CN106916872A - A kind of high-performance bio conversion prepares 11 α, the method for 17 alpha-dihydroxy progesterone - Google Patents
A kind of high-performance bio conversion prepares 11 α, the method for 17 alpha-dihydroxy progesterone Download PDFInfo
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Abstract
11 α, the method for 17 alpha-dihydroxy progesterone are prepared the invention provides a kind of conversion of high-performance bio.The method with 17 Alpha-hydroxy progesterone be raw material, by adding carbon source in biotransformation, so as to provide the carbon source of abundance for thalli growth, hydroxylase in mycelium is set to keep vigor high, extension thalline high-performance bio conversion stationary phase, higher than 2% (w/v), 11 α of preparation, the conversion yield of 17 alpha-dihydroxy progesterone reaches more than 90% to 17 Alpha-hydroxy progesterone inventorys in alloing fermentation medium.17 Alpha-hydroxy progesterone optimum charging ratio scopes can reach 98% for the conversion yield of the alpha-dihydroxy progesterone of 2-20%, 11 α, 17 in fermentation medium.The problems such as low caused equipment output capacity of reactant rate of charge is low, pollutant discharge amount is big is efficiently solved, output efficiency is improve, production cost is reduced.
Description
Technical field
The invention belongs to the preparation field of steroid, it is related to a kind of bioconversion to prepare 11 α, the method for 17 alpha-dihydroxy progesterone, more particularly to a kind of high-performance bio conversion prepares 11 α, the method for 17 alpha-dihydroxy progesterone.
Background technology
11 α, 17 alpha-dihydroxy progesterone (referred to as double Hydroxyprogesterones), it is a kind of important corticoid pharmaceutical intermediate, is the important intermediate for producing the steroid hormone class medicines such as hydrocortisone, dexamethasone, metacortandracin, betamethasone for the synthesis of corticoid medicine.
17 Alpha-hydroxy progesterone, the Alpha-hydroxy of full name 17-pregnant alkene-3 of steroid-4,20- diketone, also known as hydroxyprogesterone, is a kind of odorless, white or off-white color crystalline powder, is insoluble in water, dissolves in part organic solvent such as methyl alcohol, acetonitrile, chloroform, acetone etc..
11 α are converted into by 17 Alpha-hydroxy progesterone, the molecular structure of 17 alpha-dihydroxy progesterone is：
The C11 hydroxylatings of steroidal are that various cortins synthesize an indispensable step.Nineteen fifty-two Peterson and Murray carry out hydroxylating with bread mold (Rhizopusnigricans) to progesterone C11 earliest, and the step of progesterone one successfully changed into 11 Alpha-hydroxy progesterone, make progesterone be reduced to only need 3 steps to the process of cortisone, solve the key issue in cortin building-up process.Hydroxyl is extremely difficult for chemical synthesis on steroidal, and in addition to being easily introduced a hydroxyl on C17 of steroidal, other positions are all difficult chemically to introduce hydroxyl.Steroidal C11 hydroxylatings are the distinctive conversion reactions of microorganism, are raw material with 17 Alpha-hydroxy progesterone in recent years, and 11 α are prepared using bioconversion method, and 17 alpha-dihydroxy progesterone have been achieved with industrialization.The method does not solve 11 α singly, and 17 alpha-dihydroxy progesterone are difficult to the problem of chemical synthesis, and introduce the drug effect that hydroxyl increased corticosteroid at 11, for steroid hydroxylating provides a new way.
But steroidal compounds poorly water-soluble, solubility is generally 10-6~10-5Mol/L, mass-transfer efficiency is low in biotransformation, limits reactant rate of charge, and 11 α are prepared by raw materials through biotransformation of 17 Alpha-hydroxy progesterone in document report, during 17 alpha-dihydroxy progesterone, the mass ratio that feeds intake of the Alpha-hydroxy progesterone of reactant 17 is below 2%.Because the mass ratio that feeds intake of the Alpha-hydroxy progesterone of reactant 17 is low, cause output efficiency low, culture medium utilization rate is low, and pollutant discharge amount is big.
The content of the invention
In view of the shortcomings of the prior art, 11 α are prepared it is an object of the invention to provide a kind of bioconversion, the method for 17 alpha-dihydroxy progesterone is provided in particular in a kind of high-performance bio conversion and prepares 11 α, the method for 17 alpha-dihydroxy progesterone.
It is that, up to this purpose, the present invention uses following technical scheme：
On the one hand, the present invention provides a kind of bioconversion and prepares 11 α, and the method for 17 alpha-dihydroxy progesterone the described method comprises the following steps：
(1) prepared by inclined-plane：Aspergillus ochraceus strain is inoculated in slant medium, incubated, generation yellow spore inclined-plane is standby；
(2) seed culture：Taken from the yellow spore inclined-plane that step (1) is obtained and carried out during strain is inoculated in seed culture medium seed culture and obtain seed liquor；
(3) bioconversion：Step (2) gained seed liquor is seeded in fermentation medium, the Alpha-hydroxy progesterone of reactant 17 is added simultaneously, shaken cultivation, during shaken cultivation, carbon source and the Alpha-hydroxy progesterone of reactant 17 are added, keeps concentration of the carbon source in zymotic fluid to be not less than 0.8mg/mL, the total inventory of 17 Alpha-hydroxy progesterone is the 2-20% of fermentation medium volume, obtain 11 α, the zymotic fluid of 17 alpha-dihydroxy progesterone.
11 α, the bioconversion preparation process of 17 alpha-dihydroxy progesterone is the hydroxylase catalysis process in fermentation thalli, and the vigorous then hydroxylase catalysis vigor of thalli growth is high, and it is the key for improving transformation efficiency to keep thalline catalysis activity.The present invention in biotransformation by adding carbon source, concentration of the carbon source in zymotic fluid is kept to be not less than 0.8mg/mL, so as to provide the carbon source of abundance for thalli growth, hydroxylase in mycelium is set to keep vigor high, extend thalline high-performance bio conversion stationary phase, therefore the high-performance bio conversion to 17 Alpha-hydroxy progesterone can be realized under reactant inventory high, output efficiency is improve, production cost and gross contamination emission is reduced.
In the method for the invention, comprising the component that mass volume ratio (w/v) is following in step (1) described slant medium：Glucose 2.0-5.0%, malt extract 1.0-5.0%, peptone 0.5-3.0%, agar 2.0-2.5%, the pH value 5.0-6.0 of the slant medium are used after steam sterilizing.
Mass volume ratio described in step (1) refers to relative to the quality (g) of the component added in 100mL culture mediums, glucose 2.0-5.0% is for example included in slant medium, that is, refers to the glucose containing 2.0-5.0g in 100mL slant mediums.Hereinafter to the implication of the mass volume ratio of each composition in seed culture medium and in fermentation medium and all same herein.
In slant medium, the content of glucose can be 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.4%, 4.6% or 4.8%；The content of malt extract can be 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.3%, 2.5%, 2.8%, 3%, 3.3%, 3.5%, 3.8%, 4%, 4.3%, 4.5% or 4.8%；The content of peptone can be 0.6%, 0.8%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.3%, 2.5% or 2.8%；The content of agar can be 2.1%, 2.15%, 2.2%, 2.25%, 2.3%, 2.35%, 2.4%, 2.45%.The pH value of slant medium of the present invention is 5.0-6.0, and such as 5.1,5.2,5.3,5.4,5.5,5.6,5.7,5.8 or 5.9, the slant medium can be used through 121 DEG C of steam sterilizing 20min rears.
Preferably, step (1) the incubated temperature is 25-28 DEG C, such as 25.5 DEG C, 26 DEG C, 26.5 DEG C, 27 DEG C or 27.5 DEG C.
Preferably, step (1) the incubated time is 4-7 days, such as 4.3 days, 4.5 days, 4.8 days, 5 days, 5.3 days, 5.5 days, 5.8 days, 6 days, 6.3 days, 6.5 days or 6.8 days.
The yellow spore inclined-plane that step (1) is obtained can be saved backup in constant temperature at 4 DEG C of refrigerator, can be preserved 2-3 month.
In the method for the invention, comprising the component that mass volume ratio (w/v) is following in step (2) described seed culture medium：Glucose 2.0-5.0%, corn pulp 1.0-5.0%, peptone 1.0-5.0% and yeast extract 0.5-2.0%, the pH value of the seed culture medium is 5.5-6.5, is used after steam sterilizing.
In seed culture medium, the content of glucose can be 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, 4.2%, 4.4%, 4.6% or 4.8%；The content of corn pulp can be 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.3%, 2.5%, 2.8%, 3%, 3.3%, 3.5%, 3.8%, 4%, 4.3%, 4.5% or 4.8%；The content of peptone can be 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.3%, 2.5%, 2.8%, 3%, 3.3%, 3.5%, 3.8%, 4%, 4.3%, 4.5% or 4.8%；The content of yeast extract can be 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8% or 1.9%.The pH value of seed culture medium is 5.5-6.5, such as 5.6,5.7,5.8,5.9,6.0,6.1,6.2,6.3 or 6.4.The seed culture medium can be used through 121 DEG C of steam sterilizing 20min rears.
Preferably, step (2) described seed culture is to carry out shaken cultivation under 160-220r/min (such as 160r/min, 165r/min, 170r/min, 175r/min, 180r/min, 185r/min, 190r/min, 195r/min, 200r/min or 220r/min).
Preferably, the temperature of step (2) described seed culture is 25-32 DEG C, such as 26 DEG C, 27 DEG C, 28 DEG C, 29 DEG C, 30 DEG C or 31 DEG C.
Preferably, the time of step (2) described seed culture is 20-30h, such as 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h or 29h.
In the method for the invention, comprising the component that mass volume ratio (w/v) is following in step (3) described fermentation medium：Glucose 1.0-4.0%, corn pulp 0.5-4.0%, peptone 0.5-4.0% and yeast extract 0.5-3.0%, the pH value of the fermentation medium is 5.5-6.5, is used after steam sterilizing.
In the fermentation medium, the content of glucose can be 1.3%, 1.5%, 1.8%, 2%, 2.3%, 2.5%, 2.8%, 3%, 3.3%, 3.5% or 3.8%；The content of corn pulp can be 0.6%, 0.8%, 1%, 1.3%, 1.5%, 1.8%, 2%, 2.3%, 2.5%, 2.8%, 3%, 3.3%, 3.5% or 3.8%；The content of peptone can be 0.6%, 0.8%, 1%, 1.3%, 1.5%, 1.8%, 2%, 2.3%, 2.5%, 2.8%, 3%, 3.3%, 3.5% or 3.8%；The content of yeast extract can be 0.6%, 0.8%, 1%, 1.3%, 1.5%, 1.8%, 2%, 2.3%, 2.5% or 2.8%.The pH value of the fermentation medium is 5.5-6.5, such as 5.6,5.7,5.8,5.9,6.0,6.1,6.2,6.3 or 6.4.The seed culture medium can be used through 121 DEG C of steam sterilizing 20min rears.
Preferably, the inoculum concentration of step (3) described seed liquor is the 2-10% of fermentation medium volume, preferably such as 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 9.5%, 2-5%.
Preferably, the quality of step (3) the addition Alpha-hydroxy progesterone of reactant 17 is the 2-20% of fermentation medium volume, such as 3%, 4%, 5%, 6%, 8%, 10%, 12%, 14%, 16% or 18%.
The 17 Alpha-hydroxy progesterone for adding first are 2-20% with the inventory sum of the 17 Alpha-hydroxy progesterone then optionally added.
Preferably, step (3) shaken cultivation is carried out under the oscillation rate of 160-220r/min (such as 160r/min, 165r/min, 170r/min, 175r/min, 180r/min, 185r/min, 190r/min, 195r/min, 200r/min or 220r/min).
Preferably, the temperature of step (3) described shaken cultivation is 25-32 DEG C, such as 26 DEG C, 27 DEG C, 28 DEG C, 29 DEG C, 30 DEG C or 31 DEG C.
Preferably, step (3) described carbon source is organic carbon source and/or inorganic carbon source.
Preferably, step (3) described carbon source is selected from any one in glucose, fructose, galactolipin, maltose, lactose, sucrose, molasses, starch, pectin, cellulose, grease type, amino acid, alcohol, aldehyde, phenol, carbonate or bicarbonate or at least two combination, preferably any one in glucose, fructose, molasses, grease, alcohol or carbonate or at least two combination.
Preferably, the additional way of step (3) described carbon source is in batches continuous stream adds or adds.
In the present invention, by adding carbon source, for thalli growth provides required carbon source, hydroxylase in mycelium is set to keep vigor high, extend thalline high-performance bio conversion stationary phase such that it is able to realize the high-performance bio conversion to 17 Alpha-hydroxy progesterone under reactant inventory high.
Preferably,Carbon source is timely and appropriately added in step (3),The concentration for adding carbon source is not less than 50g/L (concentration for for example adding carbon source can be 50g/L,80g/L,100g/L,150g/L,200g/L,300g/L,500g/L or 700g/L etc.) keeping concentration of the carbon source in zymotic fluid to be not less than 0.8mg/mL,Concentration of the carbon source in zymotic fluid is for example set to be not less than 1.0mg/mL,1.5mg/mL,2mg/mL,2.5mg/mL,3mg/mL,3.5mg/mL,4mg/mL,4.5mg/mL,5mg/mL,5.5mg/mL,6mg/mL,6.5mg/mL,7mg/mL or 8mg/mL etc..
Preferably, the additional way of step (3) the Alpha-hydroxy progesterone of reactant 17 feeds intake or batch feeding or mixes flow feeding or batch feeding with carbon source for disposable.
The Alpha-hydroxy progesterone of reactant 17 can be carried out a 17 Alpha-hydroxy progesterone and fed intake disposably to feed intake that is, before inoculation in step (3), subsequently without feed supplement again；Or batch feeding or mix flow feeding or batch feeding with carbon source, no matter feed intake in which way, the total amount of feeding of the Alpha-hydroxy progesterone of reactant 17 is the 2-20% of fermentation medium volume, such as 3%, 5%, 8%, 10%, 12%, 14%, 16%, 18% or 20%.Total amount of feeding described here is 2-20g, such as 3g, 5g, 8g, 10g, 12g, 14g, 16g, 18g or 20g for the amount that the 2-20% of fermentation medium volume refers to the total reactant put into 100mL culture mediums.
Used as optimal technical scheme, bioconversion of the present invention prepares 11 α, and the method for 17 alpha-dihydroxy progesterone is comprised the following steps：
(1) prepared by inclined-plane：Aspergillus ochraceus strain is inoculated in slant medium, 25-28 DEG C incubated 4-7 days, generation yellow spore inclined-plane is standby；
(2) seed culture：Strain is taken from the yellow spore inclined-plane that step (1) is obtained to be inoculated in seed culture medium, in 25-32 DEG C, shaken cultivation 20-30h under 160-220r/min obtains seed liquor；
(3) bioconversion：Step (2) gained seed liquor is seeded in fermentation medium with the inoculum concentration of the 2-10% of fermentation medium volume, the Alpha-hydroxy progesterone of reactant 17 is added simultaneously, 160-220r/min shaken cultivations at 25-32 DEG C, during shaken cultivation, add carbon source and add the Alpha-hydroxy progesterone of reactant 17, concentration of the carbon source in zymotic fluid is kept to be not less than 0.8mg/mL, the total inventory of 17 Alpha-hydroxy progesterone is the 2-20% of fermentation medium volume, obtain 11 α, the zymotic fluid of 17 alpha-dihydroxy progesterone.
Another aspect of the present invention provides 11 α that the preparation method as described in first aspect is prepared, 17 alpha-dihydroxy progesterone.
Relative to prior art, the invention has the advantages that：
The present invention in biotransformation by adding carbon source, concentration of the carbon source in zymotic fluid is kept to be not less than 0.8mg/mL, so as to provide required carbon source for thalli growth, hydroxylase in mycelium is set to keep vigor high, extend thalline high-performance bio conversion stationary phase, allow to realize the high-performance bio conversion to 17 Alpha-hydroxy progesterone under the reactant inventory high of 2-20%, the conversion yield of 17 Alpha-hydroxy progesterone reaches more than 90%, can reach 98%, efficiently solve the low caused equipment output capacity of reactant rate of charge low, the problems such as pollutant discharge amount is big, improve output efficiency, reduce production cost.
Specific embodiment
Technical scheme is further illustrated below by specific embodiment.Those skilled in the art it will be clearly understood that the embodiment be only to aid in understand the present invention, be not construed as to concrete restriction of the invention.
Embodiment 1
In the present embodiment, 11 α are prepared by the following method, 17 alpha-dihydroxy progesterone specifically include following steps：
(1) Aspergillus ochraceus test tube strains are inoculated in slant medium (glucose 2.0%, malt extract 1.0%, peptone 0.5%, agar 2.0%, 5.0,121 DEG C of steam sterilizing 20min of pH value), 25 DEG C incubated 4 days, prepares spore inclined-plane；
(2) standby spore inclined-plane will be made to take out, a ring is taken with oese be connected to seed culture medium (glucose 2.0%, corn pulp 1.0%, peptone 1.0%, yeast extract 0.5%, pH value 5.5,121 DEG C of steam sterilizing 20min) in, 25 DEG C, 160r/min shaken cultivation 20h, the seed liquor needed for obtaining final product fermentation；
(3) seed liquor is accessed into sterilized fermentation medium (glucose 1.0%, corn pulp 0.5%, peptone 0.5%, yeast extract 0.5% by 2% inoculum concentration, pH value 5.5,121 DEG C of steam sterilizing 20min), 4% 17 Alpha-hydroxy progesterone are once added simultaneously, at 25 DEG C, 160r/min shaken cultivations, the sugar juice that concentration is 50g/L is repeatedly added during shaken cultivation, 0.8mg/mL is not less than with sugared concentration in keeping fermentation medium, obtain 11 α, the zymotic fluid of 17 alpha-dihydroxy progesterone.
Analyzed through high performance liquid chromatography (HPLC), 11 α in zymotic fluid, 17 alpha-dihydroxy progesterone conversion ratios are 94%.
Embodiment 2
In the present embodiment, 11 α are prepared by the following method, 17 alpha-dihydroxy progesterone specifically include following steps：
(1) Aspergillus ochraceus test tube strains are inoculated in slant medium (glucose 3.0%, malt extract 2.0%, peptone 1.0%, agar 2.0%, 5.3,121 DEG C of steam sterilizing 20min of pH value), 26 DEG C incubated 7 days, prepares spore inclined-plane；
(2) standby spore inclined-plane will be made to take out, a ring is taken with oese be connected to seed culture medium (glucose 3.0%, corn pulp 2.0%, peptone 2.0%, yeast extract 1.0%, pH value 5.5,121 DEG C of steam sterilizing 20min) in, 28 DEG C, 180r/min shaken cultivation 22h, the seed liquor needed for obtaining final product fermentation；
(3) seed liquor is accessed into sterilized fermentation medium (glucose 1.5%, corn pulp 1.0%, peptone 1.0%, yeast extract 0.5% by 5% inoculum concentration, pH value 6.0,121 DEG C of steam sterilizing 20min), the 17 Alpha-hydroxy progesterone that total amount is 4% are added in batches, at 28 DEG C, 180r/min shaken cultivations, stream plus concentration are the sugar juice of 200g/L during shaken cultivation, sugared concentration is not less than 2mg/mL in keeping fermentation medium, obtain 11 α, the zymotic fluid of 17 alpha-dihydroxy progesterone.
Analyzed through high performance liquid chromatography (HPLC), 11 α in zymotic fluid, 17 alpha-dihydroxy progesterone conversion ratios are 96%.
Embodiment 3
In the present embodiment, 11 α are prepared by the following method, 17 alpha-dihydroxy progesterone specifically include following steps：
(1) Aspergillus ochraceus test tube strains are inoculated in slant medium (glucose 2.5%, malt extract 1.5%, peptone 2.0%, agar 2.5%, 6.0,121 DEG C of steam sterilizing 20min of pH value), 27 DEG C incubated 6 days, prepares spore inclined-plane；
(2) standby spore inclined-plane will be made to take out, a ring is taken with oese be connected to seed culture medium (glucose 3.0%, corn pulp 1.5%, peptone 1.5%, yeast extract 1.0%, pH value 6.0,121 DEG C of steam sterilizing 20min) in, 28 DEG C, 220r/min shaken cultivation 30h, the seed liquor needed for obtaining final product fermentation.Seed liquor is accessed into sterilized fermentation medium (glucose 4.0%, corn pulp 4.0%, peptone 4.0%, yeast extract 3.0% by 3% inoculum concentration, pH value 5.5,121 DEG C of steam sterilizing 20min), at 28 DEG C, 220r/min shaken cultivations, the mixing suspension of stream plus the Alpha-hydroxy progesterone of reactant 17 and sugar juice (200g/L) during shaken cultivation, wherein the total rate of charge of reactant is 4%, sugared concentration is not less than 3mg/mL in keeping fermentation medium, obtain 11 α, the zymotic fluid of 17 alpha-dihydroxy progesterone.
Analyzed through high performance liquid chromatography (HPLC), 11 α in zymotic fluid, 17 alpha-dihydroxy progesterone conversion ratios are 98%.
Embodiment 4
In the present embodiment, 11 α are prepared by the following method, 17 alpha-dihydroxy progesterone specifically include following steps：
(1) Aspergillus ochraceus test tube strains are inoculated in slant medium (glucose 5.0%, malt extract 5.0%, peptone 3.0%, agar 2.5%, 6.0,121 DEG C of steam sterilizing 20min of pH value), 28 DEG C incubated 7 days, prepares spore inclined-plane；
(2) standby spore inclined-plane will be made to take out, a ring is taken with oese be connected to seed culture medium (glucose 5.0%, corn pulp 5.0%, peptone 5.0%, yeast extract 2.0%, pH value 6.5,121 DEG C of steam sterilizing 20min) in, 32 DEG C, 200r/min shaken cultivation 24h, the seed liquor needed for obtaining final product fermentation；
(3) seed liquor is accessed into sterilized fermentation medium (glucose 3.0%, corn pulp 2.5%, peptone 2.5%, yeast extract 1.0% by 5% inoculum concentration, pH value 6.5,121 DEG C of steam sterilizing 20min), in 26 DEG C, 200r/min shaken cultivations.The 17 Alpha-hydroxy progesterone that total amount is 6% are added in batches, at 26 DEG C, 200r/min shaken cultivations, sugared concentration is not less than 1.5mg/mL during stream plus concentration keep fermentation medium for the sugar juice of 200g/L during shaken cultivation, obtain 11 α, the zymotic fluid of 17 alpha-dihydroxy progesterone.
Analyzed through high performance liquid chromatography (HPLC), 11 α in zymotic fluid, 17 alpha-dihydroxy progesterone conversion yields are 94%.
Embodiment 5
In the present embodiment, 11 α are prepared by the following method, 17 alpha-dihydroxy progesterone specifically include following steps：
(1) Aspergillus ochraceus test tube strains are inoculated in slant medium (glucose 5.0%, malt extract 5.0%, peptone 3.0%, agar 2.5%, 6.0,121 DEG C of steam sterilizing 20min of pH value), 26 DEG C incubated 6 days, prepares spore inclined-plane；
(2) standby spore inclined-plane will be made to take out, a ring is taken with oese be connected to seed culture medium (glucose 2.5%, corn pulp 2.0%, peptone 2.0%, yeast extract 1.0%, pH value 6.0,121 DEG C of steam sterilizing 20min) in, 30 DEG C, 200r/min shaken cultivation 24h, the seed liquor needed for obtaining final product fermentation；
(3) seed liquor is accessed into sterilized fermentation medium (glucose 3.0%, corn pulp 2.5%, peptone 2.5%, yeast extract 1.0% by 6% inoculum concentration, pH value 6.5,121 DEG C of steam sterilizing 20min), at 30 DEG C, 220r/min shaken cultivations, the mixing suspension of the Alpha-hydroxy progesterone of reactant 17 and sugar juice (100g/L) is added during shaken cultivation in batches, wherein the total rate of charge of reactant is 6%, sugared concentration is not less than 2mg/mL in keeping fermentation medium, obtain 11 α, the zymotic fluid of 17 alpha-dihydroxy progesterone.
Analyzed through high performance liquid chromatography (HPLC), 11 α in zymotic fluid, 17 alpha-dihydroxy progesterone conversion yields are 95%.
Embodiment 6
In the present embodiment, 11 α are prepared by the following method, 17 alpha-dihydroxy progesterone specifically include following steps：
(1) Aspergillus ochraceus test tube strains are inoculated in slant medium (glucose 3.0%, malt extract 2.0%, peptone 1.5%, agar 2.0%, 5.5,121 DEG C of steam sterilizing 20min of pH value), 26 DEG C incubated 7 days, prepares spore inclined-plane；
(2) standby spore inclined-plane will be made to take out, a ring is taken with oese be connected to seed culture medium (glucose 2.0%, corn pulp 2.0%, peptone 2.0%, yeast extract 1.5%, pH value 6.5,121 DEG C of steam sterilizing 20min) in, 28 DEG C, 180r/min shaken cultivation 30h, the seed liquor needed for obtaining final product fermentation；
(3) seed liquor is accessed into sterilized fermentation medium (glucose 3.0%, corn pulp 1.5%, peptone 1.5%, yeast extract 1.5% by 5% inoculum concentration, pH value 6.5,121 DEG C of steam sterilizing 20min), 20% 17 Alpha-hydroxy progesterone are once added, in 28 DEG C, 180r/min shaken cultivations, certain hour adds the sugar juice that concentration is 100g/L, sugared concentration is not less than 2.5mg/mL in keeping fermentation medium, obtains 11 α, the zymotic fluid of 17 alpha-dihydroxy progesterone.
Analyzed through high performance liquid chromatography (HPLC), 11 α in zymotic fluid, 17 alpha-dihydroxy progesterone conversion yields are 97%.
Embodiment 7
In the present embodiment, 11 α are prepared by the following method, 17 alpha-dihydroxy progesterone specifically include following steps：
(1) Aspergillus ochraceus test tube strains are inoculated in slant medium (glucose 2.0%, malt extract 1.0%, peptone 1.5%, agar 2.5%, 5.5,121 DEG C of steam sterilizing 20min of pH value), 24 DEG C incubated 7 days, prepares spore inclined-plane；
(2) standby spore inclined-plane will be made to take out, a ring is taken with oese be connected to seed culture medium (glucose 1.5%, corn pulp 2.0%, peptone 2.0%, yeast extract 0.5%, pH value 6.0,121 DEG C of steam sterilizing 20min) in, 28 DEG C, 200r/min shaken cultivation 24h, the seed liquor needed for obtaining final product fermentation；
(3) seed liquor is accessed into sterilized fermentation medium (glucose 3.0%, corn pulp 1.5%, peptone 1.5%, yeast extract 1.5% by 5% inoculum concentration, pH value 6.5,121 DEG C of steam sterilizing 20min), 17 Alpha-hydroxy progesterone are added in batches, at 28 DEG C, 200r/min shaken cultivations, total rate of charge is 20%.Certain hour adds the sugar juice that concentration is 100g/L, and sugared concentration is not less than 3mg/mL in keeping fermentation medium, obtains 11 α, the zymotic fluid of 17 alpha-dihydroxy progesterone.
Analyzed through high performance liquid chromatography (HPLC), 11 α in zymotic fluid, 17 alpha-dihydroxy progesterone conversion yields are 98%.
Embodiment 8
In the present embodiment, 11 α are prepared by the following method, 17 alpha-dihydroxy progesterone specifically include following steps：
(1) Aspergillus ochraceus test tube strains are inoculated in slant medium (glucose 3.0%, malt extract 2.5%, peptone 1.5%, agar 2.0%, 5.5,121 DEG C of steam sterilizing 20min of pH value), 26 DEG C incubated 7 days, prepares spore inclined-plane；
(2) standby spore inclined-plane will be made to take out, a ring is taken with oese be connected to seed culture medium (glucose 1.5%, corn pulp 2.5%, peptone 2.5%, yeast extract 1.5%, pH value 6.5,121 DEG C of steam sterilizing 20min) in, 28 DEG C, 180r/min shaken cultivation 30h, the seed liquor needed for obtaining final product fermentation；
(3) seed liquor is accessed into sterilized fermentation medium (glucose 2.0%, corn pulp 2.5%, peptone 1.5%, yeast extract 2.0% by 5% inoculum concentration, pH value 6.5,121 DEG C of steam sterilizing 20min), at 28 DEG C, 220r/min shaken cultivations, the mixing suspension of stream plus the Alpha-hydroxy progesterone of reactant 17 and sugar juice (200g/L) during shaken cultivation, wherein the total rate of charge of reactant is 20%, sugared concentration is not less than 5mg/mL in keeping fermentation medium, obtain 11 α, the zymotic fluid of 17 alpha-dihydroxy progesterone.
Analyzed through high performance liquid chromatography (HPLC), 11 α in zymotic fluid, 17 alpha-dihydroxy progesterone conversion yields are 98%.
Applicant states that the present invention illustrates method detailed of the invention by above-described embodiment, but the invention is not limited in above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Person of ordinary skill in the field it will be clearly understood that any improvement in the present invention, addition, the selection of concrete mode to the equivalence replacement and auxiliary element of each raw material of product of the present invention etc., all fall within protection scope of the present invention and it is open within the scope of.
Claims (10)
1. a kind of bioconversion prepares 11 α, the method for 17 alpha-dihydroxy progesterone, it is characterised in that the side
Method is comprised the following steps：
(1) prepared by inclined-plane：Aspergillus ochraceus strain is inoculated in slant medium, it is incubated, generate yellow spore
Sub- inclined-plane, it is standby；
(2) seed culture：Strain is taken from the yellow spore inclined-plane that step (1) is obtained and is inoculated in seed training
Seed culture is carried out in foster base obtain seed liquor；
(3) bioconversion：Step (2) gained seed liquor is seeded in fermentation medium, while adding
The Alpha-hydroxy progesterone of reactant 17, shaken cultivation during shaken cultivation, adds carbon source and adds reaction
The Alpha-hydroxy progesterone of thing 17, keeps concentration of the carbon source in zymotic fluid to be not less than 0.8mg/mL, and 17 Alpha-hydroxies are yellow
The total inventory of body ketone is the 2-20% of fermentation medium volume, obtains 11 α, the hair of 17 alpha-dihydroxy progesterone
Zymotic fluid.
2. method according to claim 1, it is characterised in that step (1) described slant medium
In comprising the following component of mass volume ratio (w/v)：Glucose 2.0-5.0%, malt extract 1.0-5.0%, egg
White peptone 0.5-3.0% and agar 2.0-2.5%, the pH value of the slant medium is 5.0-6.0, through steam sterilizing
After use；
Preferably, step (1) the incubated temperature is 25-28 DEG C；
Preferably, step (1) the incubated time is 4-7 days.
3. method according to claim 1 and 2, it is characterised in that step (2) the seed training
Comprising the following component of mass volume ratio (w/v) in foster base：Glucose 2.0-5.0%, corn pulp 1.0-5.0%,
Peptone 1.0-5.0% and yeast extract 0.5-2.0%, the pH value of the seed culture medium is 5.5-6.5, through steam
Used after sterilizing；
Preferably, step (2) described seed culture is that shaken cultivation is carried out under 160-220r/min；
Preferably, the temperature of step (2) described seed culture is 25-32 DEG C；
Preferably, the time of step (2) described seed culture is 20-30h.
4. the method according to any one of claim 1-3, it is characterised in that step (3) is described
Comprising the component that mass volume ratio (w/v) is following in fermentation medium：Glucose 1.0-4.0%, corn pulp
0.5-4.0%, peptone 0.5-4.0% and yeast extract 0.5-3.0%, the pH value of the fermentation medium is 5.5-6.5,
Used after steam sterilizing.
5. the method according to any one of claim 1-4, it is characterised in that step (3) is described
The inoculum concentration of seed liquor is the 2-10%, preferably 2-5% of fermentation medium volume.
6. the method according to any one of claim 1-5, it is characterised in that step (3) is described
Shaken cultivation is carried out under the oscillation rate of 160-220r/min；
Preferably, the temperature of step (3) described shaken cultivation is 25-32 DEG C.
7. the method according to any one of claim 1-6, it is characterised in that step (3) is described
Carbon source is organic carbon source and/or inorganic carbon source；
Preferably, step (3) described carbon source be selected from glucose, fructose, galactolipin, maltose, lactose,
Sucrose, molasses, starch, pectin, cellulose, grease type, amino acid, alcohol, aldehyde, phenol, carbonate or
In bicarbonate any one or at least two combination, preferably glucose, fructose, molasses, grease,
In alcohol or carbonate any one or at least two combination.
8. the method according to any one of claim 1-7, it is characterised in that step (3) is described
The additional way of carbon source is in batches continuous stream adds or adds；
Preferably, the concentration for carbon source being added described in step (3) is not less than 50g/L, to keep carbon source in hair
Concentration in zymotic fluid is not less than 0.8mg/mL.
9. the method according to any one of claim 1-8, it is characterised in that institute in step (3)
The additional way of the Alpha-hydroxy progesterone of reactant 17 is stated to feed intake or batch feeding or mix with carbon source for disposable
Flow feeding or batch feeding.
10. the method according to any one of claim 1-9, it is characterised in that methods described includes
Following steps：
(1) prepared by inclined-plane：Aspergillus ochraceus strain is inoculated in slant medium, 25-28 DEG C of incubated 4-7
My god, generation yellow spore inclined-plane is standby；
(2) seed culture：Strain is taken from the yellow spore inclined-plane that step (1) is obtained and is inoculated in seed training
Support in base, in 25-32 DEG C, shaken cultivation 20-30h under 160-220r/min obtains seed liquor；
(3) bioconversion：Just step (2) gained seed liquor is with the 2-10%'s of fermentation medium volume
Inoculum concentration is seeded in fermentation medium, while the Alpha-hydroxy progesterone of reactant 17 is added, at 25-32 DEG C
160-220r/min shaken cultivations, during shaken cultivation, add carbon source and add the Alpha-hydroxy of reactant 17
Progesterone, keeps concentration of the carbon source in zymotic fluid to be not less than 0.8mg/mL, the total throwing of 17 Alpha-hydroxy progesterone
Doses is the 2-20% of fermentation medium volume, obtains 11 α, the zymotic fluid of 17 alpha-dihydroxy progesterone.
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