A method for applying enzyme solution suitable for steroidal ketoreductase catalysis

Abstract

The present invention relates to a method for applying enzyme solution suitable for enzyme catalysis of steroidal compounds. The present invention provides a process for applying enzyme solution of normal batch enzyme catalysis. The emulsifier added in the reaction system will cause the substrate and the product to form an emulsification with water. The emulsification of the product can be destroyed by adding a defoaming agent auxiliary agent, so that the product is peeled off from the emulsified state under the action of the defoaming agent auxiliary agent to form an insoluble powdery solid product. At the same time, it will not affect the substrate reaction and is also helpful to promote the reaction in the positive direction. After that, the enzyme solution is obtained by filtering to apply the batch reaction. The cost can be effectively reduced and the yield can be further improved.

Description

Technical Field

[0001] The invention relates to an enzyme solution application method suitable for steroid compound ketoreductase catalysis. Background Art

[0002] Steroidal compounds are water-insoluble cyclopentane polyhydrogen phenanthrene nucleus compounds, and their basic structure is as follows: Figure 1 As shown, they are generally divided into five categories: estrogen, androgen, progesterone, mineralocorticoid and glucocorticoid. Estrogen has the function of maintaining female secondary sexual characteristics and promoting their development, such as breast development, body shape changes, fat accumulation, pregnancy control, lactation, etc.; androgen has the function of promoting the maturation of male secondary sexual characteristics, maintaining normal sexual desire and reproductive function, and promoting sperm development and maturation; progesterone provides basic protection for the implantation of the fertilized egg and the maintenance of pregnancy; mineralocorticoids can act on the kidneys, sweat glands, salivary glands and gastrointestinal tract to maintain electrolyte and extracellular fluid balance; glucocorticoids have the function of regulating blood pressure, protein and lipid metabolism, anti-inflammatory and inhibiting immune response.

[0003] At present, the synthesis of steroidal compound drugs is mainly chemical and biological. Chemical synthesis generally requires multi-step reactions to complete, and requires the use of a large amount of organic solvents, catalysts, heating, pressurization and other conditions to participate in the reaction. It has the disadvantages of high cost, low yield, and high pollution. The biological method uses molecular biology technology to construct engineered bacteria for biotransformation to prepare steroidal intermediates. Compared with the chemical synthesis method for preparing steroidal intermediates, the preparation conditions of biocatalysis and transformation are relatively mild, and it has the advantages of low cost, low pollution, and high yield.

[0004] Enzyme catalysis belongs to biological catalytic transformation, which has the advantages of high efficiency, high specificity, mild reaction conditions and environmental friendliness. The hydroxylation of steroidal compounds is widely present in microorganisms, such as monohydroxylation of C6, C7, C11, C12 or C17 and dihydroxylation occurring at two sites simultaneously. For example, the redox reaction between the keto group CH=O and the alcohol group CH2-OH at the C17 site can be completed by 17β-hydroxysteroid dehydrogenase, and the conversion between androstenedione, a steroidal drug intermediate with low physiological activity, and testosterone and boldenone, steroidal hormone drugs with high physiological activity, etc.

[0005] Enzymes generally react with substrates in aqueous solution, and it is difficult to recover the enzymes after the reaction. The existing method for reusing enzymes is mainly enzyme immobilization technology, but this method is too cumbersome, has a high R&D cost, and most of them are only applicable to water-soluble substrates. The present invention provides an enzyme solution application method suitable for steroid compound enzyme catalysis, which is applied by applying the enzyme solution obtained after cooling and filtering after the enzyme catalysis of a normal batch. Compared with the normal batch, the applied batch only needs to add glucose and part of the enzyme to reach the substrate feeding level of the normal batch, which can effectively reduce the cost and greatly improve the yield compared with the normal batch.

[0006] Summary of the invention

[0007] The main content of the present invention is to provide an enzyme solution application method suitable for steroid compound enzyme catalysis.

[0008] In order to achieve the above purpose, the technical solution provided by the present invention is as follows:

[0009] Provided is a method for applying an enzyme solution suitable for enzyme catalysis of steroidal compounds, comprising the following steps:

[0010] (1) A normal batch enzyme catalytic reaction system is constructed. After the catalysis is completed, the temperature is lowered to 5-10° C. and filtered to obtain an enzyme solution. The normal batch enzyme catalytic reaction system is: dipotassium hydrogen phosphate 1.800-5.900 g / L, potassium dihydrogen phosphate 0.775-2.325 g / L, glucose 38.500-115.500 g / L, emulsifier 13.500-40.688 g / L, defoamer auxiliary 0.050-0.150 g / L, keto reductase 3-4 g / L, glucose dehydrogenase 0.5-1.0 g / L, NADP + 0.15-0.20g / L, steroid substrate 50-100g / L;

[0011] (2) Add 50-100 g / L of substrate, 77-154 g / L of glucose, ketoreductase and NADP to the obtained enzyme solution in sequence. + , obtaining an enzyme solution application system;

[0012] (3) The enzyme solution prepared in step (2) is applied to the reaction system to catalyze the product.

[0013] According to the above scheme, in step (2), ketoreductase and NADP + The added amount is 10%-50% of the enzyme amount in a normal batch enzyme catalytic reaction system.

[0014] According to the above scheme, the catalytic conditions are: controlling the pH at 6.75-6.95 and the temperature at 25-35°C.

[0015] According to the above scheme, the average yield of ethyl hydroxylate can reach 95%, and the product purity is ≥98.5%; the average yield of testosterone can reach 93%, and the product purity is ≥99.5%; the average yield of boldenone can reach 95%, and the product purity is ≥99.0%; the average yield of estradiol can reach 94%, and the product purity is ≥98.5%

[0016] According to the above scheme, the emulsifier can be selected from one or more of Tween 60, Tween 80, and Triton X-100. Specifically, for ethyl condensate, it can be selected from Tween 60 and Triton X-100 in combination, for androstenedione, it can be selected from Triton X-100 or Tween 60, for 1,4-androstenedione (ADD), it can be selected from Tween 60, and for estrone, it can be selected from Tween 80.

[0017] According to the above scheme, the steroid compound substrate is ethyl condensate, androstenedione, 1,4-androstenedione (ADD), and estrone. The structural formulas of ethyl condensate, androstenedione, 1,4-androstenedione (ADD), and estrone are as follows: Figure 2 shown.

[0018]

[0019] According to the above scheme, the defoamer auxiliary agent is a combination of one or more of a polyether defoamer, a polysiloxane defoamer, an esterification defoamer, and a polyoxypropylene glyceryl ether defoamer. For ethyl condensate, the defoamer auxiliary agent can be a polyoxypropylene glyceryl ether defoamer, for androstenedione, the defoamer auxiliary agent can be an esterification defoamer, for 1,4-androstenedione (ADD), the defoamer auxiliary agent can be a polyoxypropylene glyceryl ether defoamer, and for estrone, the defoamer auxiliary agent can be a polysiloxane defoamer.

[0020] The present invention provides a process for applying enzyme solution catalyzed by a normal batch of enzymes. The emulsifier added to the reaction system will cause the substrate and the product to form an emulsification with water. The emulsification of the product can be destroyed by adding a defoaming agent, so that the product is peeled off from the emulsified state under the action of the defoaming agent to form an insoluble powdery solid product. At the same time, it will not affect the substrate reaction and is also helpful to promote the reaction in the positive direction. After that, the enzyme solution is obtained by filtering and then applied to the batch reaction.

[0021] In addition, by adding a defoaming agent to a normal batch reaction system and applying a batch reaction, the product content in the filtrate of the system reaches saturation, which can reduce the effect of the use of emulsifiers in the system on the product yield, thereby reducing product losses and correspondingly increasing the yield.

[0022] For example, in the synthesis of steroid intermediate ethyl hydroxylate, the average yield in a normal enzyme reaction system is about 80%. A portion of the product in the enzyme solution is dissolved in the system and cannot be obtained. In addition, the enzyme is unstable and easily inactivated, and the feed amount cannot be further increased. Discarding the filtrate will cause a certain amount of waste.

[0023] The present invention improves the yield of the normal batch by adding a defoaming agent, reaching about 90%. At the same time, the enzyme solution obtained by filtering the normal batch is applied, and only substrate, glucose, ketoreductase and NADP are added. + (The dosage only needs 10%-50% of the normal batch), it can reach the feeding level of the normal batch, the cost is lower than before, and the yield can be further improved to about 95%, and the purity is consistent with the normal batch.

[0024] The advantages of this method are as follows:

[0025] 1. The addition of defoaming agent to the system of the present invention is conducive to product collection, and the enzyme solution can be used in parallel. The average reaction yield is greatly improved compared with the normal batch, and the same purity and impurity level as the normal batch can be achieved.

[0026] 2. To apply the enzyme solution, you only need to add substrate, glucose, part of the ketoreductase and NADP+, which can effectively reduce costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Steranine core structure of steroidal compounds

[0028] Figure 2 The structural formula of the steroidal compound substrate

[0029] Figure 3 Schematic diagram of the ethyl hydroxylase catalytic reaction of Example 1

[0030] Figure 4 HPLC spectrum of the normal batch product of Example 1

[0031] Figure 5 HPLC spectrum of the batch product applied to Example 1

[0032] Figure 6 HPLC spectrum of normal batch product of Example 2

[0033] Figure 7 HPLC spectrum of the batch product applied to Example 2

[0034] Figure 8 Schematic diagram of the testosterone enzyme catalytic reaction in Example 3

[0035] Fig. 9 HPLC spectrum of the normal batch product of Example 3

[0036] Fig.10 HPLC spectrum of the batch product applied to Example 3

[0037] Fig.11 Schematic diagram of the estradiol enzyme catalyzed reaction in Example 4

[0038] Fig.12 HPLC spectrum of the normal batch product of Example 4

[0039] Fig.13 HPLC spectrum of the batch product applied to Example 4

[0040] Fig.14 Schematic diagram of the boldenone enzyme catalyzed reaction in Example 5

[0041] Fig.15 HPLC spectrum of the normal batch product of Example 5

[0042] Fig.16 The HPLC spectrum of the batch product applied to Example 5. DETAILED DESCRIPTION

[0043] Embodiment 1:

[0044] Reaction diagram Figure 3 , weigh 30g glucose, 0.678g potassium dihydrogen phosphate, 1.605g potassium hydrogen phosphate into a three-necked flask, then add 300mL of water, stir for 0.5h, then add 1g Tween 60, 10.5g Triton, 0.04g polyoxypropylene glycerol ether defoamer, 19.5g ethyl condensate, stir, wait until the pH stabilizes at 6.85 and the temperature is 30°C, then add 0.975g ketoreductase, 0.195g glucose dehydrogenase, 48.75mg NADP + Enzyme solution for transformation.

[0045] After the catalysis was completed, the temperature was lowered to 5°C and filtered to obtain enzyme solution and crude ethyl hydroxylate. The crude product was slurried in 195 mL of water for 0.5 h, filtered, and dried to obtain 17.84 g of ethyl hydroxylate with a yield of 91.49% and a purity of 98.860% as measured by HPLC. Figure 4 .

[0046] To the obtained enzyme solution, 19.5 g of ethyl condensate, 30 g of glucose, 0.4875 g of ketoreductase and 24.375 mg of NADP were added in sequence. + The reaction started, pH stabilized at 6.85, and temperature was 30°C.

[0047] After the catalysis was completed, the temperature was lowered to 5°C and kept warm for 1 hour. The enzyme solution (used as waste liquid treatment) and the crude ethyl hydroxylate were filtered. The crude product was slurried in 195 mL of water for 0.5 hour, filtered, and dried to obtain 19.03 g of ethyl hydroxylate with a yield of 97.59%. The purity was 97.810% as measured by HPLC. Figure 5 .

[0048] Embodiment 2:

[0049] Weigh 150g glucose, 3.39g potassium dihydrogen phosphate, 8.025g potassium hydrogen phosphate into a three-necked flask, then add 1500mL of water, stir for 0.5h, then add 5g Tween 60, 52.5g Triton X-100, 0.2g polyoxypropylene glycerol ether defoamer, 97.5g ethyl condensate, stir, wait until the pH stabilizes at 6.85 and the temperature is 30°C, then add 4.875g ketoreductase, 0.975g glucose dehydrogenase, 243.75mg NADP + Enzyme solution for transformation.

[0050] After the catalysis was completed, the temperature was lowered to 10°C and filtered to obtain enzyme solution and crude ethyl hydroxylate. The crude product was slurried in 975 mL of water for 0.5 h, filtered, and dried to obtain 89.35 g of ethyl hydroxylate with a yield of 91.64% and a purity of 98.886% as measured by HPLC. Figure 6 .

[0051] To the obtained enzyme solution, 97.5 g of ethyl condensate, 150 g of glucose, 2.4375 g of ketoreductase and 121.875 mg of NADP were added in sequence. + , pH stable at 6.85, temperature 30℃.

[0052] After the catalysis was completed, the temperature was lowered to 10°C, kept warm for 1 hour, and filtered to obtain enzyme solution (for waste liquid treatment) and crude ethyl hydroxylate. The crude product was added with 975 mL of water and slurried for 0.5 hour, filtered, and dried to obtain 94.82 g of ethyl hydroxylate with a yield of 97.25%. The purity was 98.589% as measured by HPLC. Figure 7 .

[0053] Embodiment three:

[0054] Reaction diagram Figure 8 , weigh 22.5g glucose, 0.339g potassium dihydrogen phosphate, 0.8025g potassium dihydrogen phosphate and add them to a three-necked flask, then add 200mL of water, stir for 0.5h, then add 6g Triton X-100, 0.02g esterified defoamer, 15g androstenedione, stir, wait until the pH stabilizes at 6.8 and the temperature is 30℃, then add 0.75g ketoreductase, 0.15g glucose dehydrogenase, 37.5mg NADP+ Enzyme solution for transformation.

[0055] After the catalysis was completed, the temperature was lowered to 10°C and filtered to obtain enzyme solution and crude testosterone. The crude product was purified by ethyl acetate to obtain 14.06 g of testosterone with a yield of 93.73%. The purity measured by HPLC was 99.690%. Fig. 9 .

[0056] To the obtained enzyme solution, 15 g of androstenedione, 22.5 g of glucose, 0.075 g of ketoreductase and 3.75 mg of NADP were added in sequence. + , pH stable at 6.85, temperature 30℃.

[0057] After the catalysis was completed, the temperature was lowered to 10°C and filtered to obtain enzyme solution (for waste liquid treatment) and crude testosterone. The crude product was purified by ethyl acetate to obtain 14.61 g of testosterone with a yield of 97.4%. The purity measured by HPLC was 99.818%. Fig.10 .

[0058] Embodiment 4:

[0059] Reaction diagram Fig.11 , weigh 30g glucose, 0.339g potassium dihydrogen phosphate, 0.8025g potassium dihydrogen phosphate and add them into a three-necked flask, then add 300mL of water, stir for 0.5h, then add 10g Tween 80, 0.03g polysiloxane defoamer, 15g estrone, stir, wait until the pH stabilizes at 6.85 and the temperature is 30℃, then add 0.9g ketoreductase, 0.15g glucose dehydrogenase, 45mg NADP + Enzyme solution for transformation.

[0060] After the catalysis was completed, the temperature was lowered to 10°C and filtered to obtain enzyme solution and crude estradiol. The crude product was purified by ethyl acetate to obtain 13.65 g of estradiol with a yield of 91.00%. The purity measured by HPLC was 99.854%. Fig.12 .

[0061] To the obtained enzyme solution, 15 g of estrone, 30 g of glucose, 0.45 g of ketoreductase and 22.5 mg of NADP were added in sequence. + The reaction started, pH stabilized at 6.85, and temperature was 30°C.

[0062] After the catalysis was completed, the temperature was lowered to 10°C and kept warm for 0.5h. The enzyme solution (used as waste liquid treatment) and crude estradiol were filtered and purified with ethyl acetate to obtain 14.22g of estradiol with a yield of 94.80%. The purity was 99.894% as measured by HPLC. Fig.13 .

[0063] Embodiment five:

[0064] Reaction diagram Fig.14 , weigh 30g glucose, 0.339g potassium dihydrogen phosphate, 0.8025g potassium hydrogen phosphate into a three-necked flask, then add 300mL of water, stir for 0.5h, then add 10g Tween 60, 0.03g polyoxypropylene glycerol ether defoamer, 15g 1,4-androstenedione, stir, wait until the pH stabilizes at 6.85 and the temperature is 30℃, then add 0.9g ketoreductase, 0.15g glucose dehydrogenase, 45mg NADP + Enzyme solution for transformation.

[0065] After the catalysis was completed, the temperature was lowered to 10°C and filtered to obtain enzyme solution and crude boldenone. The crude product was purified by ethyl acetate to obtain 13.98 g boldenone with a yield of 93.20%. The purity measured by HPLC was 99.703%. Fig.15 .

[0066] To the obtained enzyme solution, 15 g of 1,4-androstenedione, 30 g of glucose, 0.27 g of ketoreductase and 22.5 mg of NADP were added in sequence. + The reaction started, pH stabilized at 6.85, and temperature was 30°C.

[0067] After the catalysis was completed, the temperature was lowered to 10°C and kept warm for 0.5h. The enzyme solution (used as waste liquid treatment) and crude boldenone were obtained by filtration. The crude product was purified by ethyl acetate to obtain 14.35g boldenone with a yield of 95.67%. The purity was 99.688% as measured by HPLC. Fig.16 .

Claims

1. A method for applying an enzyme solution suitable for steroidal compound enzymatic catalysis, comprising the following steps: (1) Build a normal batch enzyme catalytic reaction system. After the catalysis is completed, cool it to 5-10°C and filter to obtain an enzyme solution. The normal batch enzyme catalytic reaction system is: dipotassium hydrogen phosphate 1.800-5.900 g / L, potassium dihydrogen phosphate 0.775-2.325 g / L, glucose 38.500-115.500 g / L, emulsifier 13.500-40.688 g / L, defoamer auxiliary 0.050-0.150 g / L, keto reductase 3-4 g / L, glucose dehydrogenase 0.5-1.0 g / L, NADP + 0.15-0.20 g / L, steroidal compound substrate 50-100 g / L, the steroidal compound substrate is ethyl condensate, androstenedione, 1,4-androstenedione or estrone, and the structural formulas of ethyl condensate, androstenedione, 1,4-androstenedione and estrone are as follows: For the ethyl condensate, the emulsifier is a combination of Tween 60 and Triton X-100, and the defoaming agent adjuvant is a polyoxypropylene glycerol ether defoaming agent; For androstenedione, the emulsifier is Triton X-100 or Tween 60, and the defoaming agent adjuvant is an esterified defoaming agent; For 1,4-androstenedione, the emulsifier is Tween 60, and the defoaming agent adjuvant is polyoxypropylene glycerol ether defoaming agent, For estrone, the emulsifier is Tween 80, and the defoaming agent adjuvant is a polysiloxane defoaming agent; (2) Add 50-100 g / L substrate, 77-154 g / L glucose, ketoreductase and NADP to the obtained enzyme solution in sequence. + , and obtain the enzyme solution application system, ketoreductase and NADP + The amount of enzyme added is 10%-50% of the amount of enzyme in the normal batch enzyme catalytic reaction system; (3) The enzyme solution prepared in step (2) is applied to the reaction system to catalyze the product.

2. The enzyme solution application method according to claim 1, characterized in that: The catalytic conditions are: controlling the pH value at 6.75-6.95 and the temperature at 25-35°C.

3. The enzyme solution application method according to claim 1, characterized in that: The average yield of ethyl hydroxylate can reach 95%, and the product purity is ≥98.5%; the average yield of testosterone can reach 93%, and the product purity is ≥99.5%; the average yield of boldenone can reach 95%, and the product purity is ≥99.0%; the average yield of estradiol can reach 94%, and the product purity is ≥98.5%.

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