Method for converting echinocandin B by using microbial enzyme

An echinocandin, conversion technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., to achieve the effects of easy separation and purification, improved yield and purity, and high molar conversion rate

Active Publication Date: 2014-07-09
SHANGHAI INST OF PHARMA IND CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this process conversion takes 20-30 hours, and the ECB deacylase is only used once

Method used

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  • Method for converting echinocandin B by using microbial enzyme
  • Method for converting echinocandin B by using microbial enzyme
  • Method for converting echinocandin B by using microbial enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] 1), fermentation of echinocandin B deacylase

[0040] Strains: Actinoplanes utahensis NRRL12052, purchased from the American Agricultural Research Culture Collection.

[0041] Medium formula:

[0042] Slant medium: yeast powder 0.3%, malt extract powder 0.3%, peptone 0.5%, glucose 1.0%, agar 2.5%, pH 7.0-7.2, cultured at 30°C for 5-7 days

[0043] Seed medium: sucrose 2.5%, oat flour 2%, yeast powder 0.25%, K 2 HPO 4 0.1%, KCl 0.05%, MgSO 4 ·7H 2 O 0.05%, FeSO 4 ·7H 2 O 0.0002%, pH=6.8, cultured at 30°C for 4-5 days

[0044] Fermentation medium: 2.5% sucrose, 1.2% peanut meal powder, K 2 HPO 4 0.1%, MgSO 4 ·7H 2 O0.3%, pH=6.8, cultivated at 30°C for 3-4 days

[0045] 2), after the fermentation is completed, add Na to the fermentation broth 2 HPO 4 12H 2 O and NaH 2 PO 4 2H 2 The O concentrations were 6.10 g / L and 5.8 g / L, respectively, and the pH was adjusted to 6.0 with HCl, sonicated for 1 hour, and the supernatant was collected by centrifugation, wh...

Embodiment 2

[0052] 1), fermentation of echinocandin B deacylase

[0053] Strains: Actinoplanes utahensis NRRL12052, purchased from the American Agricultural Research Culture Collection.

[0054] Medium formula:

[0055] Slant medium: yeast powder 0.3%, malt extract powder 0.3%, peptone 0.5%, glucose 1.0%, agar 2.5%, pH 7.0-7.2, cultured at 30°C for 5-7 days

[0056] Seed medium: sucrose 2.5%, oat flour 2%, yeast powder 0.25%, K 2 HPO 4 0.1%, KCl 0.05%, MgSO 4 ·7H 2 O 0.05%, FeSO 4 ·7H 2 O 0.0002%, pH=6.8, cultured at 30°C for 4-5 days

[0057] Fermentation medium: 2.5% sucrose, 1.2% peanut meal powder, K 2 HPO 4 0.1%, MgSO 4 ·7H 2 O0.3%, pH=6.8, cultivated at 30°C for 3-4 days

[0058] 2), after the fermentation is completed, add Na to the fermentation broth 2 HPO 4 12H 2 O and NaH 2 PO 4 2H 2 The O concentrations were 18.20 g / L and 15.15 g / L respectively, the pH was adjusted to 7.0 with NaOH, ultrasonicated for 2 hours, and the supernatant was obtained by centrifugation...

Embodiment 3

[0065] 1), fermentation of echinocandin B deacylase

[0066] Strains: Actinoplanes utahensis NRRL12052, purchased from the American Agricultural Research Culture Collection.

[0067] Medium formula:

[0068] Slant medium: yeast powder 0.3%, malt extract powder 0.3%, peptone 0.5%, glucose 1.0%, agar 2.5%, pH 7.0-7.2, cultured at 30°C for 5-7 days

[0069] Seed medium: sucrose 2.5%, oat flour 2%, yeast powder 0.25%, K 2 HPO 4 0.1%, KCl 0.05%, MgSO 4 ·7H 2 O 0.05%, FeSO 4 ·7H 2 O 0.0002%, pH=6.8, cultured at 30°C for 4-5 days

[0070] Fermentation medium: 2.5% sucrose, 1.2% peanut meal powder, K 2 HPO 4 0.1%, MgSO 4 ·7H 2 O0.3%, pH=6.8, cultivated at 30°C for 3-4 days

[0071] 2), after the fermentation is completed, add Na to the fermentation broth 2 HPO 4 12H 2 O and NaH 2 PO 4 2H 2 The O concentrations were 12.20 g / L and 10.15 g / L respectively, the pH was adjusted to 6.8, ultrasonication was performed for 1.5 h, and the supernatant was obtained by centrifugat...

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Abstract

The invention provides a method for converting echinocandin B (ECB) by using microbial enzyme. The method comprises the steps that: (1) microbes are fermented, such that echinocandin B deacylase is obtained; (2) when fermentation is finished, phosphate is added into the fermentation broth; ultrasonic processing is carried out; and supernatant is obtained by centrifugation, and the supernatant is echinocandin B deacylase crude enzyme solution; (3) the crude enzyme solution is purified, such that echinocandin B deacylase enzyme solution is obtained; (4) echinocandin B is dissolved in ethanol, and is mixed with the deacylase enzyme solution; mixing and stirring are carried out, such that the material is converted into echinocandins B nucleus; when conversion is finished, filtering is carried out; and the filtrate is obtained; and (5) the filtrate is processed by using macroporous adsorption resin; echinocandins B nucleus is absorbed on resin; and echinocandins B nucleus is eluted by using an organic solvent. According to the invention, a molar conversion rate is high, a conversion time is short, an obtained product is easy to separate and to purify, and ECB deacylase can be repeatedly used.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, more specifically, the invention relates to a method for converting echinocandin B into echinocandin B mother nucleus by microbial enzymes. Background technique [0002] Since the 1970s, the frequency and types of fungal infections have been increasing, especially in immunosuppressed patients including HIV-infected patients, organ transplant recipients, cancer patients and those with other diseases who are undergoing immunotherapy of patients. Until the successful development of imidazole and triazole antifungal drugs in the late 1980s and 1990s, fungal infections could be effectively and safely controlled clinically. Although these drugs have played an important role in the control of clinical deep fungal infections, due to the poor selectivity of these drugs, the emergence of drug-resistant fungi, and the low sensitivity to fungi such as Aspergillus and Candida albicans, the incidence of deep...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/04C12R1/045
Inventor 李继安陈详阮建昌林慧敏陈代杰沈剑锋阮霞琴石飞燕董华成
Owner SHANGHAI INST OF PHARMA IND CO LTD
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