A kind of preparation method and application of immobilized cyclic lipopeptide deacylase

A technology of cyclolipopeptide deacylase and enzyme activity, which is applied in the field of preparation of immobilized cyclolipopeptide deacylase, can solve the problems of low recovery rate of immobilized enzyme activity, enzyme loss, increased cost, etc., and save enzyme purification Equipment, Enhanced Selective Binding, High Transformation Efficiency

Active Publication Date: 2018-10-16
HANGZHOU HUADONG MEDICINE GRP PHARMA RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This is because there are many impurities such as pigments, amino acids, cell fragments, and secondary metabolites in the fermentation broth; the concentration of the enzyme is relatively low relative to various impurities, and the impurities will inhibit the activity of the immobilized carrier; at the same time, the impurities contained in the impurities The active group will compete for the binding site of the hydrophilic carrier, resulting in a low recovery rate of immobilized enzyme activity
[0007] However, the enzyme purification process will cause enzyme loss, and additional equipment is required, increasing the cost

Method used

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  • A kind of preparation method and application of immobilized cyclic lipopeptide deacylase
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  • A kind of preparation method and application of immobilized cyclic lipopeptide deacylase

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Experimental program
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Effect test

Embodiment 1

[0026] The acquisition of embodiment 1 cyclolipopeptide deacylase solution

[0027] 1.1 Cyclic lipopeptide deacylase produced by Actinoplanes utahensis

[0028] Using the Actinomycetes uthaensis IFO-13244 strain, according to the fermentation method described in Example 1 of the patent US5376634, the fermentation culture was carried out to obtain 150 L of mycelium culture solution. Then filter through a Buchner funnel covered with filter paper, collect 120 L of filtrate containing cyclolipidase deacylase, that is, the free cyclolipopeptide deacylase solution, collect the filtrate, and detect by HPLC, the enzyme activity is 0.864 × 10 5 U.

[0029] 1.2 Cyclic lipopeptide deacylase produced by Streptomyces sp.

[0030] Streptomyces strain No. 6907 was used to carry out fermentation culture according to the fermentation method described in Example 1 of patent WO97 / 32975 to obtain 150 L of mycelium culture solution. Then filter through a Buchner funnel covered with filter paper...

Embodiment 2

[0033] The selection of embodiment 2 different types of epoxide carriers

[0034] Take the free cyclolipopeptide deacylase solution prepared in 1.1 of Example 1 above, add 1g Eupergit CM or LX1000-EP respectively, stir at 25°C for 24 hours, collect the immobilized cyclolipopeptide deacylase by filtration, and wash with pure water three times , dried at room temperature for 3 hours, and finally carried out the enzyme activity assay.

[0035] Table 1 The immobilization of different types of epoxide carriers to the original free enzyme solution

[0036]

[0037] It can be seen from Table 1 that the recovery rate of the vector LX1000-EP is higher than that of the vector EupergitCM under the condition of the same enzyme dosage.

[0038] Table 2 The immobilization of the original free enzyme solution by the epoxide carrier LX1000-EP

[0039]

[0040] It can be seen from Table 2 that the immobilization of the free enzyme can be realized when the mixing ratio of the free cyclo...

Embodiment 3

[0042] The optimization of aqueous solution polarity in the immobilization process of embodiment 3

[0043]Take 1 L of the free cycloaliphatic peptide deacylase solution prepared in 1.1 of Example 1, and add different types of organic solvents (including ethanol, isopropanol, n-hexane, DMSO, acetone, DMF, tetrahydrofuran, pyridine, acetonitrile) respectively. 50mL, 100mL, 200mL or 300mL. Continue to add 5 g of LX1000-EP carrier respectively, stir at 25°C for 24 hours, collect the immobilized cyclolipopeptide deacylase by filtration, wash with pure water three times, dry at room temperature for 3 hours, and finally measure the enzyme activity.

[0044] Table 3 Cyclic lipopeptide deacylase immobilization in different organic solvents

[0045]

[0046]

[0047] As can be seen from Table 3, in the free cycloaliphatic peptide deacylase solution, adding different types of organic solvents, including ethanol, isopropanol, n-hexane, DMSO, acetone, DMF, tetrahydrofuran, pyridine...

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Abstract

The invention discloses a preparation method of immobilized cyclic lipopeptide deacylase. A cyclic lipopeptide deacylase solution is directly mixed with a porous hydrophily enzyme carrier without purification, one or more kinds of organic solutions and inorganic salt are added, and then the immobilized cyclic lipopeptide deacylase is obtained. The invention further discloses application of the method in industrial production of micafungin parent nucleuses and anidulafungin parent nucleuses. The preparation method is simple in technology, enzyme purification equipment is omitted, the recovery rate of enzyme activities is obviously increased in the immobilization process, and immobilized enzymes can be repeatedly used. The enzyme method for converting micafungin and anidulafungin intermediates has higher concentration of reaction substrates when compared with a fermentation conversion method, a few product impurities are generated, and the conversion efficiency is high.

Description

technical field [0001] The invention belongs to the technical field of enzyme immobilization in biotechnology; specifically, the invention relates to a preparation method and application of an immobilized cycloaliphatic peptide deacylase. technical background [0002] Echinocandin antibiotics are a class of natural products discovered in the 1970s that can inhibit β-1,3-glucan synthase activity and have good antifungal activity. Their basic structure is shown in Formula 1. Due to the R of natural echinocandins 2 Fatty acid side chains have a certain degree of hemolytic toxicity in the human body. Therefore, micafungin and anifungin and other antifungal drugs that have been on the market all remove the original fatty acid side chains of natural compounds and replace them with different ones. fatty acid side chains. Since the process of removing side chains is difficult to complete by chemical methods, cyclolipopeptide deacylase (Deacylase) is generally used for biotransform...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/08C12P21/04
Inventor 冯国栋陈振明陈晓霞赖敦岳周硕朱健
Owner HANGZHOU HUADONG MEDICINE GRP PHARMA RES INST
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