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Method for preparing micafungin sodium precursor

A polymer and cross-linking enzyme technology, applied in the direction of peptides, fermentation, etc., can solve the problems of complicated separation and purification steps, slow conversion speed, poor stability, etc., and achieve the effect of simple operation, high conversion speed and low cost

Pending Publication Date: 2018-08-31
BRIGHTGENE BIO MEDICAL TECH (SUZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Chinese patent CN106544382 discloses a method for converting FR901379 into FR179642. This method significantly improves the conversion efficiency of the enzyme by controlling the initial concentration of FR901379 in the conversion liquid and controlling the temperature and pH during the conversion process, and the conversion rate can be as high as 83%. , but the method disclosed in this patent is to collect bacteria for transformation. There are a large number of microorganisms in the transformation system, the transformation speed is slow, the stability of the enzyme is poor, the separation and purification steps are complicated, and the cost is high
[0016] The enzymes used in the currently reported cross-linked enzyme polymers include hydrolases, lyases, and oxidoreductases. At present, there are no reports on the fermentation of semi-synthetic drugs, and there are no reports on the application of this technology to deacylases.

Method used

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  • Method for preparing micafungin sodium precursor
  • Method for preparing micafungin sodium precursor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Fermentation of deacylase-producing strains and crude enzyme preparation

[0035] Bacterial species: Streptomyces albus (Streptomyces albus), preservation number: ATCC21725; -80°C cryopreservation tube;

[0036] Seed medium: 0.5% hot-fried soybean cake powder (w / v, the same below), 0.5% yeast powder, 0.5% peptone, 1% glucose, pH about 6.8-7.2, cultured at 30°C for 1-2 days;

[0037] Fermentation medium: hot fried soybean cake powder 1%, yeast powder 1%, peptone 1%, glucose 3%, sodium chloride 0.5%, magnesium sulfate heptahydrate 0.2%, dipotassium hydrogen phosphate 0.1%, pH about 6.8-7.2 , cultured at 30°C for 2 to 3 days;

[0038] Inoculate the genetically engineered strain of Streptomyces albicans on the seed medium, culture at 30°C for 1-2 days, then inoculate 5% of the fermentation volume into the fermentation medium, and cultivate at 30°C for 2-3 days;

[0039] After the fermentation is completed, filter the fermented liquid to obtain the supernatant, a...

Embodiment 2

[0040] Embodiment 2 Preparation of FR901379

[0041] Coleophoma empetri strains were grown in seed medium. The seed culture medium is glucose 10g / L, soluble starch 30g / L, cottonseed cake powder 20g / L, dry corn steep liquor 5g / L, dipotassium hydrogen phosphate 2g / L, calcium carbonate 3g / L, defoamer 1g / L.

[0042] Prepare an appropriate amount of seed medium, sterilize at 120°C for 30 minutes, cool to 24-26°C, insert 48h seed-age shake flask seeds at an inoculum volume ratio of 0.5-1% of the seed medium volume, and then culture at 24-26°C for 48h .

[0043] Transfer the cultured seeds to 12000L fermentation medium at 120°C for 30 minutes and cool to 24-26°C at a ratio of 2%. The composition of the fermentation medium is as follows: fructose 140. Corn gluten powder 10, casein 6, yeast peptone 7, magnesium sulfate 2, dipotassium hydrogen phosphate 0.5, calcium carbonate 3, defoamer 0.5, and the rest is water.

[0044] During the fermentation process, the temperature is 24-26°...

Embodiment 3

[0046] Example 3 Deacylase cross-linking transformation to produce micafungin core

[0047] Prepare 1000 L of phosphate buffer solution in the reaction tank, containing 2.24 g / L of potassium dihydrogen phosphate and 1.24 g / L of disodium hydrogen phosphate, and adjust the pH to 6.0 with hydrochloric acid or sodium hydroxide.

[0048] Add 20kg of crude enzyme to the phosphate buffer solution, blow in air at a flow rate of 200L per minute, then add 5mmol / L of metaldehyde, and stir at 30°C for 2 hours at a speed of 50rpm.

[0049] Add 20kg of micafungin precursor FR901379 to the reaction solution, stir and react at 30°C for 6-24 hours, the stirring speed is 100rpm, and monitor the micafungin precursor FR901379 and micafungin in the conversion system by HPLC during the conversion process Mother nucleus concentration, when the concentration of micafungin precursor FR901379 no longer decreases and the concentration of micafungin mother nucleus no longer increases, the conversion ends...

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Abstract

The invention discloses a brand new method for transforming FR901379 into FR179642. According to the method, a cross-linked enzyme aggregation technology is applied to a process of transforming FR901379 into FR179642. The method comprises the following steps: preparing deacylase into deacylase cross-linked enzyme aggregate, and transforming the FR901379 into FR179642 by the cross-linked enzyme aggregate. According to the method, a molar conversion ratio of transforming the FR901379 into FR179642 can reach 92% or higher, the production process is simplified, and the cost is reduced.

Description

technical field [0001] The invention relates to the field of microbial fermentation, in particular to the preparation of antifungal fermented semi-synthetic drug precursors, and more specifically to the preparation method of micafungin sodium precursor FR179642. Background technique [0002] In recent years, due to the increasing number of immunocompromised patients, the incidence of fungal infections has increased significantly, especially the incidence and mortality of deep fungal infections. Echinocandin antibiotics are a group of natural products discovered in the 1970s, with similar cyclic polypeptide cores and different fatty acid side chains, which can non-competitively inhibit fungal cell wall β-1,3-glucan synthesis Enzyme activity, so as to achieve the purpose of antifungal. Compared with traditional antifungal drugs, this type of drug has a unique mechanism of action, low toxicity and side effects, and has strong antibacterial activity against some azole and ampho...

Claims

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Application Information

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IPC IPC(8): C12P21/04
CPCC07K7/56
Inventor 袁建栋别一
Owner BRIGHTGENE BIO MEDICAL TECH (SUZHOU) CO LTD
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