SI9300047A - Microbiological method for preparation of lovostatin and/or mevinoline acid - Google Patents

Abstract

A process is proposed for the biotechnological production of lovastatin and/or mevinolin acid using basidiomycetes fungi from the genus Pleurotus, such as Pleurotus ostreatus, Pleurotus sapidus and Pleurotus saca, preferably Pleurotus sapidus G 24 and Pleurotus saca G 23, which are deposited in the microbiological collection of Kemijski institut Ljubljana, Slovenia (MZKIBK). These fungi are cultivated by the submerged or surface method or the fruit bodies of the said fungi are used, to inoculate the production phase 5 to 10% of the broth is removed sterile, the cultivation in the production phase takes place submerged or on the surface, after the end of the production phase the pH of the broth is adjusted to 3.0 so that the product is obtained in lactone and acid form, or to pH 7.7 so that only the acid is obtained, methanol is added, and the product is extracted by shaking, after the extraction the mycelium is filtered off, and lovastatin and/or mevinolin acid are obtained in the filtrate.

Description

(57) We propose a new process for the biotechnological preparation of lovastatin and / or mevinolinic acid using fungi of the genus Pleurotus, such as Pleurotus ostreatus, Pleurotus sapidus and Pleurotus saca, preferably Pleurotus sapidus G 24 and Pleurotus saca G 23, deposited in the Microbiology Institute, Ljubljana (MZKIBK), grown under submerged or surface process, or use the fruits of said fungi to sterilize 5 to 10% of the broth for grafting of the production phase and to adjust the broth in the production phase submergedly or superficially. 3.0 to give the product as lactone and acid, respectively. at pH 7.7 to give acid only, methanol was added and the product was shaken, filtered after extraction of the mycelium and lovastatin and / or mevinolinic acid were obtained in the filtrate.

KRKA, Medicines Factory, p.o. Institute of Chemistry, Ljubljana

PROCEDURE FOR THE BIOTECHNOLOGICAL PREPARATION OF LOVASTATIN AND / OR MEVINOLINIC ACIDS

The present invention relates to a novel process for the biotechnological preparation of lovastatin and / or mevinolinic acid using fungi of the genus Pleurotus.

Lovastatin and related substances (compactin, mevastatin, simvastatin, epstatin) inhibit cholesterol synthesis by inhibiting the step that determines the rate of cholesterol biosynthesis, i.e., the conversion of hydroxy methyl glutaryl coenzyme A to mevalonate, which is catalyzed by HMG-CoA reductase (JA TobAert, JA Tob G. Hitzenberger, WR Kukovetz, IB Holmes, KH Jones, Atherosclerosis 41, 61 - 65, 1982). The active form of the drug is beta hydroxy acid, which is similar in part to the HMG-CoA molecule and therefore acts as a competitive inhibitor. (G. Zubay, Biochemistry, Addison Wesley Publishing Company, 584, 1984).

Lovastatin is used as a medicine for hypercholesterolemia and for the prevention of ischemic heart disease. It decreases the concentration of total, LDL and VLDL cholesterol in humans (S. M. Grundy, G. L. Vega, Journal of Lipid Research 26, 1464 - 1475, 1985).

The first inhibitor of a series of related compounds was the metabolite of the fungus Penicillium citrinum (ML - 236B) and was discovered by the Japanese in 1976. It was later shown to be identical to the fungicidal compound compactin. Another related compound, monacolin K, which is a metabolite of the Monascus ruber fungus, was discovered by the same researchers in 1979 and the use of this fungus and substance production process was first patented by Sankyo. Independent of the Japanese company Sankyo, researchers at Merck in America discovered and patented mevinolin, under the other name lovastatin. It is a secondary metabolite of the fungus Aspergillus terreus, which has been found to be identical in composition to monacolin K.

In addition to these metabolites, they subsequently acquired a number of related derivatives, which were formed by biotransformation with the help of various microorganisms, respectively. with chemical synthesis. (A. Endo, Journal of Medicinal Chemistry 28, 4, 401 405, 1985). In addition to hypocholesterolemic activity, lovastatin also has a fungicidal, herbicidal and cytostatic activity (S. Schindler, T. Bach, H. K. Lichtenthaler, Naturforsch. 40c, 208 - 214, 1985).

In addition to the fungi mentioned above, they are from the literature as potential producers of lovastatin, respectively. The following fungi are also known for related substances: Penicillium brevicompactum, Pythium ultimum, Hypomyces chrysospermus, Paecylomyces sp., Eupenicillium sp., Trichoderma longibrachiatum, T. pseudokoningii, Phoma sp., Doratomyces nanus and A. Gymnoicuso umbrinus, K. (A. Yamada, R. Shimoda, H. Takeshima, J. Antibiot. 49, 11, 1609 - 1610, 1986).

Submerged aerobic fermentation is used in the above procedures. The medium must contain a source of assimilative carbon, nitrogen, inorganic salts and other nutrients. The fungi are grown at a temperature between 20 and 37 ° C, the pH of the culture medium is between 6 and 8. After fermentation, lovastatin is isolated by extraction with organic solvents.

DESCRIPTION OF THE SOLUTION WITHIN THE INVENTION

The present invention is used for the biosynthesis of lovastatin by a basidiomycetic fungus of the genus Pleurotus, which has not yet been mentioned as a potential producer of this inhibitor. Suitable species are P. ostreatus, P. sapidus and P. saca, which have been deposited in the microbiological collection of the Institute of Chemistry, Ljubljana (MZKIBK). The most active fungi from this collection, however, are Pleurotus sapidus (G 24) and Pleurotus saca (G 23). Other species of this genus are also capable of synthesizing lovastatin. Their use for this purpose is the object of the present invention.

The morphological characteristics of the G 23 and G 24 organisms are characteristic of the Pleurotus genus. The criteria for taxonomic determination are specified in The Biology and Cultivation of Edible Mushrooms, by S. T. Chang, W. A. Haynes, Eds. Academic Press, New York, 1978.

The fungi are stored in the form of live culture on an oblique beer agar, which is inoculated every 6 months, respectively. under paraffin oil, which can last for many years. According to the present invention, lovastatin can be obtained with these submerged, respectively. surface process, or just from the fruits of said fungi. For the biosynthesis of lovastatin in the surface or submerged cultivation of these fungi, the age of the culture for graft is very essential. This should be 14 days to 4 months, preferably 2.5 months. Macroscopically, they must be visible in the growth of the already rotated fruits that already sporulate. To prepare a pinch of mycelium, cut from the surface of the agar, respectively. grind it in a Warring mixer, respectively. from the agar culture on the petri dish with the cork, drill out the plugs (6 mm in diameter) and use them to vaccinate the liquid medium for the vegetative phase.

The vegetative phase medium contains corn steep liquor (CSL), oatmeal, glucose, tomato meal, trace elements (B. Buckland, K. Gbewonyo, T. Hallada, L. Kaplan and P. Masurekar, Novel Microbial Products for Medicine and Agriculture, AL Demain, GA Somkuti, JC Hunter-Cevera, HW Rossmoore, Eds. Society for Industrial Microbiology, Elsevier, Amsterdam, 1989, pp. 161-169) and tap water. The pH is 7.0. The medium was sterilized for 20 minutes at 121 ° C, then cooled to 28-35 ° C and vaccinated with the fungus. Use 3 to 10 ml of mycelium suspension, preferably 5 (one tube / 25 ml of sterile water) per 80 ml of medium, respectively. 3 - 10 stoppers, preferably 5, per 80 ml of vegetative medium.

In the vegetative phase, the fungus is grown on a shaker at 150 - 250 rpm, preferably 200 rpm, at a temperature of 25 to 35 ° C, preferably 30 ° C, 5 to 10 days, preferably 6 days.

For the vaccination of the production phase, 5 to 10% of the broth is sterile and the residue can be used as a protein feed additive after drying.

The production medium consists of complex carbon and nitrogen sources and minerals [glycerol, beer yeast, oatmeal, tomato meal, glucose, Na-acetate, (NH ₄ ) ₂ SO4, straw]. Growing in the production phase can be submerged or surface. In the case of submerged cultivation, the conditions are the same as for vegetative culture. In the case of surface fermentation, the bottles are allowed to stand at a temperature of 20 - 35 ° C, preferably 30 ° C, in a thermostat of 7-14 days, preferably 9 - 12 days.

In order to obtain lovastatin from the fruits, the fungi are ground in a mixer together with water and then worked in the same way as after the end of the submerged, respectively. surface fermentations. After the production phase is complete, the broth is adjusted to pH 7.7, the same volume of methanol is added and the product is extracted by shaking for 30 hours at 30 ° C. After the mycelium extraction, it is filtered off. The filtrate gives lovastatin as an acid. This form can be converted by means of organic solvents into a lactone form used as an active ingredient in the pharmaceutical industry.

The filtered mycelium, resp. after being washed with water and dried, the ground fruits can be used as a protein supplement to the feed.

The invention is explained by way of example examples, which should not be construed by any means.

Example 1

Surface fermentation

Vegetative phase

The stalk is prepared by scraping a 2.5-month-old mycelium of Pleurotus saca G 23 (MZKIBK) and grafting a vegetative medium with the following composition (g / 100 ml):

0.5 g of corn irrigation fluid (CSL), 1 g of oat flour, 1 g of glucose, 4 g of tomato paste, 1 ml of trace element solution and tap water up to 100 ml. pH is 7.0.

Autoclave the medium for 121 minutes at 121 ° C.

In the vegetative phase, the fungus is grown under shaken culture in 500 ml Erlenmeyer bottles containing 80 ml of substrate for 6 days at 30 ° C and 200 rpm. We produce the production medium with 5 - 10% of the vegetative medium.

Production phase

Autoclave the production medium for 20 minutes at 121 ° C. The production medium has the following composition (g / 100 ml):

g of straw, 1 g of glucose, mineral salts solution up to 100 ml, pH - 7,0.

In the production phase, the fungus is cultured in surface culture in Erlenmeyeq bottles containing 40 ml of substrate at room temperature, with occasional manual agitation, for 12 to 16 days.

After fermentation is complete, add again the amount of methanol and adjust the pH of the broth to 3.0. The extraction is performed on a shaker at 220 rpm and 30 ° C. The slurry was then filtered to give 8 mg / l of product, 7.1 mg / ml as lovastatin and 0.9 mg / l as mevinolic acid in the filtrate.

Example 2

Fermentation in shaking culture

Vegetative phase

The inoculum is prepared by scraping the mycelium of a 2.5-month-old fungal culture of the genus Pleurotus sapidus G 24 (MZKIBK) and grafting a vegetative medium which is autoclaved for 20 minutes at 121 ° C. Vegetative medium composition (g / 100 ml):

0.5 g corn irrigation fluid (CSL), 1.0 g oatmeal, 1.0 g glucose, 4.0 g tomato meal, 1.0 ml trace element solution, tap water up to 100 ml. pH is 7.0.

In the vegetative phase, the fungus is grown for 6 days in a shaken culture in 500 ml Erlenmeyer bottles containing 80 ml of culture medium on a shaker at 30 ° C and 200 rpm.

We produce the production medium with 5 - 10% of the vegetative medium.

Production phase

Autoclave the production medium for 20 minutes at 121 ° C. The production medium has the following composition (g / 100 ml):

g glycerol, 4 g beer yeast, 4 g oatmeal, 1.5 g tomato meal, 5 g glucose, 3 g Na-acetate, 0.5 g (NH ^ SC ^, 1 g straw.

During the production phase, the fungus is grown for 9 days in a shaken culture in 500 ml Erlenmeyer bottles containing 40 ml of medium on a shaker at 30 ° C and 200 rpm.

After fermentation is complete, the pH of the broth is adjusted to 3.0 and the amount of methanol is added again. The extraction is carried out for 1 hour at 30 ° C and 220 rpm. The slurry was then filtered to give 20 mg / l of product, 0.6 mg / l in the form of mevinolinic acid and 19.4 mg / l in the form of lovastatin.

Example 3

Extraction of lovastatin from fruiting grounds

The commercially available fresh fruits of Pleurotus ostreatus are ground in a minimum volume of water and the same volume of methanol is added (methanol: water = 1: 1). The extraction is performed on a shaker at 30 ° C and 220 rpm for at least one hour. The slurry was then filtered and the filtrate adjusted to pH 3.0. 45 kg of product, 2.03 mg / l in the form of mevinolinic acid and 42.97 mg / l in the form of lovastatin are obtained in 1 kg of fresh fruits.

KRKA, a drug factory.

Institute of Chemistry, Ljubljana

Claims (2)

A method for the biotechnological preparation of lovastatin and / or mevinolinic acid, characterized in that the basidiomycetes fungi of the genus Pleurotus, such as Pleurotus ostreatus, Pleurotus sapidus and Pleurotus saca, preferably Pleurotus sapidus G 24 and Pleurotus saca G 23, deposited by Kemijlo Institute, Ljubljana (MZKIBK), grown under sub-freezing or surface treatment, or use the fruits of said fungi to grow the fungus on a shaker at 150 - 250 rpm at 25 to 35 ° C for 5 to 10 days in the vegetative phase. to sterilize 5 to 10% of the vegetative phase broth for vaccination of the production phase, that the production submerged medium consists of complex carbon and nitrogen sources and a solution of mineral substances, that the cultivation in the production phase takes place submerged or surface, in the case of submerged cultivation conditions are the same as for vegetative culture, with surface fermentation the temperature is 20 to 35 ° C, within 7 - 14 days, after adjust the pH to 3.0 to complete the product in the form of lactone and acid, respectively. at pH 7.7 to give acid only, methanol was added and the product was shaken, filtered after extraction of the mycelium and lovastatin and / or mevinolinic acid were obtained in the filtrate. A method according to claim 1, characterized in that, when extracting lovastatin from the fruit plants, the fungal fruits are ground in a mixer together with water and then acted in the same way as after the end of the submerged, respectively. surface fermentations. KRKA, Medicines Factory, p.o. Institute of Chemistry, Ljubljana ABSTRACT