RU1264579C - Method of producing 5'-inosine acid - Google Patents

Description

The invention relates to the microbiological industry, in particular the production of compounds related to nucleic acids, and concerns a method for producing 5-nosioic acid (5-IMP) by microbiological synthesis.

5-IMP is a valuable reagent used in various fields of experimental biology, serves as a starting compound for the synthesis of anomalous purine derivatives used as anticancer drugs, and also finds application in the food industry as a highly effective flavoring seasoning.

The aim of the invention is to increase the yield of the target product.

The strain used in the proposed method is Brevibacterlum ammonfagenes 8I2 Genetics-107 obtained from strain Br. ammoniagenes VNIIgenetics-225-5 directional by breeding. It differs from the parent strain in its mercury resistance to mercaptopurine riboside and has an elevated 5-1LMP biosynthesis level.

Cultural and morphological characteristics of the strain VNIIgenetika-.107. Gram-positive wand, does not form a dispute. On agar medium Hottinger and m septon agar forms colonies of round shape, smooth, with a smooth edge, pale yellow color, with a diameter of 1.0-1.5 mm. The strain is required for growth of vadenine. The strain used in the described method is deposited in the Central Museum of Cultures of Industrial Microorganisms of the Institute of Scientific Research Institute of Genetics and has the registration number CMPM-B-3172.

The nutrient medium in the proposed method contains monosubstituted phosphate potassium phosphate as a source of phospholate instead of a mixture of potassium phosphate monosubstituted and disubstituted, which eliminates the additional operation of acidification of the medium before sterilization.

In the described method, under optimal fermentation conditions, the level of accumulation of 5-IMP reaches 16.3-26.8 g / l in 144 hours of fermentation, compared with 10.3 g / l in the known method. At the same time, the rate of accumulation of 5-IMP is 0.18 g / l.h, and in the prototype, 0.07 g / l.h, the economic utilization of glucose is 19.1% instead of 10.3% in the prototype. In this connection, despite a certain increase in the cost of the medium, associated with an increase in the concentration of its components, the advantages of the proposed method are not reduced.

The method is carried out as follows.

The culture of strain Br. ammoniagenes are grown on Hottinger's agar medium for 24 hours at 28 ° C, transferred to flasks with inoculated medium, and grown with constant stirring and aeration for 18-24 hours at 28-30 ° C. Then the seed is transferred to the fermentation medium in an amount of 10 vol.%. The fermentation process is carried out in flasks on a rocking chair (240-350 rpm) with aeration and stirring, providing an oxygen dissolution rate of 2.6-3.8 g of Ol-h. The accumulation of 5-IMP in the culture fluid reaches 16.3 26.8 g / l for 144 hours of fermentation.

L i eme r 1. Culture of the strain Br. ammoniagenes ВНИМгенетика-107 from Hottinger's beveled agar is transferred to a liquid inoculum of the following composition, wt%:

Technical Glucose 2.00

Peptone1,00

Yeast Extract 1.00

NaCt.0.25

WaterEverything

Seed material is grown in Erlenmeyer flasks with a capacity of 750 ml, containing 75 ml of medium, at a rocking chair making 240 rpm, for 24 hours at 28-30 ° C. Inoculant nt is transferred in an amount of 10 vol.% To the fermentation culture medium of the following composition, wt. .%: Technical glucose 15.0 Corn extract (EC) 6.0 KH2P043.0

MgS041.0

Urea. 0.84

WaterEverything

The medium is sterilized at 0.8 atm for 30 minutes. Urea is sterilized separately in the form of a 40% solution at 0.5 atm for 30 minutes. After sterilization, the pH of the fermentation medium is adjusted with 5 N KOH solution to 7.5 and the urea solution is added.

The fermentation process is carried out in 750-ml Erlenmeira flasks, containing 30 ML of medium, on a rocking chair at 350 rpm, which, with aeration and stirring, corresponds to an oxygen dissolution rate of 3.8 g Oa / l-h at 35 ° C. After 144 hours of fermentation, 26.8 g / l of 5-IMP accumulates in the culture fluid.

Examples of the method with other parameters, i.e. on the environments of another

composition and under other modes of cultivation are shown in table

Accumulation of IMP depending on the composition of the medium

As can be seen from the table, the maximum level of accumulation of IMP 26.8 g / l is achieved using nutrient medium containing 15% glucose, 6% corn extract and 0.84% urea. A decrease in the concentration of these components leads to a significant inhibition of the accumulation of IMP. The increase in the termination of the medium is inefficient and unprofitable, since this leads to its appreciation and to a decrease in the yield of the target product.

The maximum yield of IMP on the medium of optimal composition is achieved under the conditions of an intensive supply of oxygen to the medium and at a certain, rather narrow temperature range.

Figure 1 shows the dependence of IMP accumulation on aeration conditions; Fig. 2 shows the dependence of IMP accumulation on cultivation temperature.

As can be seen from .fig. 1, the highest yield of IMP is observed with aeration, corresponding to an oxygen dissolution rate of 2.6-3.8 g 02 / lh. Increasing the degree of aerov1 above this level is not possible for technical reasons.

As can be seen from figure 2, the most active formation of IMP occurs at 3235С. Reducing the temperature to 28 ° C causes a decrease in the yield of IMP. Increasing the temperature to 37 ° С sharply inhibits the process of biosynthesis.

(56) Patent of France No. 1465166, class, C 12 R 19/30, published. 1970. 25 USSR Author's Certificate No. 615130. cl. C 12 R 19/30, 1975.

Claims (1)

Invention Formula CnOCQB of production of b-inosinic acid, including the cultivation of microorganisms - producers of the species Brevibacterlum ammontagenes on an aqueous nutrient medium containing a source of carbon, nitrogen, mineral salts and growth factors under aeration conditions, isolation and purification, characterized in that, in order to increase output the target product, as a producer use strain Brevibacterium 30 ammonlagene s All-Russian Research Institute of Genetics-107, and the nutrient medium contains as a carbon source technical glucose in an amount of 12-15 wt.%, As a nitrogen source - urea in a quantity of 0.72 - 0.84 wt.%, As a source growth factors - corn extract in the amount of 5-6 wt.%, while the cultivation is carried out at 32 - 35 ° C, and aeration is carried out with the dissolution rate of oxygen 2.6 - 3.8 g 02 / l -h. 0.05-0.5 wt.% Palladium.