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Echinocandin biotransformation method

An echinocandin, biotransformation technology, applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., can solve the problems of affecting the transformation effect, reducing the transformation effect, cell damage, etc., to shorten the transformation cycle, reduce Production cost, effect of increased substrate concentration

Active Publication Date: 2014-09-10
ZHEJIANG HISUN PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to solve this problem, the researchers considered adding a co-solvent. For example, in the early research, the researchers added ethanol as a co-solvent to the fermentation system, and the concentration must exceed 10% to have a good effect, and the solvent concentration reached a certain level. After a certain degree, it will inhibit the enzyme activity and reduce the conversion effect
There are also researchers who use dimethyl sulfoxide (DMSO) as a solvent. When the amount of DMSO added is greater than 15%, the substrate concentration of 0.5g / L can be reached. For whole cells, the presence of DMSO has a very damaging effect on cells. Significantly, severely impact conversion performance
Some researchers also immobilized ECB on the resin and then converted it with free deacylase, but it is obviously not practical for the deacylase mainly located in the cell

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Cultivation and preparation of whole cell catalyst

[0034] The microorganism is Actinoplanes utahensis AS 4.1543.

[0035] Slant medium: Chase-Peptone Agar

[0036] Seed medium (%): glucose 2, maltodextrin 1, cottonseed cake powder 0.5, yeast powder 1, peptone 0.5, CaCO 3 0.1.

[0037] Fermentation medium (%): sucrose 4, cottonseed cake powder 3, yeast powder 1, K 2 HPO 4 0.1, CaCO 3 0.2, KCl 0.2, MgSO 4 ·7H 2 O 0.1.

[0038] Culture method: culture on slant at 28°C for 7-10d, pick up 0.5×1cm colony with inoculation shovel and inoculate in 25mL / 250mL seed medium, culture at 28°C, 200-250r / min for 48-60h, as fermented seeds . The above-mentioned seeds are inoculated in the fermentation medium according to the inoculum amount (v / v) of 2-10%, and cultured for 72-120 hours at 27-32° C. and 200-250 r / min.

[0039] Preparation of the whole-cell catalyst: centrifuge the fermented broth obtained from fermentation, collect the cells, and then wash severa...

Embodiment 4

[0050] Example 4: Conversion method of Echinocandin B (ECB) in dimethyl-β-cyclodextrin system

[0051] Dissolve dimethyl-β-cyclodextrin in 50L acetic acid buffer solution with pH 6 concentration of 0.2M, fully dissolve until the solution is clear and transparent, the concentration of cyclodextrin is 12g / L, and grind 100g of ECB into powder Disperse in the above solutions respectively, and stir for 60 min with a magnetic stirrer at 1000 r / min to fully dissolve ECB. The whole-cell catalyst described in Example 1 was added at a concentration equivalent to 5 g dry cells / L, and after transformation at 25° C. for 48 hours, acetic acid was added to adjust the pH to 3 to terminate the transformation.

[0052] Take the transformation solution at 12000r / min, centrifuge for 5min, take the supernatant for HPLC analysis, use ODS C18 (8×100mm), mobile phase A: 3% acetonitrile / 0.5% NH 4 h 2 PO 4 , mobile phase B: 50% acetonitrile / 0.5% NH 4 h 2 PO 4 , Elution condition: 5% B+95% A 3min,...

Embodiment 5

[0053] Example 5: Conversion method of aculeacin A in β-cyclodextrin system

[0054] Dissolve β-cyclodextrin in 50L pH 5 0.1M phosphate buffer solution, fully dissolve until the solution is clear and transparent. Disperse in the above solution, and stir with a magnetic stirrer at 1000r / min for 60min to fully dissolve aculeacin A. The whole-cell catalyst described in Example 2 was added at a concentration equivalent to 4 g dry cells / L, and after transformation at 40° C. for 48 hours, acetic acid was added to adjust the pH to 3 to terminate the transformation.

[0055] Get the conversion solution at 12000r / min, centrifuge for 5min, get the supernatant HPLC analysis, (analysis method is the same as in Example 4), and calculate the conversion rate according to the consumption of aculeacin A to be 90%.

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Abstract

The invention discloses an echinocandin biotransformation method using an actinomycete whole cell or fermentation broth as a catalyst. The invention also provides an echinocandin biotransformation method using cyclodextrin and its derivative or other cosolvents. A transformation system involved in the invention is characterized in that the system can effectively improve the solubility of echinocandin compounds in an aqueous solution, improve the utilization rate and the reaction rate of substrates, and improve the transformation rate of products. Actinoplanes involved in the invention and their mutant strains are in a nutritional medium containing an assimilable carbon and nitrogen source and can generate whole-cell deacylated enzymes under ventilation conditions, and the whole cell deacylated enzymes can be used for the echinocandin biotransformation.

Description

technical field [0001] The invention relates to the field of biotransformation, in particular to a method for improving the biotransformation efficiency of echinocandins by using cyclodextrin and its derivatives or other cosolvents. Background technique [0002] Echinocandin antibiotics are a class of natural products discovered in the 1970s that can inhibit the enzymatic activity of β-1,3-glucan synthase, and have good antifungal activity. The basic structural formula is shown in Formula 1. Since natural echinocandins have R 2 The side chains of fatty acids in the human body have certain hemolytic toxicity in the human body. Therefore, antifungal drugs such as micafungin and anidungin that are already on the market, and aminocandin, which is still in clinical research, all need to re-modify the side chains of fatty acids. The premise of this modification is that the original fatty acid side chain needs to be removed. This process is difficult to complete by chemical method...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P21/04C12R1/045C12R1/465
Inventor 滕云陈正杰朱进伟陈晓静胡海潮
Owner ZHEJIANG HISUN PHARMA CO LTD
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