The method of biosynthesis of cephalosporin with

 

(57) Abstract:

The invention relates to the field of biotechnology and relates to a method of biosynthesis of cephalosporin C in liquid nutrient medium with the use of culture Acremonium chrysogenum. The proposed method includes the cultivation of a producer in a liquid nutrient medium containing sources of carbon, nitrogen, mineral salts, vegetable oil under conditions of aeration and mixing, regulating the concentration of ammonia nitrogen and pH of the culture fluid. As a source of polysaccharide nutrient medium contains hydrolyzed corn or potato starch, obtained by treating starch enzyme complex drug Miloserdiya with a specific activity of 600-3000 U/g Mass fraction of starch hydrolysate in the medium - 8-10% based on the starch. The process of biosynthesis lead during the first 30 hours at a temperature of 28-29°C, followed by a change in temperature and the process of biosynthesis at 24-25°C. the Process of antibioticaccutane ends at 135-140 h of growth activity in the culture fluid 22500-24000 IU/ml the use of the invention allows to increase the activity of antibioticaccutane with 20000-22000 IU/ml to 22500-24000 the ti biotechnology, in particular the production of antibiotics, and relates to a method of biosynthesis of cephalosporin C-the main raw materials for the production of 7-aminocephalosporanic acid is an intermediate in the synthesis of semi-synthetic antibiotics, such as Cefotaxime, Ceftriaxone, Cefazolin and other

Known methods of biosynthesis of cephalosporin With carried out by culturing producer Acremonium chrysogenum in a liquid nutrient medium containing sources of carbon, nitrogen and mineral salts. Carbon-containing components of the nutrient medium (primarily mono - and polysaccharides) play a crucial role in the process of antibioticaccutane, acting as structural elements and energy source.

Known methods of biosynthesis of cephalosporin C, where as the carbon sources used mainly sucrose /Pat. U.S. No. 3926973, 1973, U.S. Pat. U.S. No. 3826729, 1973, U.S. Pat. U.S. No. 3816257, 1974, U.S. Pat. Korea No. 9104371, 1991/. According to other data/Pat. Korea No. 9604037, 1993, U.S. Pat. Germany No. 4127648 C1, 1993/ as the source of carbon used mainly glucose or glucose-fructose mixture. According to U.S. Pat. U.S. No. 4533632, 1982, the main carbon source is lactose.

The disadvantage of the above methods is used for the processing of vegetable raw materials.

Known as carbon sources polysaccharides corn or potato starch, significantly reduces the environment in which carry out the biosynthesis and activates the process of antibioticaccutane of cephalosporin C /Pat. U.S. No. 3825473, 1971, Pat. Russia №1605512, 1995/.

The closest in technical essence and the achieved effect is the way of the biosynthesis of cephalosporin C, described in U.S. Pat. Russia №1605512, 1995 (prototype). According to the prototype cultivation producer Acremonium chrysogenum is carried out in a liquid nutrient medium containing, wt.%: corn starch (reducing substances) 4,5-5,5; soy flour 0,01-0,08; extract of corn 8-11; vegetable oil 0,15-0,35; ammonium sulphate at 0.7-1.4; phosphoric acid potassium one-deputizing of 0.5-0.7; magnesium sulfate 0.3 to 0.4; copper sulfate 0,0017; zinc sulfate 0,014-0,016; manganese sulfate 0,002-0,004; iron sulfate 0,003-0,006; antifoam 0,01-0,2, water the rest and when the ratio of the mass fraction of organic sources of nitrogen and the mass fraction of polysaccharides in the medium of 1:0.4 to 0.6. The fermentation process lead during the first 30 h until the set volume fraction of wet biomass 15-25% at a temperature of 27-29°C, followed by a change of pace the growth is 20000-22000 IU/ml

The disadvantages of the method on the prototype.

1. Use during cultivation in liquid nutrient medium polysaccharides corn starch or corn flour, which belong to trudnostyami sources of carbon, resulting in a large time consuming process of biosynthesis-140-150 hours

2. Not a high level of activity of a cephalosporin With in the culture fluid (20000-22000 IU/ml).

The aim of the proposed method is.

- increased activity of cephalosporin C in the culture fluid with 20000-22000 IU/ml to 22500-24000 IU/ml

- reducing the time consuming process of biosynthesis with 140-150 hours before 135-140 PM

The goal in the present method is achieved through identified in the research of cultural properties Acremonium chrysogenum to form in the process of biosynthesis of cephalosporin With high activity in liquid medium containing sources of carbon partially hydrolyzed corn or potato starch. Partially hydrolyzed starch is formed in the processing of starch complex enzyme preparation aminocoumarin (brand G3x or GH). Aminocoumarin is a complex enzyme preparation is Christmas protease - up to 10 U/g, beta glucanase - up to 250 IU/g, the cellulase is about 2 U/g, klientu - up to 10 U/G. Aminocoumarin G3x and GH standardizes on alpha-amylase (3.2.1.1.alpha-1,4-glucarpidase) up to 600 to 1000 conventional units for G3x and between 1000 to 3000 conventional units for GH. Consumption 1-2 conditional units per 1 g of starch. Hydrolysis of starch by Miloserdiya leads to the formation of mainly starch mixture over low molecular weight oligosaccharides, which provides an easier assimilation of culture data carbon components, shorten biosynthesis and increase antibioticaccutane to 22500-24000 IU/ml

The research also found that the optimal concentration of enzyme hydrolysate in the cultivation process, providing the level of activity 22500-24000 IU/ml in the culture fluid at 135-140 h is the mass fraction of the last 8-10% (based on starch). The decrease in the mass fraction of the hydrolysate in the medium leads to a decrease in the level of antibioticaccutane less 22500 IU/ml Increase in the mass fraction of the hydrolyzate more than 10% leads to an unreasonable increase in the cost of the nutrient medium without additional growth activity and does not exceed the above level 24000 IU/ml

Distinctive and liquid nutrient medium hydrolysates of corn or potato starch, obtained by treating starch enzyme complex drug Miloserdiya.

2. Use in the cultivation of culture Acremonium chrysogenum starch hydrolysates with a mass fraction in the medium 8-10% (based on starch).

The inventive method provides

1. The reduction in the duration of the process of biosynthesis with 140-150 hours before 135-140 PM

2. The increased activity of the culture fluid with 20000-22000 IU/ml to 22500-24000 IU/ml

3. By reducing processing time and increasing the activity achieved by reducing energy costs and lowering the cost of the finished product.

The following examples illustrate the invention.

EXAMPLE 1

stage 1 (receipt of enzymatic hydrolysate of starch).

In the apparatus for cultivating culture Acremonium chrysogenum with a capacity of 3.0 m3pour half of the calculated amount of water, loaded with stirring to 120 kg of corn or potato starch. Then add when mixing calcium chloride based 0.7 g per 1 kg of starch (calculated on the anhydrous product). Make complex enzymatic preparation of aminocoumarin G3x with specific activity of the CT). The temperature in the apparatus was raised to (70±5)°C and maintained at this temperature for 30 minutes the temperature was Then rapidly increased to 110°C and incubated for 3-5 minutes to inactivate the enzyme. Get the enzymatic starch hydrolysis. The resulting hydrolyzate is used in the preparation of the nutrient medium.

stage 2 (preparation of the nutrient medium and maintaining the process of biosynthesis).

Received on the 1st stage, the hydrolysate is cooled to 60°C load all the necessary components and brought to a volume of 1500 l of water. Mass fraction of loaded components,%:

1. The enzyme hydrolyzed corn

(potato) starch

(based on starch) 8,0

2. Corn extract 8,0

3. Soy flour 0,085

4. Glucose 0,5

5. Chalk 1,0

6. Ammonium sulfate 1,7

7. Vegetable oil 0,3

8. Potassium phosphate

one-deputizing 0,7

9. Trace elements (total) 0,715

10. Water Up to 1500 l

After sterilization, the medium in the apparatus with a nutrient medium is passed vegetative seed culture Acremonium chrysogenum, having a pH value of 7.0 pH and volume of Reda.

The cultivation process is conducted at the following parameters: temperature (28±1)°With from 0 to 35 h, then till the end of the process (25±1)°C. Aeration and peremeshivanie exercise at maximum intensity breathing, maintaining a positive pressure device (0,06±0,01) MPa and the partial pressure of dissolved oxygen not less than 30% of saturation. The pH value is maintained at the level (6,0±0,1) pH by dosing the ammonia water solution of ammonium sulfate and vegetable oil. Dosing of a solution of ammonium sulfate carried out so that the mass fraction of ammonium nitrogen was in the range (0,05±0,1)%. Dosing vegetable oil is carried out so that the partial pressure of dissolved oxygen was below 30% of saturation. After 140 h of cultivation activity of the culture liquid is 22500 IU/ml

EXAMPLE 2

stage 1 (receipt of enzymatic hydrolysate of starch).

In the apparatus for cultivating culture Acremonium chrysogenum with a capacity of 3.0 m3pour half of the calculated amount of water, loaded with stirring to 150 kg of corn or potato starch. Then add when mixing calcium chloride based 0.7 g per 1 kg of the collapse of eskay activity of 1000 U/g or GH with a specific activity of 2000 U/g at the rate of 2 Units/g starch (calculated on the anhydrous product). The temperature in the apparatus was raised to (70±5)°C and maintained at this temperature for 30 minutes the temperature was Then rapidly increased to 110°C and incubated for 3-5 minutes to inactivate the enzyme. Get the enzymatic starch hydrolysis. The resulting hydrolyzate is used in the preparation of the nutrient medium.

stage 2 (preparation of the nutrient medium and maintaining the process of biosynthesis).

Received on the 1st stage, the hydrolysate is cooled to 60°C load all the necessary components and brought to a volume of 1500 l of water. Mass fraction of loaded components, %:

1. The enzyme hydrolyzed corn

(potato) starch

(based on starch) 10,0

2. Corn extract 8,0

3. Soy flour 0,085

4. Glucose 0,5

5. Chalk 1,0

6. Ammonium sulfate 1,7

7. Vegetable oil 0,3

8. Potassium phosphate one-deputizing 0,7

9. Trace elements (total) 0,715

10. Water Up to 1500 l

After sterilization, the medium in the apparatus with a nutrient medium is passed vegetative seed culture Acremonium chrysogenum, having a pH value of 7.2 pH and volume fraction Mois is SS="ptx2">The cultivation process is conducted at the following parameters: temperature (28±1)°With from 0 to 35 h, then till the end of the process (25±1)°C. Aeration and stirring is carried out at maximum intensity breathing, maintaining a positive pressure device (0,06±0,01) MPa and the partial pressure of dissolved oxygen not less than 30% of saturation. The pH value is maintained at the level (6,0±0,1) pH by dosing the ammonia water solution of ammonium sulfate and vegetable oil. Dosing of a solution of ammonium sulfate carried out so that the mass fraction of ammonium nitrogen was in the range (0,05±0,1)%. Dosing vegetable oil is carried out so that the partial pressure of dissolved oxygen was below 30% of saturation. Over 135 hours of cultivation activity of the culture liquid is 24000 IU/ml

EXAMPLE 3.

stage 1 (receipt of enzymatic hydrolysate of starch).

In a device for cultivation of Acremonium chrysogenum with a capacity of 3.0 m3pour half of the calculated amount of water, loaded with stirring to 135 kg of corn or potato starch. Then add when mixing calcium chloride based 0.7 g per 1 kg of starch (in terms of ¢ 800 U/g or GH with a specific activity of 2500 U/g at the rate of 2 Units/g starch (calculated on the anhydrous product). The temperature in the apparatus was raised to (70±5)°C and maintained at this temperature for 30 minutes the temperature was Then rapidly increased to 110°C and incubated for 3-5 minutes to inactivate the enzyme. Get the enzymatic starch hydrolysis. The resulting hydrolyzate is used in the preparation of the nutrient medium.

stage 2 (preparation of the nutrient medium and maintaining the process of biosynthesis).

Received on the 1st stage, the hydrolysate is cooled to 60°C load all the necessary components and brought to a volume of 1500 l of water. Mass fraction of loaded components, %:

1. The enzyme hydrolyzed corn

(potato) starch

(based on starch) 9,0

2. Corn extract 8,0

3. Soy flour 0,085

4. Glucose 0,5

5. Chalk 1,0

6. Ammonium sulfate 1,7

7. Vegetable oil 0,3

8. Potassium phosphate one-deputizing 0,7

9. Trace elements (total) 0,715

10. Water Up to 1500 l

After sterilization, the medium in the apparatus with a nutrient medium is passed vegetative seed culture Acremoniurn chrysogenum, having a pH value of 7.2 pH and volume fraction Mois is SS="ptx2">The cultivation process is conducted at the following parameters: temperature (28±1)°With from 0 to 35 h, then till the end of the process (25±1)°C. Aeration and stirring is carried out at maximum intensity breathing, maintaining a positive pressure device (0,06±0,01) MPa and the partial pressure of dissolved oxygen not less than 30% of saturation. The pH value is maintained at the level (6,0±0,1) pH by dosing the ammonia water solution of ammonium sulfate and vegetable oil. Dosing of a solution of ammonium sulfate carried out so that the mass fraction of ammonium nitrogen was in the range (0,05±0,1)%. Dosing vegetable oil is carried out so that the partial pressure of dissolved oxygen was below 30% of saturation. After 138 h of cultivation activity of the culture liquid is 23200 IU/ml

The method of biosynthesis of cephalosporin C by culturing producer Acremonium chrysogenum in a liquid nutrient medium containing a source of polysaccharide, corn extract, soy flour, ammonium sulphate, potassium phosphate one-deputizing, magnesium sulfate, chalk, copper sulfate, zinc sulfate, manganese sulfate, iron sulfate, vegetable oil and water, in Usmani temperature, wherein the cultivation is carried out in a nutrient medium containing a source of hydrolyzed polysaccharide complex enzyme drug Miloserdiya, the specific activity of 600-3000 conditional U/g of corn or potato starch, while the mass fraction of starch hydrolysate in the medium is 8-10%, calculated on the starch.



 

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