Preparation method of anidulafungin precursor

A polymer, echinocandin technology, applied in the direction of peptide, fermentation, etc., can solve the problems of negative effect of deacylase activity, cumbersome extraction process of crude enzyme, unfavorable scale-up production, etc., achieve low cost, improve conversion speed and enzyme stable and easy to operate

Pending Publication Date: 2018-08-17
BRIGHTGENE BIO MEDICAL TECH (SUZHOU) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of this method is that the crude enzyme extraction process is cumbersome, which is not conducive to industrial scale-up production; the extraction of crude enzyme by rotary steaming is easy to cause protein denaturation due to high temperature, and the activity of deacylase has a negative impact
[0012] 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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1 produces the fermentation of deacylase bacterial strain and crude enzyme preparation

[0031] Bacterial species: Streptomyces albus (Streptomyces albus), preservation number: ATCC21725; -80°C cryopreservation tube;

[0032] 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;

[0033] 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;

[0034] 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;

[0035] After the fermentation is completed, filter the fermented liquid to obtain the s...

Embodiment 2

[0036] Example 2 Deacylase cross-linking conversion to produce echinocandin B nucleus

[0037] 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.

[0038] 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.

[0039] Add 20 mmol / L of glutaraldehyde to the reaction solution, stir at 30° C. for 2 hours, and the stirring speed is 100 rpm.

[0040] Add 10% echinocandin B dimethyl sulfoxide solution containing 20kg echinocandin B to the reaction solution, stir and react at 30°C for 12 to 48 hours, the stirring speed is 150rpm, and the echinocandin B in the conversion system is monitored by HPLC during the conversion process. The concentrati...

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PUM

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Abstract

The invention discloses a novel method of converting echinocandin B into echinocandin B parent nucleus. A crosslinked enzyme aggregation technique is applied to the process of conversion of echinocandin B into echinocandin B parent nucleus. The method comprises: preparing deacylase crosslinked enzyme aggregate with deacylase; allowing the crosslinked enzyme aggregate to convert echinocandin B intoechinocandin B parent nucleus. The preparation method enables the molar conversion rate to reach 85% to 90%; the production process is simplified; the cost is lowered.

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 the preparation of anifungin precursor echinocandin B core. 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 amphote...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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