阅读说明：本技术 一种利用混合溶剂减少乙基双酮11a羟化过程中杂质的方法 (Method for reducing impurities in hydroxylation process of ethyl diketone 11a by using mixed solvent ) 是由 陈海林 蔡啸 宋盟军 应娟 狄飞飞 汪洋 胡甜 于 2020-05-27 设计创作，主要内容包括：本发明涉及一种利用混合溶剂减少乙基双酮11a羟化过程中杂质的方法,包括如下步骤：将绿僵菌菌种培养获得绿僵菌菌体,收集菌体；收集菌体后投料转化：将底物DL-18-甲基-40雌烯-3,17-二酮采用由DMF和DMSO组成的混合溶剂溶解,将收集的菌体投入到底物中,将底物转化为11a-羟基-18-甲基雌甾-4-烯-3,17-二酮。本发明方法可明显减少乙基双酮生物羟化产物中杂质的生成,尤其是10a-OH乙基双酮和6β-乙基双酮的生成,提升了转化率,并简化了后处理工艺,提高了平均收率。(The invention relates to a method for reducing impurities in a hydroxylation process of ethyl diketone 11a by using a mixed solvent, which comprises the following steps: culturing a metarhizium strain to obtain metarhizium thalli, and collecting the thalli; and (3) feeding and converting after the thalli are collected: dissolving a substrate DL-18-methyl-40 estrene-3, 17-dione in a mixed solvent consisting of DMF and DMSO, putting the collected thalli into the substrate, and converting the substrate into 11 a-hydroxy-18-methylestra-4-ene-3, 17-dione. The method can obviously reduce the generation of impurities in the biological hydroxylation product of the ethyl diketone, particularly the generation of 10a-OH ethyl diketone and 6 beta-ethyl diketone, improves the conversion rate, simplifies the post-treatment process and improves the average yield.)

1. A method for reducing impurities in the hydroxylation process of ethyl diketone 11a by using a mixed solvent is characterized in that: the method comprises the following steps:

(1) culturing a metarhizium strain to obtain metarhizium thalli, and collecting the thalli;

(2) and (3) feeding and converting after the thalli are collected: dissolving a substrate DL-18-methyl-40 estrene-3, 17-dione in a mixed solvent consisting of DMF and DMSO, putting the collected thalli into the substrate, and converting the substrate into 11 a-hydroxy-18-methylestra-4-ene-3, 17-dione.

2. The method of claim 1, wherein: the conversion time of the step (2) is 24-50 h.

3. The method of claim 1, wherein: the conversion temperature of the step (2) is 20-40 ℃.

4. The method of claim 1, wherein: in the step (2), the volume ratio of DMF: DMSO is 1-20: 1.

5. the method of claim 1, wherein: the dissolving temperature of the substrate is 40-90 ℃, and the amount of the used mixed solvent is 1-20 times of the feeding amount of the substrate.

6. The method of claim 1, wherein: the mass ratio of the substrate to the thallus is 1-10: 1.

7. the method of claim 1, wherein: the culture method in the step (1) comprises the following steps:

(1.1) inoculating metarhizium anisopliae to a Potato Dextrose Agar (PDA) culture medium for culture;

(1.2) inoculating the strain into a shake flask filled with a liquid culture medium under an aseptic condition, and performing rotary shaking culture to obtain a seed solution;

and (1.3) transferring the cultured shake flask seed solution to a small pot for culture to obtain thalli.

8. The method of claim 7, wherein: in the step (1.1), the culture temperature of the green muscardine strain is 25-32 ℃, and the culture time is 3-10 days;

in the step (1.2), the rotation speed of shaking table culture is 100-;

in the step (1.3), the culture time of the small pot is 16-48h, the culture temperature is 25-32 ℃, and the ventilation volume is 0.2-1.2 vvm.

9. The method of claim 7, wherein: the liquid culture medium comprises 7.5-10 g/L glucose, 7.5-10 g/L soybean meal and 3.5-5 g/L silkworm chrysalis meal.

10. The method of claim 1, wherein: the conversion rate of 11a-OH ethyl diketone reaches 60 percent, and the yield reaches 50 percent.

Technical Field

The invention relates to a method for reducing impurities in a hydroxylation process of ethyl diketone 11a by using a mixed solvent.

Background

Disclosure of Invention

The invention aims to provide a simple and effective method for reducing impurities in the hydroxylation process of ethyl diketone 11a by using a mixed solvent.

In order to achieve the above object, the technical solution provided by the present invention is as follows:

a method for reducing impurities in a hydroxylation process of ethyl diketone 11a by using a mixed solvent comprises the following steps:

(1) culturing a metarhizium strain to obtain metarhizium thalli, and collecting the thalli;

(2) and (3) feeding and converting after the thalli are collected: dissolving a substrate DL-18-methyl-40 estrene-3, 17-dione in a mixed solvent consisting of DMF and DMSO, putting the collected thalli into the substrate, and converting the substrate into 11 a-hydroxy-18-methylestra-4-ene-3, 17-dione.

According to the scheme, the conversion time of the step (2) is 24-50 h.

According to the scheme, the conversion temperature of the step (2) is 20-40 ℃.

According to the scheme, in the step (2), the volume ratio of DMF: DMSO is 1-20: 1.

according to the scheme, the dissolving temperature of the substrate is 40-90 ℃, and the amount of the used mixed solvent is 1-20 times of the feeding amount of the substrate.

According to the scheme, the mass ratio of the substrate to the thallus is 1-10: 1.

according to the scheme, the culture method in the step (1) comprises the following steps:

(1.1) inoculating metarhizium anisopliae to a Potato Dextrose Agar (PDA) culture medium for culture;

(1.2) inoculating the strain into a shake flask filled with a liquid culture medium under an aseptic condition, and performing rotary shaking culture to obtain a seed solution;

and (1.3) transferring the cultured shake flask seed solution to a small pot for culture to obtain thalli.

According to the scheme, the culture temperature of the green muscardine strain in the step (1.1) is 25-32 ℃, and the culture time is 3-10 days.

According to the scheme, the liquid culture medium comprises 7.5-10 g/L glucose, 7.5-10 g/L soybean meal and 3.5-5 g/L silkworm chrysalis meal.

According to the scheme, in the step (1.2), the rotation speed of shaking culture is 100-200rpm, the culture temperature is 25-32 ℃, and the culture time is 10-20 h.

According to the scheme, in the step (1.3), the culture time of the small pot is 16-48h, the culture temperature is 25-32 ℃, and the ventilation volume is 0.2-1.2 vvm.

According to the scheme, the conversion rate of the 11a-OH ethyl diketone obtained by the treatment of the scheme reaches 60%, the yield reaches 50%, and the product purity is more than or equal to 99.5%.

According to the invention, by utilizing the mixed solvent of DMF and DMSO in a certain ratio, the metabolic pathway of hydroxylation of Metarrhizium anisopliae is optimized, the generation of impurities is reduced, particularly the generation of 10a-OH ethyl diketone and 6 beta-ethyl diketone, the conversion rate is improved, the post-treatment process is simplified, the average yield is improved, and the product purity is high and can reach 99.9%.

In the process of synthesizing 11a-OH ethyl diketone by ethyl diketone biocatalysis, the following impurities are mainly generated: 6 beta-OH ethyl diketone, 10a-OH ethyl diketone and 6 beta, 10a-OH ethyl diketone, wherein the 10a-OH ethyl diketone is difficult to separate in the actual industrial production due to the similar properties with the 11a-OH ethyl diketone, in the experimental process, the mixed solvent of DMF and DMSO is used for carrying out charging conversion, the generation amount of 10a-OH ethyl diketone and 6 beta-ethyl diketone is found to be remarkably reduced, the yield of 11a-OH ethyl diketone is improved to a certain extent, and the obtained conversion solution is concentrated and crystallized to obtain the 11a-OH ethyl diketone with the purity of more than or equal to 99.5 percent.

The invention has the following beneficial effects:

the method can obviously reduce the generation of impurities in the biological hydroxylation product of the ethyl diketone, particularly the generation of 10a-OH ethyl diketone and 6 beta-ethyl diketone, improves the conversion rate, simplifies the post-treatment process, improves the average yield, has high substrate conversion rate, and has the conversion rate of 60 percent and the yield of 50 percent.

The operation is simple, special separation and purification equipment is not needed in the product post-treatment and separation process, and qualified products can be obtained through normal concentration and crystallization.

Drawings

FIG. 1 is a process flow diagram of the present invention.

Detailed Description

Comparative example one:

inoculating Metarrhizium anisopliae to potato glucose agar (PDA) culture medium, and culturing at 25 deg.C for 3-10 days.

Transferring a loop to a shake culture medium for culture at a shake culture rotation speed of 150rpm at a culture temperature of 27 ℃ in a liquid culture medium of 8.5g/L glucose, 8.5g/L soybean meal and 4g/L silkworm chrysalis meal for 14 h.

The culture time in a small pot is 16h, the culture temperature is 27 ℃, and the ventilation volume is 0.1 vvm.

And (2) collecting thalli, feeding and converting, wherein the mass ratio of a substrate to the thalli is 1:3, the substrate DL-18-methyl-40 estrene-3, 17-diketone is dissolved by adopting 500mL of DMF (dimethyl formamide) solvent, the temperature is 40 ℃, the conversion is carried out for 24h, the feeding concentration is 4g/L, the feeding amount is 52g, the conversion is carried out for 24h at the temperature of 24 ℃, the temperature is increased to 70 ℃, the maintenance is carried out for 5min, and the reaction is stopped. After the thalli are obtained by suction filtration, 500ml ethyl acetate is used for extraction for 30min, the extraction is repeated for three times, the combined extraction liquid is concentrated and crystallized, the conversion rate is 52.2 percent, the contents of known impurities 6 beta-OH ethyl diketone and 10a-OH ethyl diketone are respectively 8.5 percent, 7.2 percent and 18.5 percent, the qualified product is obtained after purification, and the yield is 43.2 percent.

Comparative example two:

inoculating Metarrhizium anisopliae into potato glucose agar (PDA) culture medium, and culturing at 28 deg.C for 3-10 days.

Transferring a loop to a shake culture medium for culture at a shake culture rotation speed of 150rpm at a culture temperature of 27 ℃ in a liquid culture medium of 8.5g/L glucose, 8.5g/L soybean meal and 4g/L silkworm chrysalis meal for 14 h.

The culture time in small pot was 16h, the culture temperature was 27 ℃ and the aeration rate was 0.2 vvm.

And (2) collecting thalli, feeding and converting, wherein the mass ratio of a substrate to the thalli is 1:5, the substrate DL-18-methyl-40 estrene-3, 17-diketone is dissolved by 500mL of DMSO solvent, the temperature is 50 ℃, the conversion is carried out for 24h, the feeding concentration is 4g/L, the feeding amount is 58g, the conversion is carried out for 24h at the temperature of 24 ℃, the temperature is increased to 70 ℃, the maintenance is carried out for 5min, and the reaction is stopped. After the thalli are obtained by suction filtration, the thalli are extracted for 30min by using 500ml of ethyl acetate, the extraction is repeated for three times, the combined extract is concentrated and crystallized, the conversion rate is 54.9 percent, the contents of known impurities 6 beta-OH ethyl diketone and 10a-OH ethyl diketone are respectively 7.6 percent, 8.3 percent and 19.7 percent, and the qualified product 27.96g is obtained after purification, and the yield is 48.2 percent.