CN101570775B - Method for preparing 3 beta, 7 beta-dihydroxy-15 beta, 16 beta-methylene-5-androstene-17-ketone by microbial transformation method - Google Patents
Method for preparing 3 beta, 7 beta-dihydroxy-15 beta, 16 beta-methylene-5-androstene-17-ketone by microbial transformation method Download PDFInfo
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Abstract
The invention provides a method for preparing 3 beta, 7 beta-dihydroxy-15 beta, 16 beta-methylene-5-androstene-17-ketone by a microbial transformation method. The method uses Botryodiplodia malorum CBS13450 as a strain and comprises the steps of preparing a resting cell transformation system and transforming a substrate, wherein the resting cell transformation system comprises a buffer solution which is selected from a phosphate buffer solution, an acetate buffer solution, a phosphate-citric acid buffer solution, a monopotassium phosphate-sodium hydroxide buffer solution or a citric acid-sodium hydroxide-hydrochloric acid buffer solution. Under the optimal transformation conditions, the system has a transformation result that 0.3 percent of substrate is fully transformed within 45 hours.
Description
Technical field
The invention belongs to the microbial transformation field, be specifically related to a kind ofly utilize mikrobe with 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone changes into 3 β, 7 beta-dihydroxyies-15 β, the method for 16 β-methylene radical-5-androstene-17-ketone.
Background technology
3 β, 7 beta-dihydroxyies-15 β, 16 β-methylene radical-5-androstene-17-ketone is a kind of important medicine intermediate, can be used for synthetic contraceptive bian drospirenone and hydragog(ue) spirorenone.3 β, 7 beta-dihydroxyies-15 β, the synthetic of 16 β-methylene radical-5-androstene-17-ketone mainly utilizes microbial transformation 3 beta-hydroxies-15 β, and 16 β-methylene radical-5-androstene-17-ketone forms.Document Steroid hydroxylations with Botryodiplodiamalorum and Colletotrichum lini (steroids 71 (2006) 429-434) and patent UnitedStates Patent 4 614 616 (Sep.30; 1986) all report Botryodiplodia malorum and can transform 3 beta-hydroxies-15 β; 16 β-methylene radical-5-androstene-17-ketone is 3 β; 7 beta-dihydroxyies-15 β, 16 β-methylene radical-5-androstene-17-ketone.
The conversion of resting cells method is meant under suitable substratum and culture condition, thalline is fully grown after, the separating, washing thalline is suspended in the suitable damping fluid then, adds substrate again and carries out conversion reaction.Compare with conversion in the direct fermentation liquid, this method Yi Ban has following advantage: need under sterile state, not operate, condition is controlled easily; Can freely change the ratio of substrate and biomass in the reaction solution; Can shorten the reaction times, the influence of material to transforming that produces in material in the eliminating substratum and the thalli growth process reduces by product; Impurity is less in the liquid that transforms, and the separation and purification ratio is easier to.
Adopt direct conversion method in Patent 4 614 616 (Sep.30, the 1986) literary composition, the result who obtains is for complete with 0.2% substrate conversion in 48 hours.
And Steroid hydroxylations with Botryodiplodia malorum andColletotrichum lini (steroids 71 (a 2006) 429-434) literary composition use is the conversion of resting cells method; Damping fluid that it is used and part conversion condition are unclear, the result who obtains be 72 hours 0.1% substrate conversion is complete.
Summary of the invention
This paper invents a kind of high-level efficiency and transforms 3 beta-hydroxies-15 β; 16 β-methylene radical-5-androstene-17-ketone is 3 β; 7 beta-dihydroxyies-15 β; The conversion of resting cells method of 16 β-methylene radical-5-androstene-17-ketone, this method is a bacterial classification with Botryodiplodia malorum CBS13450, comprises the step of preparation conversion of resting cells system and substrate conversion; Wherein said conversion of resting cells system comprises damping fluid, and said damping fluid is selected from phosphate buffered saline buffer, acetate buffer solution, phosphoric acid salt-citrate buffer solution, potassium primary phosphate-sodium hydrate buffer solution or Hydrocerol A-sodium hydroxide-hydrochloride buffer.
Wherein said damping fluid is preferably potassium primary phosphate-sodium hydrate buffer solution.
The concentration of potassium primary phosphate-sodium hydrate buffer solution is 0.01N-0.1N, preferred 0.02N; The pH value of potassium primary phosphate-sodium hydrate buffer solution is 4.0-9.0, preferred 6.0.
The condition of wherein said fermentation culture is: liquid amount is 10-100ml/250ml (substratum/shake bottle), preferred 40ml/250ml (substratum/shake bottle); Rotating speed is 100-300r/min, preferred 250r/min; Temperature is 0-50 ℃, preferred 25-35 ℃, and more preferably 30 ℃.
Biotransformation method of the present invention is with 3 beta-hydroxies-15 β, and 16 β-methylene radical-5-androstene-17-ketone is converted into 3 β, 7 beta-dihydroxyies-15 β, and the concrete steps of the method for 16 β-methylene radical-5-androstene-17-ketone are following:
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, and mycelium gets mycelia totally with deionized water wash, the gained mycelia is suspended in prepare in the damping fluid; Substrate 3 beta-hydroxies-15 β of adding 0.3%; 16 β-methylene radical-5-androstene-17-ketone add 0.5% glucose, are 10-100ml/250ml (substratum/shake bottle) in liquid amount; Rotating speed is 100-300r/min, and temperature is to begin under the 0-50 ℃ of condition to transform.
The present invention adopts the bio-transformation of conversion of resting cells method to synthesize 3 β, 7 beta-dihydroxyies-15 β, and 16 β-methylene radical-5-androstene-17-ketone, compared with prior art, the present invention obviously shortens transformation time, has improved transformation efficiency.
Embodiment
Embodiment 1
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.02N for preparing; PH value 6.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 200r/min; Temperature is 0 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 35%.
Embodiment 2
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.02N for preparing; PH value 6.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 200r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 95%.
Embodiment 3
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.02N for preparing; PH value 6.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 200r/min; Temperature is 50 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 41%.
Embodiment 4
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.02N for preparing; PH value 6.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 100r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 79%.
Embodiment 5
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.02N for preparing; PH value 6.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 250r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 97%.
Embodiment 6
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.02N for preparing; PH value 6.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 300r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 90%.
Embodiment 7
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 40ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.02N for preparing; PH value 6.0) in (40ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 250r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 99%.
Embodiment 8
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 100ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.02N for preparing; PH value 6.0) in (100ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 250r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 75%.
Embodiment 9
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 10ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.02N for preparing; PH value 6.0) in (10ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 250r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 95%.
Embodiment 10
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 40ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.02N for preparing; PH value 6.0) in (40ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 250r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 99%.
Embodiment 11
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.01N for preparing; PH value 6.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 250r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 85%.
Embodiment 12
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.1N for preparing; PH value 6.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 250r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 64%.
Embodiment 13
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.02N for preparing; PH value 4.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 250r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 83%.
Embodiment 14
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.02N for preparing; PH value 9.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 250r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 70%.
Embodiment 15
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.02N for preparing; PH value 8.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 250r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 34%.
Embodiment 16
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.02N for preparing; PH value 6.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 50r/min; Temperature is 30 ℃, and 5 transform 45h under this condition, and the transformation efficiency of substrate is 41%.
Embodiment 17
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the potassium primary phosphate-sodium hydrate buffer solution (0.2N for preparing; PH value 6.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 200r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 29%.
Embodiment 18
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the phosphate buffered saline buffer (0.05N for preparing; PH value 6.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 200r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 73%.
Embodiment 19
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the acetate buffer (0.05N for preparing; PH value 6.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 200r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 67%.
Embodiment 20
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the Sodium phosphate, dibasic-citrate buffer solution (0.05N for preparing; PH value 6.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 200r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 85%.
Embodiment 21
28 ℃ cultivated down to be inoculated in after mycelia washes with sterilized water on 5 to 7 days the flat board 30ml seed culture medium is housed (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2; 121 ℃ of sterilization 20min) 250ml shakes in the bottle, at rotating speed 120r/min, cultivates under 28 ℃ of conditions of temperature 2.5 days, and the gained seed is inoculated in 10% inoculum size that (prescription is: glucose 10g/L, soybean cake powder 10g/L, pH6.2 in the fermention medium; 121 ℃ of sterilization 20min), liquid amount is 30ml/250ml (substratum/shake bottle), at rotating speed 200r/min, 28 ℃ of temperature cultivated 24 hours down sophisticated fermented liquid mycelium.
Fermented liquid is through centrifugal sophisticated mycelium, mycelium with deionized water wash 3 times must be totally mycelia, the gained mycelia is suspended in the Hydrocerol A-sodium hydroxide-hydrochloride buffer (0.05N for preparing; PH value 6.0) in (30ml), adds 0.3% substrate 3 beta-hydroxies-15 β, 16 β-methylene radical-5-androstene-17-ketone; The glucose of adding 0.5% begins to transform, and rotating speed is 200r/min; Temperature is 30 ℃, under this condition, transforms 45h, and the transformation efficiency of substrate is 68%.
Claims (13)
1. microbe transformation method prepares 3 β, 7 beta-dihydroxyies-15 β, the method for 16 β-methylene radical-5-androstene-17-ketone; This method is a bacterial classification with Botryodiplodia malorum CBS13450; The step that comprises preparation conversion of resting cells system and substrate conversion, wherein said conversion of resting cells system comprises damping fluid, and said damping fluid is potassium primary phosphate-sodium hydrate buffer solution; The concentration of said potassium primary phosphate-sodium hydrate buffer solution is 0.01N-0.1N; The pH value is 4.0-9.0, and the condition of said substrate conversion is: rotating speed is 100-300r/min, and temperature is 0-50 ℃.
2. method according to claim 1, the concentration of wherein said potassium primary phosphate-sodium hydrate buffer solution are 0.02N.
3. method according to claim 1, the pH value of wherein said potassium primary phosphate-sodium hydrate buffer solution is 6.0.
4. method according to claim 1, the condition of wherein said substrate conversion is: liquid amount is that 10-100ml substratum/250ml shakes bottle.
5. method according to claim 4, wherein said liquid amount are that 40ml substratum/250ml shakes bottle.
6. method according to claim 1, wherein said rotating speed are 250r/min.
7. method according to claim 1, wherein said temperature are 25-35 ℃.
8. method according to claim 7, wherein said temperature are 30 ℃.
9. according to the arbitrary described method of claim 1 to 4, wherein said damping fluid is that 0.02N, pH value are potassium primary phosphate-sodium hydrate buffer solution of 6.0, and liquid amount is that 30ml substratum/250ml shakes bottle, and rotating speed is 250r/min, and temperature is 30 ℃.
10. according to the arbitrary described method of claim 1 to 4, wherein said damping fluid is that 0.02N, pH value are potassium primary phosphate-sodium hydrate buffer solution of 6.0, and liquid amount is that 40ml substratum/250ml shakes bottle, and rotating speed is 250r/min, and temperature is 30 ℃.
11. microbe transformation method prepares 3 β, 7 beta-dihydroxyies-15 β, the method for 16 β-methylene radical-5-androstene-17-ketone; This method is a bacterial classification with Botryodiplodia malorum CBS13450; The step that comprises preparation conversion of resting cells system and substrate conversion, wherein said conversion of resting cells system comprises damping fluid, and said damping fluid is an acetate buffer solution; The concentration of said acetate buffer solution is 0.05N; The pH value is 6.0, and the condition of said substrate conversion is: rotating speed is 200r/min, and temperature is 30 ℃.
12. microbe transformation method prepares 3 β, 7 beta-dihydroxyies-15 β, the method for 16 β-methylene radical-5-androstene-17-ketone; This method is a bacterial classification with Botryodiplodia malorum CBS13450; The step that comprises preparation conversion of resting cells system and substrate conversion, wherein said conversion of resting cells system comprises damping fluid, and said damping fluid is phosphoric acid salt-citrate buffer solution; The concentration of said phosphoric acid salt-citrate buffer solution is 0.02N or 0.05N; The pH value is 6.0, and the condition of said substrate conversion is: rotating speed is 200r/min, and temperature is 30 ℃.
13. microbe transformation method prepares 3 β, 7 beta-dihydroxyies-15 β, the method for 16 β-methylene radical-5-androstene-17-ketone; This method is a bacterial classification with Botryodiplodia malorum CBS13450; The step that comprises preparation conversion of resting cells system and substrate conversion, wherein said conversion of resting cells system comprises damping fluid, and said damping fluid is Hydrocerol A-sodium hydroxide-hydrochloride buffer; The concentration of said Hydrocerol A-sodium hydroxide-hydrochloride buffer is 0.05N; The pH value is 6.0, and the condition of said substrate conversion is: rotating speed is 200r/min, and temperature is 30 ℃.
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| US4614616A (en) * | 1980-11-03 | 1986-09-30 | Schering Aktiengesellschaft | Process for preparing 3β7β-dihydroxy-Δ5 -steroids |
| CN101085990A (en) * | 2007-06-27 | 2007-12-12 | 华东理工大学 | Method for preparing chiral aryl secondary alcohol |
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| US4614616A (en) * | 1980-11-03 | 1986-09-30 | Schering Aktiengesellschaft | Process for preparing 3β7β-dihydroxy-Δ5 -steroids |
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