CN101570775B - Method for preparing 3 beta, 7 beta-dihydroxy-15 beta, 16 beta-methylene-5-androstene-17-ketone by microbial transformation method - Google Patents

Abstract

The invention provides a method for preparing 3β, 7β-dihydroxy-15β, 16β-methylene-5-androsten-17-one by microbial transformation method. The method uses Botryodiplodia malorum CBS13450 as the strain, including preparing resting cells Transformation system and the step of substrate transformation, wherein said quiescent cell transformation system comprises a buffer, said buffer is selected from phosphate buffer, acetate buffer, phosphate-citric acid buffer, dihydrogen phosphate Potassium-sodium hydroxide buffer or citric acid-sodium hydroxide-hydrochloric acid buffer. The conversion result of this system under the optimal conversion conditions was that 0.3% of the substrate was completely converted within 45 hours.

Description

Method for preparing 3β, 7β-dihydroxy-15β, 16β-methylene-5-androsten-17-one by microbial transformation method

technical field

The invention belongs to the field of microbial transformation, in particular to a method for converting 3β-hydroxy-15β, 16β-methylene-5-androsten-17-one into 3β, 7β-dihydroxy-15β, 16β-methylene by using microorganisms - The method of 5-androsten-17-one.

Background technique

3β, 7β-dihydroxy-15β, 16β-methylene-5-androsten-17-one is an important pharmaceutical intermediate, which can be used in the synthesis of contraceptive drospirenone and diuretic spirone. The synthesis of 3β, 7β-dihydroxy-15β, 16β-methylene-5-androsten-17-one mainly utilizes microbial conversion of 3β-hydroxy-15β, 16β-methylene-5-androsten-17-one and become. The literature Steroid hydroxylations with Botryodiplodiamalorum and Colletotrichum lini (steroids 71 (2006) 429-434) and the patent United States Patent 4 614 616 (Sep.30, 1986) have reported that Botryodiplodia malorum can convert 3β-hydroxyl-15β, 16β-methylene -5-Androsten-17-one is 3β,7β-dihydroxy-15β,16β-methylene-5-androsten-17-one.

The resting cell transformation method refers to that under the appropriate medium and culture conditions, after the bacteria are fully grown, the bacteria are separated and washed, then suspended in an appropriate buffer, and then the substrate is added to carry out the transformation reaction. Compared with the conversion in direct fermentation broth, this method generally has the following advantages: it does not need to be operated in a sterile state, and the conditions are easy to control; it can freely change the ratio of the substrate and the amount of bacteria in the reaction solution; The reaction time eliminates the influence of the substances in the medium and the substances produced during the growth of the bacteria on the transformation, and reduces the by-products; the transformed liquid has less impurities and is easier to separate and purify.

Patent 4 614 616 (Sep.30, 1986) adopted the direct conversion method in the article, and the result obtained was that 0.2% of the substrate was converted completely within 48 hours.

However, Steroid hydroxylations with Botryodiplodia malorum and Colletotrichum lini (steroids 71 (2006) 429-434) uses a static cell transformation method, and the buffer used and some transformation conditions are not clear. The result obtained is that 0.1% of Substrate conversion is complete.

Contents of the invention

In this paper, a high-efficiency conversion of 3β-hydroxy-15β, 16β-methylene-5-androsten-17-one to 3β, 7β-dihydroxy-15β, 16β-methylene-5-androsten-17- A quiescent cell transformation method for ketones, the method uses Botryodiplodia malorum CBS13450 as a bacterial species, comprising the steps of preparing a quiescent cell transformation system and substrate transformation, wherein the quiescent cell transformation system comprises a buffer, and the buffer is selected from From phosphate buffer, acetate buffer, phosphate-citric acid buffer, potassium dihydrogen phosphate-sodium hydroxide buffer or citric acid-sodium hydroxide-hydrochloric acid buffer.

Wherein the buffer is preferably potassium dihydrogen phosphate-sodium hydroxide buffer.

The concentration of potassium dihydrogen phosphate-sodium hydroxide buffer solution is 0.01N-0.1N, preferably 0.02N; the pH value of potassium dihydrogen phosphate-sodium hydroxide buffer solution is 4.0-9.0, preferably 6.0.

Wherein the condition of the fermentation culture is: the filling volume is 10-100ml/250ml (substratum/shake flask), preferably 40ml/250ml (substratum/shake flask); the rotating speed is 100-300r/min, preferably 250r/min ; The temperature is 0-50°C, preferably 25-35°C, more preferably 30°C.

The biotransformation method of the present invention converts 3β-hydroxy-15β, 16β-methylene-5-androsten-17-one into 3β, 7β-dihydroxy-15β, 16β-methylene-5-androstene The specific steps of the method of -17-ketone are as follows:

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelium, and the mycelium is washed with deionized water to obtain clean mycelium, the obtained mycelium is suspended in the prepared buffer solution, and 0.3% of the substrate 3β-hydroxyl-15β, 16β -Methylene-5-androsten-17-one, add 0.5% glucose, the filling volume is 10-100ml/250ml (culture medium/shake flask), the rotating speed is 100-300r/min, and the temperature is 0- Conversion started at 50 °C.

The present invention adopts resting cell transformation method to biotransform and synthesize 3β, 7β-dihydroxy-15β, 16β-methylene-5-androsten-17-one. Compared with the prior art, the present invention obviously shortens the conversion time and improves the conversion efficiency.

Detailed ways

Example 1

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.02N, pH value 6.0) (30ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start conversion, the speed is 200r/min , the temperature is 0°C, under this condition, the transformation takes 45h, and the conversion rate of the substrate is 35%.

Example 2

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.02N, pH value 6.0) (30ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start conversion, the speed is 200r/min , the temperature was 30°C, and the transformation was carried out for 45h under this condition, and the conversion rate of the substrate was 95%.

Example 3

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.02N, pH value 6.0) (30ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start conversion, the speed is 200r/min , the temperature was 50°C, and the transformation was carried out for 45h under this condition, and the conversion rate of the substrate was 41%.

Example 4

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.02N, pH value 6.0) (30ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start conversion, the speed is 100r/min , the temperature was 30°C, and the transformation was carried out for 45h under this condition, and the conversion rate of the substrate was 79%.

Example 5

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.02N, pH value 6.0) (30ml), add 0.3% substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start conversion, the speed is 250r/min , the temperature was 30°C, and the transformation was carried out for 45h under this condition, and the conversion rate of the substrate was 97%.

Example 6

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.02N, pH value 6.0) (30ml), add 0.3% of the substrate 3β-hydroxy-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start the conversion, and the rotation speed is 300r/min , the temperature was 30°C, and the transformation was carried out for 45h under this condition, and the conversion rate of the substrate was 90%.

Example 7

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 40ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.02N, pH value 6.0) (40ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start the conversion, and the rotation speed is 250r/min , the temperature was 30°C, and the transformation was carried out for 45h under this condition, and the conversion rate of the substrate was 99%.

Example 8

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 100ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.02N, pH value 6.0) (100ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start the conversion, and the rotation speed is 250r/min , the temperature was 30°C, and the transformation was carried out for 45h under this condition, and the conversion rate of the substrate was 75%.

Example 9

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the liquid volume is 10ml/250ml (culture medium/shake flask), and the mature fermentation broth mycelium is obtained by culturing at a speed of 200r/min and a temperature of 28°C for 24 hours .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.02N, pH value 6.0) (10ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% of glucose, start conversion, the speed is 250r/min , the temperature was 30°C, and the transformation was carried out for 45h under this condition, and the conversion rate of the substrate was 95%.

Example 10

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 40ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.02N, pH value 6.0) (40ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start the conversion, and the rotation speed is 250r/min , the temperature was 30°C, and the transformation was carried out for 45h under this condition, and the conversion rate of the substrate was 99%.

Example 11

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid was centrifuged to obtain mature mycelia, and the mycelium was washed 3 times with deionized water to obtain clean mycelia, and the obtained mycelium was suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.01N, pH value 6.0) (30ml), add 0.3% substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start conversion, the speed is 250r/min , the temperature was 30°C, and the transformation was carried out for 45h under this condition, and the conversion rate of the substrate was 85%.

Example 12

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.1N, pH value 6.0) (30ml), add 0.3% substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start conversion, the speed is 250r/min , the temperature was 30°C, and the transformation was carried out for 45h under this condition, and the conversion rate of the substrate was 64%.

Example 13

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.02N, pH value 4.0) (30ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start the conversion, and the rotation speed is 250r/min , the temperature was 30°C, and the conversion was carried out for 45 hours under this condition, and the conversion rate of the substrate was 83%.

Example 14

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.02N, pH value 9.0) (30ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start conversion, the speed is 250r/min , the temperature was 30°C, and the transformation was carried out for 45h under this condition, and the conversion rate of the substrate was 70%.

Example 15

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.02N, pH value 8.0) (30ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start the conversion, the speed is 250r/min , the temperature was 30°C, and the transformation was carried out for 45h under this condition, and the conversion rate of the substrate was 34%.

Example 16

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.02N, pH value 6.0) (30ml), add 0.3% substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start the conversion, the speed is 50r/min , the temperature was 30°C, 5 was transformed under this condition for 45h, and the conversion rate of the substrate was 41%.

Example 17

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the gained mycelium is suspended in the prepared potassium dihydrogen phosphate-sodium hydroxide buffer solution (0.2N, pH value 6.0) (30ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start conversion, the speed is 200r/min , the temperature was 30°C, and the transformation was carried out for 45h under this condition, and the conversion rate of the substrate was 29%.

Example 18

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the gained mycelium is suspended in a prepared phosphate buffer (0.05N, pH value 6.0) ( 30ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start conversion, the speed is 200r/min, the temperature is 30°C, Under these conditions, the transformation was carried out for 45 hours, and the conversion rate of the substrate was 73%.

Example 19

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelium, and the mycelium is washed 3 times with deionized water to obtain clean mycelium, and the obtained mycelium is suspended in the prepared acetate buffer solution (0.05N, pH value 6.0) (30ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start conversion, the rotation speed is 200r/min, and the temperature is 30°C , Transformed under this condition for 45h, the conversion rate of the substrate was 67%.

Example 20

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid was centrifuged to obtain mature mycelia, and the mycelium was washed 3 times with deionized water to obtain clean mycelia, and the gained mycelium was suspended in disodium hydrogen phosphate-citric acid buffer (0.05N, pH Value 6.0) (30ml), add 0.3% substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start conversion, the speed is 200r/min, The temperature was 30°C, and the conversion was carried out for 45 hours under this condition, and the conversion rate of the substrate was 85%.

Example 21

Wash the mycelium on the plate cultured at 28°C for 5 to 7 days with sterile water and inoculate it in 30ml of seed medium (formulation: glucose 10g/L, bean cake powder 10g/L, pH6.2; sterilized at 121°C) Bacteria 20min) in the 250ml shaking flask, at rotating speed 120r/min, cultivate 2.5 days under the condition of 28 ℃ of temperature, gained seed is inoculated in the fermentation medium with 10% inoculum size (prescription is: glucose 10g/L, bean cake powder 10g /L, pH6.2; sterilized at 121°C for 20min), the volume of liquid is 30ml/250ml (medium/shake flask), at a speed of 200r/min, cultivated at a temperature of 28°C for 24 hours to obtain mature fermentation broth mycelia .

The fermented liquid is centrifuged to obtain mature mycelia, and the mycelium is washed 3 times with deionized water to obtain clean mycelia, and the resulting mycelium is suspended in a prepared citric acid-sodium hydroxide-hydrochloric acid buffer solution (0.05N, pH value 6.0) (30ml), add 0.3% of the substrate 3β-hydroxyl-15β, 16β-methylene-5-androsten-17-one, add 0.5% glucose, start conversion, the speed is 200r/min , the temperature was 30°C, and the conversion was carried out for 45 hours under this condition, and the conversion rate of the substrate was 68%.

Claims (13)

1. Microbial transformation method to prepare 3β, 7β-dihydroxy-15β, the method of 16β-methylene-5-androsten-17-one, the method uses Botryodiplodia malorum CBS13450 as bacterial species, including preparation of resting cell transformation system and The step of substrate conversion, wherein said quiescent cell conversion system comprises buffer, said buffer is potassium dihydrogen phosphate-sodium hydroxide buffer, and the concentration of said potassium dihydrogen phosphate-sodium hydroxide buffer is 0.01N-0.1N, the pH value is 4.0-9.0, the conditions for the conversion of the substrate are: the rotation speed is 100-300r/min, and the temperature is 0-50°C. 2. The method according to claim 1, wherein the concentration of the potassium dihydrogen phosphate-sodium hydroxide buffer solution is 0.02N. 3. The method according to claim 1, wherein the pH value of the potassium dihydrogen phosphate-sodium hydroxide buffer is 6.0. 4. The method according to claim 1, wherein the conditions for the conversion of the substrate are: the filling volume is 10-100ml culture medium/250ml shake flask. 5. The method according to claim 4, wherein the liquid filling volume is 40ml culture medium/250ml shake flask. 6. The method according to claim 1, wherein said rotating speed is 250r/min. 7. The method according to claim 1, wherein said temperature is 25-35°C. 8. The method according to claim 7, wherein said temperature is 30°C. 9. according to the arbitrary described method of claim 1 to 4, wherein said damping fluid is the potassium dihydrogen phosphate-sodium hydroxide buffer solution of 0.02N, pH value 6.0, and liquid filling volume is 30ml culture medium/250ml shaking bottle, the rotating speed is 250r/min, and the temperature is 30°C. 10. according to the arbitrary described method of claim 1 to 4, wherein said damping fluid is the potassium dihydrogen phosphate-sodium hydroxide buffering fluid of 0.02N, pH value 6.0, and liquid filling volume is 40ml culture medium/250ml shaking bottle, the rotating speed is 250r/min, and the temperature is 30°C. 11. A method for preparing 3β, 7β-dihydroxy-15β, 16β-methylene-5-androsten-17-one by microbial transformation method, which uses Botryodiplodia malorum CBS13450 as the strain, including the preparation of resting cell transformation system and The step of substrate conversion, wherein the quiescent cell conversion system includes a buffer, the buffer is acetate buffer solution, the concentration of the acetate buffer solution is 0.05N, and the pH value is 6.0, the The conditions for substrate conversion are: the rotation speed is 200r/min, and the temperature is 30°C. 12. A method for preparing 3β, 7β-dihydroxy-15β, 16β-methylene-5-androsten-17-one by microbial transformation method, which uses Botryodiplodia malorum CBS13450 as the strain, including the preparation of resting cell transformation system and The step of substrate conversion, wherein the quiescent cell conversion system includes a buffer, the buffer is a phosphate-citrate buffer, and the concentration of the phosphate-citrate buffer is 0.02N or 0.05N, The pH value is 6.0, and the conditions for the conversion of the substrate are: the rotation speed is 200 r/min, and the temperature is 30°C. 13. A method for preparing 3β, 7β-dihydroxy-15β, 16β-methylene-5-androsten-17-one by microbial transformation method, which uses Botryodiplodia malorum CBS13450 as the strain, including the preparation of resting cell transformation system and The step of substrate conversion, wherein said quiescent cell transformation system comprises buffer, said buffer is citric acid-sodium hydroxide-hydrochloric acid buffer, and the concentration of said citric acid-sodium hydroxide-hydrochloric acid buffer is 0.05N, pH value 6.0, the conditions for the conversion of the substrate are: the rotation speed is 200r/min, and the temperature is 30°C.