Method of cephalosporin c biosynthesis with using new acremonium chrysogenum strain, rncm no f-4081d

FIELD: medicine.

SUBSTANCE: there is offered a method of preparing the antibiotic Cephalosporin C. A new antibiotic producer Acremonium chrysogenum strain, RNCM No. F-4081D is used. Acremonium chrysogenum, RNCM No.F-4081D is cultivated on an enzymatic nutrient medium containing carbohydrate sources - glucose, corn starch, dextrin, nitrogen sources - a corn extract, Pharmamedia, ammonium sulphate, and also inorganic salts - potassium phosphate, potassium sulphate, chalk, copper sulphate, zinc sulphate, manganese sulphate, ferrous sulphate, and as vegetable fat - rapeseed oil and in addition a phosphatidylcholine and/or sitosterol.

EFFECT: twice increased antibiotic production level, reduced fermentation time.

1 tbl, 7 ex

 

The invention relates to biotechnology and can be used in the pharmaceutical industry. The technical result of the invention is to increase the level of antibiotic activity, reducing fermentation time and a reduction in the content of the precursor in the culture fluid, which is achieved by using a new strain - producer of cyclosporine With Acremonium chrysogenum BKM F-4081D, new technology biosynthesis of the antibiotic and leads in General to improvements in the profitability of industrial production of the substance of cephalosporin C.

Filamentous fungi Acremonuim chrysogenum are producers of β-lactam antibiotic cephalosporin C. Natural cephalosporin With has weak antibacterial activity, but it is a starting material for chemical synthesis of various medicinal substances with a broad spectrum antibacterial action and more resistant to β-lactamase than penicillins. Because of this β-lactam antibiotics, cephalosporin widely used in antimicrobial chemotherapy (1).

The microbiological process of formation of cephalosporin C by using a fungus-producer of the genus Acremonium widely studied and documented. Known for the ability of different strains of Acremonium chrysogenum to the biosynthesis of cephalosporin C. One of the closest is a strain of Acremonium chrsogenum 12/9.

Cultivation of the fungus Acremonium chrysogenum carried out on nutrient media containing various carbon sources, nitrogen, inorganic salts, amino acids, fats in conditions of intensive aeration and mixing. To study the influence of the composition of the nutrient media devoted a large number of studies (2, 3).

The formation of a cephalosporin With many strains of Acremonium chrysogenum is stimulated by adding in the environment of the cultivation of the amino acid methionine. One of the mechanisms of this effect on the synthesis of cephalosporin C is the role of methionine in the process of differentiation culture (4). The formation of fragmented mycelium cell type arthropod in the presence of methionine is the determining factor in the development of high-yielding crops. However, the addition of methionine to the culture medium increases the cost of the process and adversely affects the ecology of the environment by extracting mercaptan.

Closest to the claimed is a method of producing a cephalosporin C using culture Acremonium chrysogenum under cultivation producer in the liquid nutrient medium in the absence of methionine. The process of antibioticaccutane ends at 140 hours of growth activity in the culture fluid 24000 mg/ml (5). The disadvantage of this process is the duration of the fermentation at a relatively low level of activity and the culture fluid.

The task that is provided by the present invention consists in the intensification of the process of biosynthesis of cephalosporin C by use of a new strain of Acremonium chrysogenum BKM F-4081D and optimization of nutrient media under conditions of submerged cultivation. The essence of the invention lies in the fact that according to the method of microbiological synthesis of cephalosporin use With a new strain of Acremonium chrysogenum BKM F-4081D, and in the composition of nutrient media using new compounds of different chemical nature.

A new strain of Acremonium chrysogenum BKM F-4081D differs from the strain of Acremonium chrysogenum 12/9 sensitive to methionine, slow growth on solid nutrient media, accelerated biosynthesis of cephalosporin C in the cultivation of deep cultivation conditions and a higher level of antibioticaccutane. The strain of Acremonium chrysogenum BKM F-4081D culture is characterized by the following morphological characteristics:

on the medium containing bacto-peptone 10 g/l, maltose 40 g/l, malt-dry extract 20 g/l at 15 days of growth is the colony diameter of 0.7 mm, the Colonies are convex, conical, irregularly folded, the surface of young colonies needle, ivory or pink pigment in the environment do not produce aerial mycelium very poorly defined. Prototroph, strict aerobe, optimum temperature for growth on AG the global environment - 24-28°C. the Visible growth of colonies appears on the 8 days of incubation at 28°C. In the above medium with addition of 0.1% methionine for 15 days of growth, the diameter of the colonies in the middle of 0.9 mm, the Colonies are convex, slightly irregularly folded, light beige, aerial mycelium is virtually absent of pigment in the environment does not emit (table 1).

Table 1
Comparison of growth rate and biomass accumulation of strains 12/9 (parent strain) and VKM F-4081D (new strain)
StrainThe appearance of a visible colony growth on medium without methionine (day)Diameter (mm) colonies on agar medium for 15 days of growthDry biomass, fermentation in flasks (g/ml)
Medium without methionineMedium with methionineAt the seed environment, 48 hoursIn a fermentation medium, 120 hour
without methioninewith methionine
12/9 (control)4 a 3.92,70,04740,11760,1206
VKM F-4081D (new strain)82.80,90,03140,10870,1012

The specified strain obtained by multi-stage selection on media containing low concentrations of methionine: 0,3-0,01%. The strain of Acremonium chrysogenum deposited in the all-Russian collection of microorganisms (VKM) number F-4081D.

The biosynthesis of cephalosporin C, using a new strain of Acremonium chrysogenum VKM F-4081D conduct fermentation nutrient medium containing sources of carbohydrates such as glucose, corn starch, dextrin, and sources of nitrogen, such as corn extract, Pharmamedia, ammonium sulfate. Nutrient medium also contains inorganic salts such as potassium phosphate, magnesium sulfate, trace elements, and differ from those in the prototype (5) the fact that the quality of vegetable oil contains rapeseed oil. In addition, on Wednesday introduced complex organic compounds such as phospholipids and sitosterol. Phospholipids are a source of metabolic fuel, along with polyunsaturated fatty KIS is otami part of the rapeseed oil. Sitosterol, added to the fermentation medium, contributes to early differentiation of the mycelium and the formation of anthroposophically cells with shortened stage of germination hyphae. It is of great importance in industrial production since the early education of arthropod and quick change of generations improves the rheological properties of the culture fluid, primarily the viscosity, which is important for biochemical processes in the stage of fermentation and allows you to finish biosynthesis at an earlier time upon reaching a high level of antibioticaccutane. The cultivation is carried out in flasks with stirring at a temperature of 24-28°C or in the fermenter with a capacity of 20 l in conditions of controlled fermentation. The activity of the culture liquid in the unregulated process in flasks 15000-17000 µg/ml at 115-120 hour, during culturing in the fermenter 32000-34000 µg/ml 106-110 hours. The number of cyclosporine determined by high performance liquid chromatography (HPLC).

The advantages of the proposed method are:

1. A new strain of Acremonium chrysogenum BKM F-4081D with low level of education of the mycelium in the surface and deep growth conditions.

2. The increase antibioticaccutane compared to the prototype when unregulated biosynthesis of 2 RA is a, and in terms of regulation 40%.

3. The reduction in fermentation time by 20%.

4. The intermediate product desacetylrifabutin is formed With not more than 5%.

The following examples illustrate antibiotikogrammou the ability of a strain of Acremonium chrysogenum BKM F-4081D.

Example 1

Culture of a strain of Acremonium chrysogenum BKM F-4081D wash with agar medium, the suspension disintegrate, making the Erlenmeyer flask with a capacity of 0.75 liters, containing 0,04 l seed medium of the following composition, g/l:

yeast extract28,0
peptone (meat)10,0
malt-extract28,3
chalk4,0
rapeseed oil3,0 (included in each flask separately)
distilled waterthe rest of it.

The pH was adjusted before sterilization to 7.0, and 7.1 conc. NaOH.

The AMF inoculum is grown for 48 hours at 28°C on a rocking chair, making 230-240 rpm grown In seed mycelium determine the amount of biomass by centrifuging at 3000 rpm for 15 minutes Biomass is 28-30%vol.

The AMF inoculum in the number 15 is B.% contribute to the Erlenmeyer flask with a capacity of 0.75 l containing 0,04 l nutrient medium of the following composition, g/l:

corn extract93,0
corn starch32,0
Pharmamedia12
dextrin43,0
glucose4,0
Caso311,0
(NH4)2SO413,5
MgSO43,5
KH2PO44,0
CuSO4·5H2O0,017
ZnSO4·7H2O0,16
MnSO4·7H2O0,03
FeSO4·7H2O0,08
rapeseed oil30,0 (included in each flask separately)
distilled waterrest

The pH value is brought to sterilization solution of concentrated NaOH to 6.0 to 6.2.

The fermentation is carried out in a pie rocking chair, making 230-240 rpm, the first days at 28°C, then until the end of fermentation at 24°C. the Content of cephalosporin C in the culture fluid at 115 hours is 15500 ug/ml

Example # 2

For growing inoculum use the medium of the following composition, g/l:

Sucrose32,0
meat extractof 17.0
corn extract6,0
rapeseed oil3,0 (included in each flask separately)
distilled waterrest

The pH value of 7.0 prior to sterilization.

The AMF inoculum is grown for 48 hours at 28°C on a rocking chair with 230-240 rpm the Amount of biomass at 48 hours growth is 28-29%. The fermentation is carried out as indicated in example No. 1. The activity of the culture liquid at 120 hours 15000 mg/ml

Example # 3

The AMF inoculum culture Acremonium chrysogenum BKM F-4081D grow, observing the conditions of cultivation and the sowing medium as in example No. 1.

The AMF inoculum in the amount of 15% making the enzyme is operating medium of the following composition, g/l:

corn extract105,0
corn starch38,0
Pharmamedia16,0
dextrin42,0
glucose4,5
Caso311,0
sitosterol0,1
(NH4)2SO413,5
MgSO43,5
KH2PO44,0
CuSO4·5H2O0,017
ZnSO4·7H2O0,16
MnSO4·7H2O0,03
FeSO4·7H2O0,08
rapeseed oil35,0 (included in each flask separately)
distilled water rest

The pH value is brought to sterilization solution of concentrated NaOH to 6.0 to 6.2.

The fermentation is carried out according to example No. 1. The activity of the culture liquid at 115 hours growth is 16000 µg/ml.

Example No. 4

Growing inoculum culture Acremonium chrysogenum BKM F-4081D and fermentation carried out as in example 1. To use fermentation medium of the following composition, g/l:

corn extract105,0
corn starch38,0
Pharmamedia16,0
dextrin43,0
glucose4,0
Caso311,0
phosphatidylcholine1,0
(NH4)2SO413,5
MgSO43,5
KH2PO44,0
CuSO4·5H2O0,017
ZnSO4/sub> ·7H2O0,16
MnSO4·7H2O0,03
FeSO4·7H2O0,08
rapeseed oil35,0 (included in each flask separately)
distilled waterrest

The pH value is brought to sterilization solution of concentrated NaOH to 6.0 to 6.2.

The fermentation is carried out according to example No. 1. The activity of the culture liquid at 115 hours growth is 16600 mg/ml

Example No. 5

The method of fermentation is carried out according to example No. 4 containing phosphatidylcholine 2 g/l and rapeseed oil 40 g/l activity of the culture liquid at 120 hours fermentation 16400 mg/ml

Example No. 6

Culture Acremonium chrysogenum BKM F-4081D for the planting of a fermentation medium gain according to example No. 1. The fermentation is carried out as in example No. 1, the following environment, g/l:

corn extract105,0
corn starch38,0
Pharmamedia16,0
dextri the 43,0
glucose4,0
Caso311,0
phosphatidylcholine1,0
sitosterol0,1
(NH4)2SO413,5
MgSO43,5
KH2PO44,0
CuSO4·5H2O0,017
ZnSO4·7H2O0,16
MnSO4·7H2O0,03
FeSO4·7H2O0,08
rapeseed oil40,0 (included in each flask separately)
distilled waterrest

The pH value is brought to sterilization solution of concentrated NaOH to 6.0 to 6.2.

The content of cephalosporin C in the culture fluid at 115 hours of fermentation is 17000 ug/ml Contents deacetylcephalosporin is With is 4.8%.

Example No. 7

Vegetative planting material culture Acremonium chrysogenum VKM F-4081D transfer in the amount of 15% vol. in the fermenter with a capacity of 20 l containing 12 l of fermentation medium, the composition specified in example # 1. The cultivation process is carried out at the following mode: temperature 28°C from the beginning of cultivation up to 30 hours and then 24°C until the end of fermentation. Input power of 4.5 kW/hour. When foaming add the antifoam propanol B-400. Aeration and stirring is carried out so as to maintain the maximum intensity of breathing with excessive air pressure (0,04±0,01 MPa) and partial pressure of dissolved oxygen not less than 30% of saturation. The pH value at the level of 5.7±0.2 to support dosing ammonia water and 12%solutions of NaOH, (NH4)2SO4and dosing of rapeseed oil. When this mass fraction of ammonia nitrogen should be in the range of 0.04 to 0.1%, and the layer of oil on the surface when determining wet biomass by centrifuging at 3000 rpm for 15 minutes did not exceed 1 mm oil Consumption 9,06% of the volume of the loaded environment.

Over 110 hours of cultivation activity in the culture fluid, determined by HPLC, is 32000 ug/ml. Deacetylcephalosporin - 4,8%.

Example No. 8

Vegetative planting material Acremonium chrysogenum BKM F-4081D passed in number 15 on the.% in the fermenter with a capacity of 20 l, containing 12 l of fermentation medium, the composition specified in example # 6. The cultivation process is carried out at the following mode: temperature 28°C from the beginning of cultivation up to 25 hours, and then 24°C until the end of fermentation. The value of pH, the mass fraction of ammonia nitrogen, rapeseed oil and the partial pressure of dissolved oxygen is maintained within the limits specified in example # 7. The oil consumption of 8.0%. The content of cyclosporine With 106 hours fermentation is 34000 ug/ml, deacetylcephalosporin With of 4.1%.

Sources of information

1. R.P. Elander Appl. Environ. Biotechnol. 2003,v61, No. 5-6, p.385-392.

2. Shen Y-Q, Heim J., Solomon, N.A., Demain A.L. The journal of Antibiotics. May 1984, v.37, n 5, p.503-511.

3. Demain A.L., Vaishnar P. Critical Reviews in Biotechnology, 2006, v 26, n 2, page 67-82.

4. S.W. Queener, L.F. Ellis Canadian Journal of Microbiology. 1975, v 21, n 12, p.1981-1996.

5. RF patent "Method of biosynthesis of cephalosporin C, No. 2241038, November 27, 2004

The method of biosynthesis of cephalosporin C by cultivating his producer Acremonium chrysogenum on fermentation nutrient medium containing sources of carbohydrates - glucose, corn starch, dextrin, nitrogen sources - corn extract, Pharmamedia, ammonium sulfate, and inorganic salt - phosphoric acid potassium, sulfur, acidic potassium, chalk, sulfuric acid copper, sulfuric acid zinc, sulfuric acid manganese, sulfuric acid iron, characterized in that as the producer of antibiot is ka use a strain of Acremonium chrysogenum BKM F-4081D, as fermentation medium as vegetable fat contains rapeseed oil, and optionally, phosphatidylcholine and/or sitosterol.



 

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