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Method for converting echinomycin B by immobilized microbial enzyme

A technology of immobilizing microorganisms and echinocandins, which is applied in the field of bioengineering, can solve the problems of low catalytic efficiency and difficulty in maintaining stable microbial cells, and achieve the effects of high conversion efficiency, easy separation and extraction, and easy storage

Inactive Publication Date: 2019-04-09
CHENGDU YATU BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the low catalytic efficiency of this step, the microbial cells are difficult to maintain stably, making this link a bottleneck link in the production process of Anifungin

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] A method for converting echinocandin B with immobilized microbial enzymes, comprising the following steps:

[0029] (1) Fermentation of Actinomycetes Utahii, collecting the mycelium, using a cell disruptor, ultrasonically disrupting the cells, controlling the temperature, the temperature is below 10°C, and centrifuging to obtain the supernatant to obtain Acanthus acanthus Crude enzyme solution of mycocin B deacylase 300 g, the ultrasonic power is 500 W, and the ultrasonic time is 10 min;

[0030] (2) slowly add ammonium sulfate solution in the crude enzyme liquid in step (1), the concentration of ammonium sulfate solution is 20%, and the consumption of described ammonium sulfate solution is 40, obtains precipitation;

[0031] (3) The precipitation in step (2) is dissolved in buffered saline solution, the concentration of buffered saline solution is 0.05mol / L, pH6.0, and described buffered saline solution is phosphate buffered saline, the consumption of phosphate buffere...

Embodiment 2

[0036] A method for converting echinocandin B with immobilized microbial enzymes, comprising the following steps:

[0037] (1) Fermentation of Actinomycetes Utahii, collecting the mycelium, using a cell disruptor, ultrasonically disrupting the cells, controlling the temperature, the temperature is below 10°C, and centrifuging to obtain the supernatant to obtain Acanthus acanthus 400ml of crude enzyme solution of mycocin B deacylase, the ultrasonic power is 550W, and the ultrasonic time is 20min;

[0038] (2) slowly add ammonium sulfate solution in the crude enzyme liquid in step (1), the concentration of ammonium sulfate solution is 30%, and the consumption of described ammonium sulfate solution is 60ml, obtains precipitation;

[0039] (3) The precipitate in step (2) is dissolved in buffered saline solution, the concentration of buffered saline solution is 0.075mol / L, pH6.5, and described buffered saline solution is acetate buffer solution, acetate buffer solution The dosage ...

Embodiment 3

[0044] A method for converting echinocandin B with immobilized microbial enzymes, comprising the following steps:

[0045] (1) Fermentation of Actinomycetes Utahii, collecting the mycelium, using a cell disruptor, ultrasonically disrupting the cells, controlling the temperature, the temperature is below 10°C, and centrifuging to obtain the supernatant to obtain Acanthus acanthus 500ml of crude enzyme solution of mycocin B deacylase, the ultrasonic power is 600W, and the ultrasonic time is 30min;

[0046] (2) slowly add ammonium sulfate solution in the crude enzyme liquid in step (1), the concentration of ammonium sulfate solution is 40%, and the consumption of described ammonium sulfate solution is 80ml, obtains precipitation;

[0047] (3) The precipitation in step (2) is dissolved in buffered saline solution, the concentration of buffered saline solution is 0.1mol / L, pH7.0, and described buffered saline solution is phosphate buffered saline, the consumption of phosphate buffe...

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Abstract

The invention discloses a method for converting echinomycin B by an immobilized microbial enzyme, and belongs to the field of bioengineering. The method comprises the following steps: (1), fermentingactinoplanesutahensis, collecting mycelia, disrupting cells through ultrasonic disruption by using a cell disrupting device, centrifuging, taking a supernatant to obtain a crude enzyme solution of echinomycin B deacylase; (2), slowly adding an ammonium sulfate solution into the crude enzyme solution in the step (1) to obtain a precipitate; (3), dissolving the precipitate in the step (2) in a buffered salt solution; (4), adding odium alginate into the solution in the step (3), stirring uniformly, then dropwise adding a pre-cooled aqueous calcium chloride solution, placing in a refrigerator overnight, and filtering to obtain the immobilized microbial enzyme; and (5), dissolving echinomycin B in ethanol, and adding the immobilized microbial enzyme in the step (4), converting, and collecting afiltrate to obtain an echinocandin B mother nucleus solution. By the method, the conversion rate is high, the conversion time is short, a product is easy to separate, a conversion solution contains less pigments, and the immobilized enzyme is easy to store and can be reused.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for converting echinocandin B with immobilized microbial enzymes. Background technique [0002] Invasive fungal infection is one of the main causes of morbidity and death in immunocompromised or hospitalized patients. Candida is the common pathogen. Antifungal drugs commonly used in the past, such as amphotericin B, can easily cause nephrotoxicity. Azole antifungal drugs (such as fluconazole and itraconazole) are prone to interact with other drugs, which limits their clinical use. The newly developed echinocandin antifungal drugs have become a new choice for invasive fungal infections due to their good efficacy and safety. [0003] Fungins, as important members of the echinocandin family, can inhibit fungal cell wall β-(1→3)-glucan synthesis, and have strong antibacterial activity against clinically common Candida and other fungi. The listed fenjing drugs anidul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/04
CPCC07K7/56C12P21/02
Inventor 朱辉易欣曹艳茹朱春燕龙燕莫洪庞雪包希臻
Owner CHENGDU YATU BIOLOGICAL TECH
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