Fermentation method of micafungin sodium precursor FR179642

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

Inactive Publication Date: 2018-08-24
BRIGHTGENE BIO MEDICAL TECH (SUZHOU) CO LTD +1
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  • 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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  • Fermentation method of micafungin sodium precursor FR179642
  • Fermentation method of micafungin sodium precursor FR179642

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 produces the fermentation of deacylase bacterial strain and crude enzyme preparation

[0036] Bacterial species: Streptomyces albus (Streptomyces albus), preservation number: ATCC21725; -80°C cryopreservation tube;

[0037] 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;

[0038] 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;

[0039] 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;

[0040] After the fermentation is completed, filter the fermented liquid to obtain the...

Embodiment 2

[0041] Embodiment 2 Preparation of FR901379

[0042] 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.

[0043] 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 .

[0044] 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.

[0045] During the fermentation process, the temperature is 24-26°...

Embodiment 3

[0047] Example 3 Deacylase cross-linking conversion to produce micafungin core

[0048] 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.

[0049] Add 20 kg of crude enzyme and 2 kg of bovine serum albumin (BSA) to the phosphate buffer, feed air at a flow rate of 200 L per minute, stir for 30 minutes at a stirring speed of 80 rpm, and stand at 8° C. for 1 hour.

[0050] Add 20 mmol / L of glutaraldehyde to the reaction solution, stir at 30° C. for 2 hours, and the stirring speed is 100 rpm.

[0051] 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 con...

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Abstract

The invention discloses a new method of converting FR901379 into FR179642. In the method, the cross-linked enzyme aggregation technology is applied to the process of converting FR901379 into FR179642.According to the method, deacylase is prepared into a deacylase cross-linked enzyme aggregate, and the cross-linked enzyme aggregate converts FR901379 into FR179642. By means of the method, the FR901379 substrate feed concentration in the reaction system can reach 20 g/L, and the molar conversion rate can reach 90-96%; meanwhile, 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 an antifungal fermented semi-synthetic drug precursor, and more specifically to a fermentation 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 amph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/04
CPCC07K7/56
Inventor 袁建栋别一
Owner BRIGHTGENE BIO MEDICAL TECH (SUZHOU) CO LTD
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