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Microbial process for preparing pravastatin

A biotransformation and substrate technology, applied in the direction of microorganism-based methods, methods using fungi, microorganisms, etc., can solve the difficult and difficult problems of DNA technology application

Inactive Publication Date: 2002-05-01
INSTITUTE FOR DRUG RESEARCH LTD (HU)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, according to the authors, in the bacterial cytochrome P-450 monooxygenase system, not one but several proteins play a role in electron transport, which makes the application of the DNA technique more difficult
The development of a cost-effective microbial hydroxylation process for the production of pravastatin was extremely difficult and complex

Method used

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  • Microbial process for preparing pravastatin
  • Microbial process for preparing pravastatin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] A spore suspension was prepared with 5 ml of 0.9% sodium chloride solution obtained from 7-10 days of Mortierella maculata nov.spec.E-97 [NCAIM(P)F 001266] strain capable of 6β-hydroxylation of compactin Aged wort-yeast extract agar slant cultures were used to inoculate 100 ml of sterilized seed medium PI in a 500 ml Erlenmeyer flask. Composition of medium PI:

[0053] Glucose 50g

[0054] Soy flour 20g

[0055] Dissolved in 1000ml tap water.

[0056] The pH of the medium was adjusted to 7.0 prior to sterilization, followed by sterilization at 121°C for 25 minutes. The culture was shaken on a rotary shaker (250rpm, 2.5cm amplitude) for 3 days at 28°C, and then 10ml of the obtained culture was transferred to 100 ml of the obtained culture in a 500ml Erlenmeyer flask sterilized at 121°C for 25 minutes. -100ml biotransformation medium MU / 4. Composition of medium MU / 4:

[0057] Glucose 40g

[0058] Soy flour 20g

[0059] Casein-Peptone 1g

[0060] Asparagine 2g

...

Embodiment 2

[0066] In a laboratory scale fermentor with a working volume of 5 liters, prepare MU / S biotransformation medium, add medium components corresponding to a volume of 5 liters, but only add up to 4.5 liters, then sterilize at 121°C for 45 minutes, Inoculate with 500 ml of seed culture prepared according to Example 1. Composition of medium MU / 8:

[0067] Glucose 20g

[0068] Glycerin 20g

[0069] Soy flour 20g

[0070] Peptone 5g

[0071] Potassium dihydrogen phosphate 0.5g

[0072] Polypropylene glycol 2000 1g

[0073] Dissolved in 1000ml tap water. The pH of the medium was adjusted to a value of 7.0 prior to sterilization.

[0074] The fermentation was carried out for 4 days at 28°C with a stirring rate of 400 rpm and an aeration rate of 60 liters / hour from the bottom direction. On day 2 after transfection, the culture began to foam heavily, which could be reduced by adding more polypropylene glycol 2000. During the early days of fermentation (16-20 hours), the pH decre...

Embodiment 3

[0078] Biotransformation medium MU / 4 was prepared as described in Example 1 in a laboratory scale 5 liter working volume fermentor, although it loaded up to 4.5 liters, the composition of the medium was calculated at 5 liters. After sterilization at 121°C for 45 minutes, 500 ml of the seed culture prepared according to Example 1 was inoculated. Fermentation was carried out at 25°C for 4 days by using a stirring rate of 300 rpm and an aeration rate of 50 liters / hour. Biotransformation was performed as in Example 2 after adding 5 g of compactin substrate to the culture.

[0079] After completing this generative transformation, 4.9 liters of broth containing 660 μg / ml pravastatin were filtered and the isolated mycelia washed by suspending in 2 x 1 liter deionized water. The pH of the combined 5.6 liter broth filtrate was adjusted to 3.5-3.7 with 20% sulfuric acid, and the acidic filtrate was stirred with 2750 ml of ethyl acetate for 30 minutes. Subsequently, the phases are sepa...

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Abstract

The present invention relates to a new microbial process for the preparation of compound formula (I) from a compound of general formula (II) wherein R stands for an alkali metal or ammonium ion, by the submerged cultivation of a mold strain able to 6 beta -hydroxylate a compound of Formula (II) in aerobic fermentation and by the separation and purification of the product of Formula (I) formed in the course of the bioconversion. The process comprises cultivating a strain of Mortierella maculata filamentous mold species that is able to 6 beta -hydroxylate a compound of general Formula (II), on a nutrient medium containing assimilable carbon and nitrogen sources and mineral salts and separating the product formed from the fermentation broth, then isolating the compound of formula (I) and purifying the same. Novel strains of Mortierella maculata are also disclosed.

Description

Field of Invention [0001] The present invention relates to a method for preparing pravastatin, in particular to a microbial method for producing pravastatin on an industrial scale. Background of the Invention [0002] The greatest risk factor for atherosclerosis, especially coronary occlusion, is high plasma cholesterol levels. In the last two decades, 3-hydroxy-3-methylglutaryl-CoA reductase (EC.1.1.1.34), which is the key rate-limiting enzyme in cholesterol biosynthesis, has been intensively studied. Pravastatin, a compound of formula I, and other related compounds (compactin, mevinolin, simvastatin) are competitive inhibitors of HMG-CoA reductase [A. Endo et al., J. Antibiot. 29, 1346-1348 (1976) ; A. Endo et al, FEBS Lett. 72, 323-326 (1976); C. H. Kuo et al, J. Org. Chem. 48, 1991 (1983)]. [0003] Pravastatin was first isolated from dog urine by M. Tanaka et al. (unpublished results) during a study of compactin metabolism (Arai, M. et al., San...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07B53/00C07CC07C61/16C07C67/02C07C67/28C07C67/52C07C69/02C07C69/30C07C69/33C12N1/14C12PC12P7/62C12R1/645
CPCC12P7/62C12R1/645C12R2001/645C12N1/145
Inventor A·杰克A·康雅I·巴塔E·伊尔科伊G·索莫伊G·阿姆布鲁斯G·霍尔瓦斯K·阿尔布雷希特I·M·沙波J·莫泽斯尼苏托J·萨拉特A·安多尔L·比林斯克S·波罗斯I·朗M·比德洛尼伊格洛伊
Owner INSTITUTE FOR DRUG RESEARCH LTD (HU)
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