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A kind of echinocandin b microbial enzyme conversion method

An echinocandin and conversion technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of negative impact on deacylase activity, cumbersome extraction process of crude enzyme, unfavorable scale-up production, etc. Achieve the effects of less negative impact on enzyme activity, favorable separation and purification, and good commercial value

Active Publication Date: 2019-08-13
CHONGQING QIANTAI BIOLOGICAL MEDICINE +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

Method used

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  • A kind of echinocandin b microbial enzyme conversion method
  • A kind of echinocandin b microbial enzyme conversion method
  • A kind of echinocandin b microbial enzyme conversion method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Fermentation of echinocandin B deacylase-producing strains and crude enzyme preparation

[0040] Bacterial species: Streptomyces albus (Streptomyces albus), preservation number: ATCC21725; -80°C cryopreservation tube;

[0041] Seed medium: hot fried soybean cake powder 0.5%, yeast powder 0.5%, peptone 0.5%, glucose 1%, pH about 6.8~7.2, culture at 30°C for 1~2 days;

[0042] 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 2-3 days;

[0043] Inoculate the genetically engineered strain of Streptomyces albicans into the seed medium, culture at 30°C for 1-2 days, then inoculate 5% of the fermentation volume in the fermentation medium, and culture at 30°C for 2-3 days;

[0044] After the fermentation is completed, the fermentation broth is filtered to obtain a supernatant, ammonium ...

Embodiment 2

[0045] Embodiment 2 echinocandin B substrate conversion

[0046] Prepare 1000L of phosphate buffer solution in the reaction tank, containing KH 2 PO 4 2.24g / L and Na 2 HPO 4 12H 2 O 1.24g / L, adjust the pH to about 6.0 with HCl.

[0047] Dissolve echinocandin B substrate in methanol at a concentration of 10%.

[0048] Control the temperature of the reaction tank at 28~32°C, stir at 200rpm, add 30kg of crude enzyme to the reaction tank for the first time, and slowly add 30L of 10% echinocandin B methanol solution to make the content of echinocandin B in the conversion reaction solution 3kg , (that is, the content of echinocandin B (kg) is equivalent to 0.3% of the volume (L) of the transformation reaction solution), and the transformation begins.

[0049] After 4 hours of conversion, add 10 kg of crude enzyme to the reaction tank, and slowly add 30 L of 10% echinocandin B methanol solution;

[0050] After 8 hours of conversion, add 10 kg of crude enzyme to the reaction t...

Embodiment 3

[0053] Embodiment 3 echinocandin B substrate conversion

[0054] Prepare 3000L of phosphate buffer solution in the reaction tank, containing KH 2 PO 4 2.24g / L and Na 2 HPO 4 12H 2 O 1.24g / L, adjust the pH to about 6.0 with HCl;

[0055] Dissolve echinocandin B substrate in ethanol at a concentration of 10%;

[0056] Control the temperature of the reaction tank at 28-32°C, stir at 200rpm, add 120kg of crude enzyme to the reaction tank for the first time, and slowly add 120L of 10% echinocandin B ethanol solution to make the content of echinocandin B in the conversion reaction solution 12kg, start conversion;

[0057] After 6 hours of transformation, add 30 kg of crude enzyme to the reaction tank, and slowly add 90 L of 10% echinocandin B ethanol solution;

[0058] After 12 hours of transformation, add 30 kg of crude enzyme to the reaction tank, and slowly add 90 L of 10% echinocandin B ethanol solution;

[0059] After 18 hours of conversion, add 60 kg of crude enzyme t...

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Abstract

The invention discloses a microbial enzymatic conversion method for echinocandin B, and particularly relates to a method of converting the echinocandin B into an echinocandin B nucleus. The method includes performing microbial fermentation to produce echinocandin B deacylase, extracting to obtain crude enzyme, and converting through adding the crude enzyme and the echinocandin B in a batch manner. The method is high in mole conversion ratio, high in product concentration, and beneficial to separation and purification. An echinocandin B substrate can be recovered and reutilized, thus reducing a production cost.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to an echinocandin B microbial enzyme conversion method. Background technique [0002] Echinocandins are a group of natural products discovered in the late 1970s. They have similar structural features of cyclic polypeptide cores and different fatty acid side chains, and can non-competitively inhibit fungal cell wall β-1,3 - Glucan synthase activity, clinically used as an antifungal drug. [0003] Echinocandin B (ECB for short) is produced by the metabolism of Aspergilus nidulans and is one of the echinocandins. Its structure is as follows: [0004] [0005] Echinocandin B Nucleus (ECBN) is obtained by deacylase, and the cyclic peptide nucleus can be chemically modified to prepare the antifungal drug Anidulafungin (Anidulafungin). At present, for the preparation of ECB core, the cost of chemical synthesis is relatively high, and microbial transformation is mainly used. Microbi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/04C12R1/47
CPCY02P20/582
Inventor 别一郭明袁建栋
Owner CHONGQING QIANTAI BIOLOGICAL MEDICINE
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