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Method for preparing androstenedione by phytosterol biotransformation in a quaternary phosphonium salt ionic liquid/water two-phase system

A phytosterol, ionic liquid technology, applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., can solve the problems of unsatisfactory cell biocompatibility, low water solubility and toxicity of sterol substrates, and achieves a solution to the problem. Poor water solubility of substrates, improving process conversion efficiency, and saving production costs

Active Publication Date: 2021-02-05
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the degradation of phytosterol side chains by mycobacteria to obtain AD requires a 16-step reaction process catalyzed by 11 functional enzymes, which can only be completed in whole cells so far.
There are two main problems: (1) the low water solubility of the sterol substrate; (2) the inhibition or even toxicity of the hydrophobic product AD to the cell activity in the cell, and the inclusion of the substrate particle and the cell after precipitation
However, the solubility of commercial ionic liquids to sterols is still not high enough, and the biocompatibility to cells is not ideal

Method used

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  • Method for preparing androstenedione by phytosterol biotransformation in a quaternary phosphonium salt ionic liquid/water two-phase system
  • Method for preparing androstenedione by phytosterol biotransformation in a quaternary phosphonium salt ionic liquid/water two-phase system
  • Method for preparing androstenedione by phytosterol biotransformation in a quaternary phosphonium salt ionic liquid/water two-phase system

Examples

Experimental program
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Effect test

Embodiment 1

[0020] Embodiment 1: trihexyltetradecylphosphonium octanoate ionic liquid ([P 6,6,6,14 ][C 7 h 15 COO]) / buffer solution two-phase system biotransformation prepares AD, specifically as follows:

[0021] Step 1: Preparation of ionic liquid [P 6,6,6,14 ][C 7 h 15 COO]. Specifically, [P 6,6,6,14 ][Br] ethanol solution at a flow rate of 1mL min -1 By filling the chromatographic column of Dowex Monosphere 550A UPW (OH) strongly basic anion exchange resin, the corresponding base [P 6,6,6,14 ][OH] in ethanol. Mix it with an equimolar amount of octanoic acid, stir the reaction at 45°C in a round bottom flask for 12h to completely neutralize, and then remove the solvent by heating and stirring with air purging until constant weight, and obtain a transparent liquid at room temperature Ionic liquid [P 6,6,6,14 ][C 7 h 15 COO].

[0022] H NMR 1 H NMR and carbon spectroscopy 13 C NMR characterization of the synthesized [P 6,6,6,14 ][C 7 h 15 COO]. 1 H NMRδ / ppm (500MHz, DM...

Embodiment 2

[0028] Embodiment 2: trihexyltetradecylphosphonium decanoate ionic liquid ([P 6,6,6,14 ][C 9 h 19 COO]) / buffer solution biphasic system, phytosterol biotransformation prepares AD, specifically as follows: prepare ionic liquid [P by the method for embodiment 1 6,6,6,14 ][C 9 h 19 COO], and H NMR spectroscopy 1 H NMR and carbon spectroscopy 13 Characterization by C NMR.

[0029] Seed liquid culture and fermentation culture were carried out according to the method of Example 1 to prepare resting cells.

[0030] The degradation process of phytosterol side chains catalyzed by resting cells of mycobacteria in an aerobic environment was carried out in a 100mL Erlenmeyer flask with baffles. Resting cells of Mycobacterium sp. NRRL B-3683 pre-cultured and stored frozen at -20°C were taken out as biocatalysts. In order to improve the catalytic rate, 50g L -1 Wet cells and 2g L -1 Glucose was added to Tris-HCl buffer at pH 7.5 and activated at 30°C and 200rpm for 7h. Add 3g L t...

Embodiment 3

[0031] Embodiment 3: trihexyltetradecylphosphonium laurate ionic liquid ([P 6,6,6,14 ][C 11 h 23 COO]) / buffer solution two-phase system biotransformation prepares AD, specifically as follows:

[0032] Prepare ionic liquid [P by the method for embodiment 1 6,6,6,14 ][C 11 h 23 COO], and H NMR spectroscopy 1 H NMR and carbon spectroscopy 13 Characterization by C NMR.

[0033] Seed liquid culture and fermentation culture were carried out according to the method of Example 1 to prepare resting cells.

[0034] The degradation process of phytosterol side chains catalyzed by resting cells of mycobacteria in an aerobic environment was carried out in a 100mL Erlenmeyer flask with baffles. Resting cells of Mycobacterium sp. NRRL B-3683 pre-cultured and stored frozen at -20°C were taken out as biocatalysts. In order to improve the catalytic rate, 50g L -1 Wet cells and 2g L -1 Glucose was added to Tris-HCl buffer at pH 7.5 and activated at 30°C and 200rpm for 7h. Add 3g L to ...

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Abstract

The invention discloses a method for preparing androstenedione (AD) through whole-cell biotransformation of phytosterol in a quaternary phosphonium salt ionic liquid / water two-phase system. The mycobacterium Mycobacterium sp.NRRL B-3683 strain was cultured in seed solution, and then the seed solution was inoculated into the fermentation medium for culture to prepare resting cells, and the resulting resting cells were activated in Tris-HCl buffer containing glucose After incubating for 7 hours, the substrate phytosterol and 0.1vol.% hydrophobic fatty acid quaternary phosphonium salt ionic liquid were put into the ionic liquid / buffer two-phase biotransformation reaction to prepare AD. The solubility of the fatty acid quaternary phosphonium salt ionic liquid used in the present invention to the substrate phytosterol is as high as 483~616g L ‑1 , and increases with the extension of the alkyl side chain of the ionic liquid anion, which is more than 100 times higher than that of ordinary commercial ionic liquids, and can effectively solve the problem of poor water solubility of the substrate, combined with the in situ extraction of the product , improve the conversion efficiency of the process, and use very little ionic liquid material, which is conducive to saving production costs.

Description

technical field [0001] The invention relates to a method for preparing androstenedione by whole-cell biotransformation of phytosterols in a quaternary phosphonium salt ionic liquid / water two-phase system. Background technique [0002] Androst-4-ene-3,17-dione (AD) is an important steroid drug intermediate, which can be used to synthesize hundreds of steroid hormone drugs. Using abundant phytosterols in nature as substrates, the preparation of AD by biotransformation has broad application prospects. However, the degradation of phytosterol side chains by mycobacteria to obtain AD requires a 16-step reaction process catalyzed by 11 functional enzymes, which can only be completed in whole cells so far. There are two main problems: (1) the low water solubility of the sterol substrate; (2) the inhibition and even toxicity of the hydrophobic product AD to the cell activity in the cell, and the inclusion of the substrate particles and cells after precipitation. Generally, a non-aq...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P33/00C12R1/32
CPCC12P33/00
Inventor 关怡新袁俊杰肖霞肖超姚善泾
Owner ZHEJIANG UNIV