is obtained.

Example 1. The soil-dried canals of Streptomyces noursei strain SIX IA 3890b are plated onto a suitable agar medium, such as Emerson's medium. After approximately seven days of incubation at 30 ° C, areas of the size of the mycelial lawn are cut out. 2 cm and cultured them with the culture medium of the 1st stage of enhancement of the following composition,%: glucose 4,0; soy flour defatted 1,5; 0.03; NaCl 0.5; CaCOj 0.3; tap water 100 ml; pH 6.5-6.9 (before sterilization).

The medium in quantities of 50 ml is poured into 500-ml wide-necked flasks with a capacity of 500 ml, sealed with a cotton plug. Sterilization was carried out for 35 minutes by heating to 120 ° C in an autoclave.

The inoculated cultures are incubated on a shaker for 48 hours. 3 ml of inoculum is used to inoculate the fermentation medium.

For fermentation, the following nutrient media are used,%: B-30 medium - glucose 2.9; soy flour defatted 1,3; NaCl 0.5; SASO3 tap water 100 ml; pH 6.5 (before sterilization), medium B-31 - corn starch 3.0; glucose 0.3; soy flour defatted 0.7; 0.5; MgS04 7HpO 0.2; NaCl 0.5; CaCOj 0.3; tap water 100 ml; pH 6, O (before sterilization).

Fermentation medium in quantities of 80 ml is poured into 500-ml wide-necked flasks with a capacity of 500 ml and sterilized at 120 ° C for 35 minutes in an autoclave.

At the moment of the inoculation of the pre-prepared seed culture, aqueous neutral solutions with the appropriate substances are added sterile-filtered (maximum 3.0 ml of additive / 80 MP medium).

 Using eight 24-hour 2 nd stage cultures obtained by growing on a rocking chair, 150 l of a thermosterilized medium (60 min at 125 ° C) of the described composition are seeded, see Table 1. Tables 1 and 2 show the average maximum concentrations of Nourzeotri-JQ in the noble-grade fermenter obtained during 72-100 h (total capacity 200 l), equipped with

devices for supplying sterile air and mixing device. The cultivation was carried out at 26-28 0, the speed of the mixing device was 375 rpm and the aeration coefficient was 1 liter of air and 1 liter of culture liquid / min (at normal pressure).

After 20-24 hours of incubation, 100 l of this culture are used to inoculate 600 l of fermentation.

incubating the culture with a producer using agar diffusion method using Bacillus subtilis ATCC 6633 as a test microorganism.

PRI mme R 2. Vegetative seed Str. Noursei is grown as described in Example 1. 10 ml of a 48-hour culture fluid are used to inoculate the 2nd inoculum. This medium has the same composition as the 1st stage medium and its

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environment. This medium was sterilized beforehand by 1 hour heating to 180 ° C.

environment. This medium was sterilized beforehand by 1 hour heating to 180 ° C.

sterilized in the same way.

Medium in portions of 400 ml is poured over .j. (total capacity is 1000 liters; there are devices - - .4. "-,. equipment for mixing, feeding

sterile air and temperature control).

The nutrient medium B-32 used for this has the following composition,%: potato starch 3.2; glucose 2,9; Soya flour defatted 1,4; ammonium nitrate 0.7; crystalline magnesium sulfate 0.2; sodium chloride 0.1; calcium carbonate 0.6; under-55 sun oil 0,3; crystalline zinc sulfate 0.048; tap water to obtain 100 ml., .PH 5.6V, corked, wide-necked flasks with a capacity of 2500 ml. Crops are incubated at 29 ° C for 24 h on a rocking chair. 800 ml each of the seed of the 2nd stage of cultivation produced in the 2-nd stage of cultivation is used to inoculate sterile fermentation media, filled in quantities of 20 l in pre-sterilized glass fermenters of 30 l each. For this purpose, the mediums B-30 and B-31 specified in example 1 are used, which are additionally contained 6, 1 (before sterilization).

0.3% more sunflower oil is harvested

as defoaming agent. Immediately after seeding, sterile-filtered aqueous neutral solutions are given in Table. 3 substances (maximum 200 ml of additives / 20 l of medium).

Fermentation media are incubated for 120 hours at 26–28 ° C and aeration coefficient of 20 liters of air / min (at normal pressure) with stirring (440 rpm). If necessary, sterile sunflower oil is used as a defoaming agent.

Formed during fermentation, noorzeotricin is determined daily by a microbiological method, as described in Example 1.

Ir im er 3. Vegetative seed Str. Noursei is grown in the same way and using the same nutrient medium as described in examples 1 and 2.

 Using eight 24-hour 2nd stage cultures obtained by growing on a rocking chair, 150 l of thermosterilized medium (60 min at 125 ° C) of the described composition are inoculated, filled into a fermenter of a noble steel (total capacity 200 l), equipped with

environment. This medium was sterilized beforehand by 1 hour heating to 180 ° C.

6.1 (before sterilization).

Zinc sulfate is added to the fermentation medium prior to sterilization.

Cultivation of the culture was carried out for 144 hours at 26–28 ° C, a mixing device operating speed of 290 rpm and aeration coefficient of 1 liter of air and 1 liter of culture fluid / min (pressure 0.14– 0.16 MPa).

For defoaming, sterile water-containing sunflower oil is used as necessary. Formed during the fermentation process, nourthothricin is determined daily by the microbiological method as described in Example 1.

The results are shown in Table. four.

For the supply of an antibiotic, 500 liters of culture fluid with 4000 mg / l of nerurothiotricin is baked with oxalic acid to a pH value.

100 g of white Nurzeotricin-hydrochlorination of Nourzeotricin 50 are obtained.

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4.0 and separated from the solids by separation. The filtrate is then neutralized with 10.0% sulphate water, disinfected with a 30% aqueous solution of NaOH and separated again to separate additional products of flocculation. To adsorb the antibiotic, the neutralized culture filtrate is filtered at a flow rate of 70 l / h through a column filled with 70 l of WoFitatite CP-300 (sodium form). The adsorbate is washed with 50 l of deionized water, then 0, 1 n. acetic acid to the acidity pH 5.5 channel and another 15 liters of water. Elution of the antibiotic is carried out with 50 L 1.0 n. HC1 and then with deionized water, the eluate being taken separately in two fractions: fraction I before the eluate is converted to acidic values, and fraction II as the acidic eluate.

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Claims (1)

Formula images The method of obtaining the hole by cultivating the myces noursei IMET lA 38 nutrient medium, carbon content and nitrogen salts and stimulating agent, in deep-seated fishing at 25–30 ° C for 10 days, followed by a left-hand product from the filter liquid, which differs from the fact that, with the use of an antibiotic release stimulator, 0.025–0.25 mm of sodium is used as an antibiotic stimulator; , 05-2.0 mmol b or amytal, or 0.15 zinc sulphate, this stimulator is introduced into nutrient sparingly, or during processing until the end of the growth phase of the plant The neutral fraction I (about 50 l) is evaporated to the syrup directly in vacuum at a temperature of maximum 40 ° C (about 4 l). This residue is dissolved in 60 L of methanol. After filtration of the methanol solution, only 260 l of acetone is added in portions with stirring in portions; zeotricin hydrochloride. The precipitate is filtered off, washed with 13 l of a mixture of methanol: acetone (1: 3; V: V) and then another 8 l of absolute acetone and dried in vacuum over concentrated sulfuric acid or phosphorus pentoxide. 740 g of white amorphous nourzhericin hydrochloride with 950 U / mg noorzeotricin content and 5.4% sulfate ash are obtained. The 15l Bofatite L-150 anion exchanger (OH-form) is added in portions with stirring to the hydrochloric fraction II 15l until the solution is neutral. After filtering off the ion exchanger, the filtrate is concentrated and treated as fraction I, and the qualities of the solvents used are accordingly reduced. Get 100 g of white amorphous nourzhericin-hydrochloride containing nourzeotricin 500 u / mg  10.0% sulphate water,  10.0% sulphate water, Invention Formula The method of obtaining nourcing tricin by cultivating the strain Streptomyces noursei IMET lA 3890 in a liquid nutrient medium containing sources of carbon and nitrogen, mineral salts and stimulator of antibiotic formation, in deep aerobic fishing at 25-30 ° C for 3 to 10 days, followed by the separation of the target product from the filtrate of the culture fluid, characterized in that, in order to reduce the consumption of the stimulator while maintaining the output of the antibiotic, 0.025-0.25 are used as an antibiotic formation promoter mmol of sodium azide, 0.05-2.0 mmol of brencatechol or amital, or 0.15-1.75 mmol of zinc sulphate or -alanine; in this case, the stimulator is introduced either directly into the nutrient medium before sterilization, or during cultivation to completion of the exponential growth phase of n Roducent. Note. Wednesday At-30. Table 1 one) The contents of norzeotritsina µg / ml. Without additive (control) Note. Wednesday At-31. Additive concentration, mmol Sodium azide 0.125 tserentskatehin 0,500 brencatechin 1,250 1390242 12 T a b i c a 2 620 Table3 Contents of nerzeotricin, mcg / ml W-30 I Wednesday-31 1835 169 396 13 1390242. 14 Continuation of table 3 Additive concentration, mmol Zinc Sulphate 0.750 445 No additive (control) 98 Additive concentration The content of norurseotricin, µg / ml, with a duration of cultivation, h 24 G 48 G 72 I 96 I 120 I 56 210 65,200 990 2377 530 760 Contents Nowzeriotricin, mcg / mp | Wednesday Wednesday 30 Wednesday Wednesday 1860 79.0 Table4 144 4876 5020 760 865 850