SI9300657A - Process for the lovastatine preparation with aspergillus terreus - Google Patents

Abstract

Prepare a solid fermentation substrate containing 40 to 60% w / w of cereal grains, 20 to 30% w / w soybean meal, 10 to 20% w / w dry apple cider, 7.5 to 15% w / w of wheat bran, 4 to 8% w / w sucrose, 2 to 6% mass / mass of glycerol, 1 to 4% by weight of dry whey, 1 up to 2% w / w NH4NO3, 0.1 to 0.6% w / w (NH4) 2SO4,1.0 to 2.0% w / w corn syrup and 1 ^ 1.0 to 2.0% w / w beer yeast, substrate sterilized in a horizontal mixing reactor at temperature from 100 to 120 degrees Celsius for 30 to 90 degrees minutes, the sterilized substrate is dried to a humidity of 30 to 45% and at the same time cool to 30 to 40 degrees Celsius, the substrate is then inoculated with Aspergillus terreus and ferment at 30 to 40 degrees Celsius for 150 to 200 hours at times stirring for a maximum of 10 minutes / day. The process is technical and energy simple.

Description

Process for the production of lovastatin by the fungus Aspergillus teireus

The present invention relates to a novel process for the production of lovastatin. It provides a technically and energetically simpler solution for the production of lovastatin by Aspergillus teireus than the previously known submerged process, since it is first performed on a solid medium.

Lovastatin is a generic name for a compound that is chemically (according to The Merck Index, Eleventh Edition, 1989) defined as:

1,2,3,7,8,8a-Hexahydro-3,7-dimethyl-8- [2- (tetrahydro-4-hydroxy-6-oxo-2H-pyran-2yl) ethyl] -1-naphthalenyl ester [ lS- [1a (R *), 3a, Ίβ, 8 /? (2S *, 4S *), 8a / 3]] - 2-methylbutanoic acid; or (1S, 3R, 7S, 8S, 8aR) -1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8- [2 - [(2R, 4R) -tetrahydro-4hydroxy-6 -oxo-2H-pyran-2-yl] ethyl] -1-naphthalenyl (S) -2-methylbutyrate; or δ-lactone 1,2,6,7,8,8a-hexahydro-0,0-dihydroxy-2,6-dimethyl-8- (2-methyl-1-oxobutoxy) -1naphthalenheptanoic acid; or lactone 2 / 3,6a-dimethyl-8α- (2-methyl-1-oxobutoxy) -metinic acid.

Lovastatin is a group of drugs that effectively lowers blood cholesterol levels at relatively low concentrations. Increased blood cholesterol levels are a major risk factor in the development of arteriosclerosis and heart disease. Clinical studies have shown that lovastatin lowers LDL (low density lipoproteins) levels in people who have primary hypercholesterolemia or heterozygous familial hypercholesterolemia. Lovastatin also lowers blood plasma cholesterol levels in people with normal blood cholesterol levels (JAToberts, GDBell, J.Birtvvell, I.James, WRKukovetz, JSPryor, A.Buntinx, I.Holmes, J.Clin.Invest , vol. 69, 1982).

Lovastatin and its derivatives also have strong fungicidal properties. They are used to control species of the genus Penicillium sp., Aspergillus niger, Cladosporium sp., Cochliobolus miyabeanus, Helminthosporium cynodontis. In the fungicide preparations for spraying or dusting the plant, the active component is mixed with suitable agents, emulsifiers and solvents (EP 0 033 536 A2 Merck Co.).

Lovastatin specifically inhibits hydroxy-methyl-glutaryl coenzyme A reductase (HMGCoA reductase), thereby affecting plant growth, pigment accumulation, and plant sterol formation (T. J. Bach, H. K. Lichtenthaler, Naturforsch., 37c, 46-50, 1982). Lovastatin is produced in the body by the biosynthesis of two polyketide chains that are synthesized from acetate units (M.Shiao, H.Don, Proc. Natl. Sci. Counc. B. ROC, vol.11, 3, 223-231, 1987).

The main skeleton is formed by nine acetate units, which form a polyketide chain of eighteen carbon atoms. The butyrate substituent is formed from two acetate units that form a polyketide chain with four carbon atoms (A.Endo, The Journal of Antibiotics, vol.32, 852-854, 1979). The construction of hexahydronaphthalene is followed by enzymatic hydroxylation and methyl butyration on C-10 (M.Shiao, H.Don, Proc. Natl. Sci. Counc. B. ROC, vol.ll, 3, 223-231, 1987).

Lovastatin inhibits mevalonic acid biosynthesis by inhibiting microsomal 3-hydroxy-3methylglutaryl coenzyme A reductase (HMG-CoA reductase). Mevinolinic acid, an active form of lovastatin, acts as a competitive inhibitor of HMG-CoA reductase. This results in inhibition of the synthesis of mevalonic acid and thus of cholesterol.

Many microorganisms secrete lovastatin in small quantities as a product of secondary metabolism. However, for a particular strain to be used successfully in the industry, it is necessary to produce as much as possible of lovastatin per unit time and substrate, to eliminate unwanted substances and to produce genetically stable.

In 1980, Monaghan and colleagues discovered a microorganism that produced potent inhibitors of cholesterol biosynthesis in mammals. Taxonomic studies have classified this microorganism as Aspergillus teireus. They also described a similar strain and stored both cultures in the American Type Culture Collection, Rockville, Maryland bearing ATCC 20541 and ATCC 20542.

Previous publications refer exclusively to submerged fermentation of fungus on chemically defined and undefined substrates.

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However, we have now found that it is technically and energetically simpler to produce lovastatin in good yields according to a new process according to the invention in which:

prepare a solid fermentation substrate containing 40 to 60% by weight of cereal grains, 10 to 30% by weight of soybean meal, 10 to 20% by weight of dried apple marc,

7.5 to 15% w / w wheat bran, 4 to 8% w / w sucrose, 2 to 6% w / w glycerol, 1 to 4% w / w dry whey, 1 to 2% w / w NH4NO3, 0.1 to 0.6 % w / w (NH ^ SOzj, 1.0 to 2.0% w / w corn syrup and 1.0 to 2.0 w / w beer yeast, the substrate is sterilized at 100 to 120 ° C for 30 to 90 minutes, the sterilized substrate is dried to humidity 30 to 45% and at the same time cooled to 30 to 40 ° C, then inoculate the substrate with Aspergillus leireus and ferment at 30 to 40 ° C for 150 to 200 hours with occasional stirring for a maximum of 10 minutes / day. undesirable because it causes kneading and agglomeration of the substrate, but occasional mixing allows the formation of a larger contact surface for the growth of the microorganism.

The sterilization of the substrate is preferably carried out in a horizontal mixing reactor.

After fermentation is complete, the substrate is extracted with methanol and then filtration separates the substrate with mycelium from the filtrate containing the product lovastatin.

The substrate can be inoculated with the liquid inoculum according to options 1 or 2, which are listed below.

1. Option

For the inoculum, ghvo Aspergillus teireus (MZK1BK A 157) was used at a concentration of 2 to 6, preferably 4x10 ⁸ spores / 1 in 2000 ml of sterile water.

Option 2

In medium A, the culture was shaken for 24 hours at 30 ° C (303 K) and 100 rpm. Medium A contains 1 liter per liter: 5 g corn syrup, 40 g tomato meal, 12 g glucose and 10 ml inorganic salts solution. The salts contain per liter: 1000 mg FeSC> 4x7 H2O, 1000 mg MnSC> 4x4 H2O, 25 mg CuCl2X H2O, 100 mg CaCl2x2 H2O, 56 mg H3BO3, 19 mg (ΝΗ4) 6Μογθ24χ4 H / iO and 200 mg ZnSO4x7 H2O.

A spore suspension of Aspergillus niger (MZKI A 157) with a concentration of 1 to 4, preferably 2x10 ⁶ spores / ml in sterile water, is used as a plug.

500 ml of the shaken vegetative phase is sterile transferred to a mixing bioreactor, V = 10 l, where the inoculum grows for 24 hours under submerged conditions in medium B at a concentration of 5.7 g / l of dry biomass.

Medium B contains per liter: 100 g glucose, 50 g glycerol, 20 g corn syrup, 20 g beer yeast and 5 g tomato meal. The prepared fungus is used as an inoculum; V = 2000 ml for solid medium in a horizontal reactor.

The fermentation of lovastatin is carried out at a temperature of 20-37 ° C (293-310 K), preferably 3035 ° C (303-308 K) at an optimal humidity of 35-45%. The constant humidity can be controlled by means of a humidifying glass bottle with glass fillers at 30 ° C (303 K). Substrate ventilation is 1 1/1 / min during fermentation. Substrate mixing is only occasional, three times for 10 minutes daily at N = 80 rpm.

The fermentation process is carried out for 7 to 10 days under the conditions described.

The present invention is illustrated by way of example examples, which should not be construed as limiting in any way.

Example 1

The fermentation substrate contained:

44.2% by weight of cereal grains; 16.6% w / w soybean meal; 11.0% w / w of dried apple marc; 9.9% w / w wheat bran, 6.6% w / w sucrose; 4.4% w / w glycerol; 2.0% w / w dry whey; 1.3% w / w NH4NO3; 0.4% w / w (NHz ^ SC ^; 1.8% w / w corn syrup and 1.8% w / w beer yeast.

Sterilization was carried out for 60 minutes at 110 ° C by introducing superheated steam into the double jacket of the reactor and occasionally introducing steam directly into the solid substrate. The introduction of superheated water vapor was carried out at a pressure of 1.52x10 ⁵ Pa and 130 ° C in a double jacket, and occasionally introducing 2x10 minutes through the hollow axis was mixed directly into the fermentation substrate. Mixing during sterilization is uncommon.

After sterilization of the solid medium, the substrate was dried to the desired humidity of 35% by introducing dry sterile air into the fermentation substrate and simultaneously cooled with cooling water in a double jacket.

Inoculation with Aspergillus terreus (MZKIBK A 157) was carried out at 35 ° C (308 K) with 2000 ml inoculum. The inoculum was prepared according to the principle of two-step fermentation (option 2), 24 h on a shaker in medium A, and 24 h in a bioreactor under submerged conditions in medium B at a concentration of 5.7 g / l of dry biomass.

The fermentation took place 150-200, preferably 170 h. The amount of the product was 520 mg lovastatin per kilogram of starting material.

Example 2

The fermentation substrate and sterilization process were the same as those described in Example 1. Sterilization was carried out for 60 minutes at 110 ° C (383 K), introducing superheated steam into the reactor double jacket and occasionally introducing steam directly into the solid substrate.

After drying with sterile air and cooling to 35 ° C (308 K), the substrate was inoculated with a suspension of Aspergillus terreus spores (MZKIBK A 157) at a concentration of 4x10 ^with spores in 2000 ml of sterile water (option 1).

The fermentation took 180 hours. The amount of the product was 600 mg lovastatin per kilogram of starting material.

Claims (1)

Process for the production of lovastatin by Aspergillus teireus, characterized in that a solid fermentation substrate is prepared containing 40 to 60% by weight of cereal grains, 10 to 30% by weight of soya flour, 10 to 20% by weight of dried apple cider tropin, 7.5 to 15% w / w wheat bran, 4 to 8% w / w sucrose, 2 to 6% w / w glycerol, 1 to 4% w / w dry whey, 1 to 2% w / w NH4NO3, 0.1 to 0.6 % w / w (NHz ^ SOzp 1.0 to 2.0% w / w corn syrup and 1.0 to 2.0% w / w beer yeast, the substrate is sterilized in a horizontal mixing reactor at 100 to 120 ° C for 30 to 90 minutes, sterilized substrate dried to 30 to 45% humidity and cooled to 30 to 40 ° C at the same time, then the substrate was inoculated with Aspergillus terreus and fermented at 30 to 40 ° C for 150 to 200 hours with occasional stirring for a maximum of 10 minutes / day.