CN110885869A - Method for producing 7 α -hydroxy-dehydroepiandrosterone by microbial transformation - Google Patents
Method for producing 7 α -hydroxy-dehydroepiandrosterone by microbial transformation Download PDFInfo
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- CN110885869A CN110885869A CN201911344459.2A CN201911344459A CN110885869A CN 110885869 A CN110885869 A CN 110885869A CN 201911344459 A CN201911344459 A CN 201911344459A CN 110885869 A CN110885869 A CN 110885869A
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Abstract
The invention belongs to the technical field of microbial steroid conversion, and discloses a biosynthesis method of 7 α -OH-DHEA, which relates to a production strain of Absidia coerulea (Absidia coerulea), specifically Absidia coerulea CGMCC14124, wherein a production raw material is Dehydroepiandrosterone (DHEA). The DHEA is biologically converted by utilizing the selective hydroxylation capacity of the strain, a target product 7 α -OH-DHEA can be effectively synthesized, when the feeding concentration of a substrate DHEA is up to 5.5g/L, the generation rate of the target product reaches 45.5%, the method realizes green and environment-friendly production, and provides a foundation for the industrial production of steroid medicines.
Description
The technical field is as follows:
the invention relates to the technical field of microbial steroid conversion and industrial fungi, in particular to a method for preparing 7 α -OH-DHEA by converting DHEA through Absidia coerulea to generate 7 α -OH-DHEA.
Background art:
dehydroepiandrosterone (DHEA) is an important active substance in a natural organism and has an important regulation effect on vital metabolic activity, and a hydroxylation product 7 α -OH-DHEA is medically used for controlling weight and treating Alzheimer's disease, has wide application, is high-efficiency and low-toxicity, is an important steroid medicament, and has great market demand and great application value.
At present, dehydroepiandrosterone acetate is used as a raw material for chemically synthesizing 7 α -OH-DHEA in industry, two main methods are to obtain a target product through bromination, isomerization, esterification and hydrolysis, the process is complicated, acyloxy is introduced into the position 7 α by substituting the free radical of benzoyl peroxide tert-butyl ester, and the product is obtained through hydrolysis.
The microbial transformation has many advantages compared with chemical synthesis medicines, the microbial transformation can generate a plurality of substances with unique chemical structures, the chemical synthesis way is difficult to obtain, the microbial growth cycle is short, the microbial transformation can be easily regulated and controlled, and the industrial production can be realized, the existing strains for transforming DHEA7 α hydroxylation mainly comprise colletotrichum lini, mucor racemosus, gibberella and the like, but the problems of low feeding concentration, more byproducts and the like exist, and the industrial production requirements cannot be met.
The invention content is as follows:
in order to achieve the purpose, the invention provides a process for producing 7 α -OH-DHEA by high-efficiency biotransformation, in particular to a biotransformation process for carrying out DHEA7 α hydroxylation under the condition of high feeding concentration by using a strain of Laurella coerulea with excellent performance, wherein the purpose of the invention is realized by the following technical scheme:
the Absidia coerulea strain AL-172(Absidia coerulea) with excellent properties has the microbial preservation number of CGMCC14124, the preservation time of 2017, 5 and 11 months, the preservation unit is China general microbiological culture Collection center, and the method for converting DHEA into 7 α -OH-DHEA by applying the strain comprises the following steps:
transferring the Absidia coerulea AL-172 seed solution into a fermentation medium with the inoculation amount of 10% -15%, fermenting and culturing at 25-30 ℃ for 5-8h at 200r/min, then firstly using 0.3-1.0g/L DHEA to induce, when the culture pH is 3.8-4.2, adjusting the pH of the fermentation liquid to 5.6-6.0, simultaneously adding the DHEA with the concentration of 2-5g/L into the fermentation medium, converting for 5-7h, then adjusting the pH of the fermentation liquid to 5.4-6.0 again, supplementing residual substrates to make the total feeding concentration reach 3-8g/L, and sampling at regular time during the period, wherein the total conversion period is 48 h. Extracting the fermentation liquor by using an organic solvent, taking supernatant, and detecting the conversion condition by using high performance liquid chromatography.
The seed culture medium comprises the following components (g/L): 12 parts of corn steep liquor, 10.5 parts of glucose, 5 parts of ammonium sulfate and 2-3 parts of yeast extract; water, pH 6.5. The fermentation medium had the following composition (g/L): 12 parts of corn steep liquor, 11 parts of glucose, 5 parts of ammonium sulfate and 2-3 parts of yeast extract; deionized water, pH 6.5.
The culture conditions of the seed liquid are as follows: 1ml of spore suspension is inoculated in a seed culture medium and cultured under the conditions of 26-30 ℃ and the rotating speed of 170-220r/min until the pH value of a culture solution reaches 3.8-4.4.
The sporeThe suspension was prepared as follows: washing slant hypha with sterile water, pouring suspension containing large amount of spores into a triangular flask attached with 4 layers of sterilized gauze and filled with glass beads, and shaking sufficiently to obtain a concentrate with a concentration of 3-10 × 107Spore suspension of one/ml.
Preferably, the substrate DHEA is converted to a final concentration of 5-6g/L.
Preferably, DHEA is added in an amount of 0.4-0.6g/L when the substrate DHEA is added for induction.
The method has the beneficial effects that the problem of low feeding concentration in the DHEA7 α hydroxylation reaction is solved, and when the feeding concentration of the substrate DHEA is up to 5.5g/L, the generation rate of the target product reaches 45.5%.
Description of the drawings:
FIG. 1 is a schematic diagram of Absidia coerulea CGMCC14124 for producing 7 α -hydroxy-dehydroepiandrosterone.
FIG. 2 shows NMR H spectrum of product 7 α -hydroxy-dehydroepiandrosterone.
FIG. 3 is a graph of the process for the production of 7 α -hydroxy-dehydroepiandrosterone at high feed concentration (5.5 g/L).
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present patent and are not intended to limit the present invention.
Example 1 transformation of high concentration of DHEA by Absidia coerulea AL-172
(1) Preparation of Absidia coerulea spore suspension: washing hypha on the inclined plane with proper amount of sterile water under sterile condition, filtering the suspension containing spores with 4 layers of gauze, pouring into a triangular flask containing glass beads, and shaking sufficiently to mix the spore suspension.
(2) Seed culture: inoculating the Absidia coerulea spore suspension into a seed culture medium, and culturing at 27 ℃ and the rotation speed of 200r/min until the pH value reaches 4.1-4.2 to prepare the Absidia coerulea seed solution.
The seed culture medium comprises the following components: 12g/L of corn steep liquor, 10g/L of glucose, 2g/L of yeast extract, 5g/L of ammonium sulfate and water, and the pH value is 6.5.
(3) Fermentation culture and transformation: inoculating the seed solution into a fermentation medium with 12% inoculum size, culturing the pH of the fermentation broth to 3.8 under the conditions of 27 ℃ and 180r/min, adjusting the pH of the fermentation broth to 5.6 by NaOH, adding 5.5g/LDHEA into the fermentation medium at one time, and converting for 24h, wherein the components of the fermentation medium are as follows: 12g/L of corn steep liquor, 10g/L of glucose, 2g/L of yeast extract, 5g/L of ammonium sulfate and deionized water, and the pH value is 6.5.
In this example, the feeding concentration of DHEA was 2g/L, after the conversion was completed, the productivity of the target product was 43%, one of the byproducts was found to be easy to be separated later, and the strain could effectively convert DHEA to produce the target product 7 α -OH-DHEA, indicating that the target product 7 α -OH-DHEA could be produced by this method.
Example 2 transformation of high concentration DHEA by Absidia coerulea AL-172
(1) Seed culture
Inoculating 1ml of the Absidia coerulea spore suspension into a seed culture medium, and culturing at 28 ℃ and the rotation speed of 200r/min until the pH value reaches 4.1-4.2 to prepare the Absidia coerulea seed solution.
The seed culture medium comprises the following components: 12g/L of corn steep liquor, 10g/L of glucose, 2g/L of yeast extract, 5g/L of ammonium sulfate and water, and the pH value is 6.5. The spore suspension was as described in example 1.
(2) Biotransformation
When the pH of the seed liquid is 3.8, inoculating the seed liquid into a fermentation culture medium by 12 percent of inoculation amount, performing fermentation culture at 28 ℃ for 5 hours at 180r/min, then inducing by using 0.5g/L of DHEA, when the pH of the culture liquid is 3.6, adjusting the pH of the fermentation liquid to 5.6, adding 5g/L of substrate DHEA, and converting for 48 hours.
The fermentation medium comprises the following components: 12g/L of corn steep liquor, 10g/L of glucose, 2.5g/L of yeast extract, 5g/L of ammonium sulfate and water, and the pH value is 6.5.
In the embodiment, the total feeding concentration of the substrate DHEA is 5.5g/L, and the generation rate of the target product 7 α -OH-DHEA reaches 43% after the strain is transformed for 48 hours.
Example 3 transformation of high concentration DHEA by Absidia coerulea AL-172
(1) Seed culture
Inoculating 1ml of the Absidia coerulea spore suspension into a seed culture medium, and culturing at 28 ℃ and the rotation speed of 200r/min until the pH value reaches 4.1-4.2 to prepare the Absidia coerulea seed solution.
The seed culture medium comprises the following components: 12g/L of corn steep liquor, 10g/L of glucose, 2.5g/L of yeast extract, 5g/L of ammonium sulfate and water, and the pH value is 6.5. The spore suspension was as described in example 1.
(2) Biotransformation
When the pH of the seed liquid is 3.8, inoculating the seed liquid into a fermentation culture medium by 12 percent of inoculation amount, performing fermentation culture at 28 ℃ for 5 hours at 180r/min, then inducing by using 0.5g/L of DHEA, when the culture pH is 3.6, adjusting the pH of the fermentation liquid to 5.6, adding 3g/L of substrate DHEA, adjusting the pH of the fermentation liquid to 5.6 again after 6 hours of conversion, adding 2g/L of DHEA to supplement the rest of the substrate, wherein the total addition amount of the substrate reaches 5.5g/L, and the conversion lasts for 48 hours.
The fermentation medium comprises the following components: 12g/L of corn steep liquor, 10g/L of glucose, 2g/L of yeast extract, 5g/L of ammonium sulfate and water, and the pH value is 6.5.
In the embodiment, the total feeding concentration of the substrate DHEA is 5.5g/L, and the generation rate of the target product 7 α -OH-DHEA reaches 45.5 percent after the strain is transformed for 48 hours.
The above specific embodiments are further detailed for the present invention, but are merely illustrative and not limitative of the present invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention. Therefore, the protection scope of this patent shall be subject to the appended claims.
Claims (5)
1. A method for producing 7 α -OH-DHEA by using microorganisms is characterized in that Absidia coerulea is taken as a production strain, and a product 7 α -OH-DHEA is obtained by converting a substrate DHEA after spore suspension preparation, seed culture, fermentation culture and conversion condition improvement, and the method comprises the following specific steps:
transferring the Absidia coerulea AL-172 seed solution into a fermentation medium with the inoculation amount of 10% -15%, fermenting and culturing at 25-30 ℃ for 5-8h at 200r/min, then using 0.3-1.0g/L DHEA for induction, adjusting the pH of the fermentation liquid to 5.6-6.0 when the pH of the culture is 3.8-4.2, simultaneously adding 2-5g/L DHEA into the fermentation medium, converting for 5-7h, adjusting the pH of the fermentation liquid to 5.4-6.0 again, supplementing residual substrates to make the total feeding concentration reach 3-8g/L, and continuing to convert. Sampling the fermentation liquor at regular time in the conversion process, and measuring the product generation rate.
2. The method for producing 7 α -OH-DHEA by using a microorganism as claimed in claim, wherein the specific strain is Absidia coerulea (Absidia coerulea) AL-172, with the accession number of CGMCC 14124.
3. The method of claim 1 wherein the seed medium component (g/L) is corn steep liquor 12, glucose 10.5, ammonium sulfate 5, yeast extract 2-3, water, pH 6.5.
Fermentation medium composition (g/L): 12 parts of corn steep liquor, 11 parts of glucose, 5 parts of ammonium sulfate and 2-3 parts of yeast extract; water, pH 6.5.
4. The method for producing 7 α -OH-DHEA by using the microorganism as claimed in claim 1, wherein the seed culture solution is prepared by inoculating 1mL of Absidia coerulea spore suspension into a seed culture medium (30/250mL triangular flask), and culturing at 26-30 ℃ and 180-210r/min until the pH reaches 3.6-4.0.
5. The method of claim 1, wherein the spore suspension is prepared by washing slant spores of the strain with sterile water, filtering with 6 layers of gauze, shaking, counting with a hemocytometer, and adjusting the concentration of the spore suspension to 3-10X 107one/mL.
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Citations (5)
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| CN107475131A (en) * | 2017-09-20 | 2017-12-15 | 天津科技大学 | One plant of Absidia is mould and its applies |
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