CN114292892A - Method for producing androstadienedione by virtue of Arthrobacter simplex fermentation - Google Patents
Method for producing androstadienedione by virtue of Arthrobacter simplex fermentation Download PDFInfo
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Abstract
The invention belongs to the technical field of preparation of steroid hormone medicaments, and particularly relates to a method for producing androstadienedione by fermentation of Arthrobacter simplex, which mainly comprises the steps of optimizing components of a fermentation culture medium for whole-cell transformation of Arthrobacter simplex, a feeding point and a substrate to obtain an ADD crude product with high conversion rate, and extracting and refining the ADD crude product by acetone-water mixing to obtain an ADD fine product. The invention effectively solves the problems of low conversion efficiency, difficult separation of byproducts, complex process, unsuitability for industrial mass production and the like in the prior art, improves the fermentation feeding concentration to 30-60g/L and improves the conversion rate to more than 97 percent.
Description
Technical Field
The invention belongs to the technical field of preparation of steroid hormone medicaments, and particularly relates to an efficient method for producing Androstadienedione (ADD) by fermenting Arthrobacter simplex CPCC 140451, in particular to optimization of components of a used culture medium, optimization of a feeding point and pretreatment of a substrate.
Background
The steroid medicine has strong pharmacological actions of resisting infection, allergy, virus and shock, and is an important medicine in clinic. Most of raw materials for early synthesis of steroid drugs are directly extracted from animal tissue fluid, so that the recovery rate is low, the cost is high, and the production requirement cannot be met; the later-stage synthesis method provides rich raw materials for the industrial production of steroid medicines by using dioscin as a starting raw material and through a chemical synthesis method, but along with the increasing exhaustion of dioscin, more abundant resources and methods are urgently needed for substitution; the occurrence of phytosterol brings new changes to the steroid field, the phytosterol is obtained by producing leftovers of waste oil and various crops, important steroid drug intermediates Androstenedione (AD) and Androstadienedione (ADD) are obtained by utilizing a biological selective side chain degradation technology, and the androstenedione and androstadienedione are further prepared to obtain the steroid drug, wherein the ADD is an important starting material for synthesizing estrogens.
The main methods for preparing ADD at present are: firstly, the mycobacteria are adopted to degrade the phytosterol to obtain AD and ADD, such as US patent USP4345029 and Chinese patent CN102477404A, but the technologies have the defects of long fermentation period (150h) and low conversion efficiency, the conversion rate is only up to 70%, and meanwhile, a large amount of byproducts such as AD, phytosterol and testosterone are generated, so that the subsequent separation and refining are not facilitated. Secondly, oxidizing and dehydrogenating the Arthrobacter simplex, the mycobacteria, the Bacillus subtilis and the Nocardia simplex by using AD to generate ADD, such as Chinese patent documents CN103266161A, CN106191190A, CN104531746A and CN108456666A, Chinese invention document CN106636160A, wherein the process advantages are obviously higher than those of the first method, but as most of the strains are transgenic recombinant strains, uncontrollable property exists in amplification production, and the method is not suitable for industrial production; the Chinese patent document CN110656147A adopts simple Nocardia bacterial liquid as an enzyme source, and adds cosolvent soybean oil during feeding, so that the auxiliary material cost is increased, the post-treatment process is complicated, and the industrial production is not suitable.
Disclosure of Invention
In view of the problems and disadvantages of the prior art, the present invention aims to provide an efficient method for producing Androstadienedione (ADD) by fermenting Arthrobacter simplex CPCC 140451. By the systematic optimization of the Arthrobacter simplex whole-cell process and the optimization of substrate pretreatment, the fermentation feeding concentration is expected to be improved to 60g/L, the conversion rate is improved to 97%, the yield of a fine product reaches 91%, and the quality of the fine product reaches more than 99%.
In order to achieve the purpose of the invention, the inventor provides the following technical scheme:
a method for producing Androstadienedione (ADD) by Arthrobacter simplex fermentation takes Arthrobacter simplex CPCC 140451 as a fermentation strain, and AD is efficiently converted into ADD by optimizing a simple Arthrobacter whole-cell fermentation system and optimizing substrate (AD) pretreatment, wherein the optimizing of the whole-cell fermentation system comprises optimizing fermentation medium components and feeding point bacterial concentration.
Specifically, the method mainly optimizes the components, feeding points and substrates of a fermentation medium for whole-cell transformation of Arthrobacter simplex to obtain an ADD crude product with high transformation rate, and then extracts and refines the ADD crude product through acetone-water mixing to obtain an ADD fine product. The concentration of the fermentation feed is increased to 30-60g/L, and the conversion rate is increased to more than 97%.
The inventor researches and discovers that through systematic research on the fermentation of the Arthrobacter simplex, the conversion rate of ADD can be greatly improved by using an optimized system of the Arthrobacter simplex, the fermentation conversion rate can reach more than 97 percent, meanwhile, the AD is treated by adopting a substrate pretreatment process, the limitation of adding different cosolvents to the feeding concentration of the substrate in the steroid dehydrogenation process is removed, the feeding substrate concentration of the reaction can reach 60g/L, meanwhile, the impurities of the converted product are few, the separation and refining are simple, the solvent consumption is low, and the process route has a greater cost advantage compared with the fermentation route from phytosterol to ADD, and is suitable for industrial production.
Preferably, in the present invention, the fermentation medium component comprises glucose: 10-30g/L, soy peptone: 3-7g/L, corn steep liquor: 10-30g/L, potassium dihydrogen phosphate: 1-5g/L, yeast extract: 2-4g/L, manganese chloride: 0.003g/L, ferrous sulfate: 0.03g/L, Tween-80: 0.04-0.2% and molinate: 0.04-0.4%, and the pH of the fermentation medium is 7.0.
Preferably, in the invention, the concentration of the bacteria at the feeding point is 30-50%. The fermentation conversion conditions are optimized, namely the bacterial concentration at the feeding point is as follows: on the basis of optimizing the components of a culture medium, the influence of the bacterial concentration of a feeding point on the AD conversion rate is considered, the light transmittance (namely the bacterial concentration) is measured by utilizing a spectrophotometer at 620nm in the middle and later stages of the logarithmic growth period of the thalli, the feeding is respectively carried out at several time points when the bacterial concentration is measured to be 20%, 30%, 40% and 50%, under the condition that other conditions are not changed, the feeding is carried out, the AD conversion rate is measured at the fermentation end point, and the feeding concentration is preferably 30% -50%.
Preferably, in the present invention, the substrate (AD) pretreatment comprises pretreatment of the substrate AD with methanol, acetone, ethyl acetate or water elution.
More preferably, in the present invention, the pretreatment of the substrate (AD) is carried out by: the methanol pretreatment method comprises dissolving substrate AD at 65-67 deg.C by volume of 10-12v, concentrating under reduced pressure at 45 deg.C or below and vacuum degree of 0.06-0.08Mpa to obtain methanol pretreated substrate; the acetone pretreatment method comprises dissolving substrate AD at 55-57 deg.C by volume of 10-12v, concentrating under reduced pressure at 45 deg.C or below and vacuum degree of 0.06-0.08Mpa to obtain acetone pretreated substrate; dissolving substrate AD at 74-76 deg.C by volume of 10-12v, concentrating under reduced pressure at 45 deg.C or below and vacuum degree of 0.06-0.08Mpa to obtain pretreated substrate; the water separation pretreatment method comprises the steps of dissolving a substrate AD clearly by adopting a DMF solvent with the volume of 1-1.5v, slowly dripping the substrate AD into a water phase with the volume of 5-6v, starting stirring, cooling to below 10 ℃ after dripping is finished, and filtering to obtain the water separation pretreatment substrate.
Most preferably, in the invention, the substrate AD pretreatment is performed by using methanol, and the feeding concentration is 30-60 g/L.
(1) Effect of different pre-treated substrates on the conversion of AD to ADD. Performing a pretreatment substrate feeding experiment on the basis of optimizing the composition of a culture medium and culture conditions, wherein the feeding concentration is 30g/L, the substrates are respectively ethyl acetate pretreatment, methanol pretreatment, acetone pretreatment and water precipitation pretreatment, after fermentation is carried out for 72 hours, the conversion rate of AD is measured at the fermentation end point, and methanol is preferably used for pretreating the substrates.
(2) Influence of different feed concentrations of the same pretreatment substrate on the conversion rate of whole-cell conversion of AD to ADD determines that the pretreatment of the substrate by methanol is preferable. On the basis of determining a methanol pretreatment substrate, increasing the concentration of a feeding substrate, wherein the feeding concentration is respectively 30g/L, 60g/L and 80g/L, and after fermentation conversion is carried out for 72 hours, measuring the conversion rate of AD at a fermentation end point to improve the maximum feeding concentration for producing ADD by completely converting AD, wherein the feeding concentration is preferably 30-60 g/L.
Preferably, in the present invention, the method further comprises the step of controlling the air flow rate in the microorganism growth stage to be 3.0-5.0m3The stirring speed is 200-400r/min, and the temperature is 31-34 ℃; the air flow rate of the microbial conversion stage is 1.0-3.0m3The stirring speed is 300-500r/min, the temperature is 33-35 ℃, and the conversion time is 72-96 h.
Preferably, in the invention, the method further comprises the steps of extracting and refining the crude ADD, and the method comprises the following steps: extracting and concentrating the crude fermentation product by using a 10-12v volume of 85% acetone-water mixed solvent, wherein the yield of the crude product is more than 97%; and refining the crude product by using 5-6v of 85% acetone-water mixed solvent to obtain a refined product, wherein the HPLC purity of ADD in the refined product is more than 99%, and the yield of the refined product is more than 90%. On the basis of fermentation optimization, a fermentation filter cake with high conversion rate is obtained through optimization of fermentation conditions, the HPLC ratio of ADD to AD in the fermentation filter cake is 98:1, the fermentation filter cake is extracted by using an acetone-water mixed solvent, and a crude product after extraction is refined by using the acetone-water mixed solvent again, so that a high-purity ADD refined product is obtained, the HPLC content of the refined product is more than or equal to 99%, and the yield of the refined product is more than 90%.
In a preferred embodiment of the invention: the fermentation medium comprises the following components: 10-30g/L, soy peptone: 3-7g/L, corn steep liquor: 10-30g/L, potassium dihydrogen phosphate: 1-5g/L, yeast extract: 2-4g/L, manganese chloride: 0.003g/L, ferrous sulfate: 0.03g/L, Tween-80: 0.04-0.2%, molinate: 0.04-0.4%, the pH of the fermentation medium is 7.0, and the concentration of the bacteria at the feeding point is 30-50%; the preferable substrate pretreatment method is methanol pretreatment; the feeding concentration AD is 60g/L, and the yield of ADD reaches 57 g/L; the yield of ADD after the fermentation product is extracted and refined reaches 91 percent, and the HPLC purity of ADD is more than 99 percent.
Compared with the prior art, the invention has the advantages that:
the method takes Arthrobacter simplex CPCC 140451 as a fermentation strain, and optimizes components of a fermentation medium, culture conditions, substrate pretreatment and the like, so that the feeding concentration reaches 30-60g/L, and the conversion rate reaches more than 97%. By optimizing a whole-cell fermentation system of Arthrobacter simplex and substrate pretreatment feeding, the whole-cell conversion of AD to produce ADD has more advantages compared with the route of sterol to produce ADD, and is more suitable for industrial application.
Drawings
FIG. 1 is a crystal pattern diagram of a water-analyzed pretreated substrate (photographing device: OLYMPUS CX31, software: Mingmei microscopic digital measurement and analysis system V1.0).
FIG. 2 is a crystal pattern diagram of the ethyl acetate pretreated substrate (photographing device: OLYMPUS CX31, software: Mingmei microscopic digital measurement analysis system V1.0).
FIG. 3 is a crystal pattern diagram of the acetone pretreated substrate (shooting equipment: OLYMPUS CX31, software: Mingmei microscopic digital measurement and analysis system V1.0).
FIG. 4 is a crystal pattern diagram of a methanol pretreated substrate according to the present invention (photographing device: OLYMPUS CX31, software: Mingmei microscopic digital measurement and analysis system V1.0).
FIG. 5 is a hydrographic pretreatment substrate conversion HPLC profile of example 1 of the present invention.
FIG. 6 is an HPLC chromatogram of substrate conversion for methanol pretreatment in example 5 of the present invention.
FIG. 7 is an HPLC chromatogram of acetone pretreatment substrate conversion according to example 6 of the present invention.
FIG. 8 is an HPLC chromatogram of conversion of an ethyl acetate pretreated substrate according to example 7 of the present invention.
Detailed Description
The present invention will be described in more detail with reference to examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
In the invention, all parts and percentages are weight units, and all equipment, raw materials and the like can be purchased from the market or are commonly used in the industry, if not specified. The methods in the following examples are conventional in the art unless otherwise specified.
Description of the drawings:
the production strain is Arthrobacter simplex CPCC 140451 purchased from China pharmaceutical microorganism strain preservation management center, and originally comes from Anyang second pharmaceutical factory in Henan province in China in 1989.
Example 1
Fermentation medium components: glucose: 10g/L, peptone: 3g/L, corn steep liquor: 10g/L, potassium dihydrogen phosphate: 1g/L, yeast extract: 1g/L, manganese chloride: 0.003g/L, 0.03g/L ferrous sulfate, 0.04g/L tween, 0.04g/L natural sodium chloride, initial pH of fermentation 7.0, and water analysis pretreatment AD substrate as a substrate.
The fermentation process is as follows: first-order seed culture, adding a culture medium into a jacketed 50L fermentation tank, wherein the charging coefficient is 0.6, sterilizing, cooling to 33 ℃, inoculating Arthrobacter simplex, inoculating 10 percent of inoculum size, and fermenting in two stages: firstly, a thallus growth stage: after inoculation, the air flow was adjusted to 3.0m3And h, stirring at the speed of 200r/min, controlling the temperature at 33 ℃, culturing for 9h, monitoring the bacterial concentration to be 20%, and feeding. ② microbial transformation stage: the feeding concentration is 10g/L, and the air flow is adjusted to be 1.0m3The stirring speed is 300r/min, the temperature is 33 ℃, the conversion time is 72h, and the conversion rate is 86.95%.
Extraction and refining: centrifuging the fermentation liquor, extracting the fermentation filter cake by using 85% acetone-water mixed solvent, drying the extracted crude product, and refining by using 85% acetone-water mixed solvent again to obtain the fine product ADD, wherein the yield of the fine product is 69%, and the content of the fine product is more than 99%.
Example 2
Fermentation medium components: glucose: 10g/L, peptone: 3g/L, corn steep liquor: 10g/L, potassium dihydrogen phosphate: 1g/L, yeast extract: 1g/L, manganese chloride: 0.003g/L, 0.03g/L ferrous sulfate, 0.04g/L tween, 0.04g/L natural sodium chloride, initial pH of fermentation 7.0, and water analysis pretreatment AD substrate as a substrate.
The fermentation process is as follows: first-order seed culture, adding a culture medium into a jacketed 50L fermentation tank, wherein the charging coefficient is 0.6, sterilizing, cooling to 33 ℃, inoculating Arthrobacter simplex, inoculating 10 percent of inoculum size, and fermenting in two stages: firstly, a thallus growth stage: after inoculation, the air flow was adjusted to 3.0m3And h, stirring at the speed of 200r/min, controlling the temperature at 33 ℃, culturing for 8h, monitoring the bacterial concentration to be 30%, and feeding. ② microbial transformation stage: the feeding concentration is 10g/L, and the air flow is adjusted to be 1.0m3The stirring speed is 300r/min, the temperature is 33 ℃, the conversion time is 72h, and the conversion rate is 92.99 percent.
Extraction and refining: centrifuging the fermentation liquor, extracting the fermentation filter cake by using 85% acetone-water mixed solvent, drying the extracted crude product, and refining by using 85% acetone-water mixed solvent again to obtain the fine product ADD, wherein the yield of the fine product is 75%, and the content of the fine product is more than 99%.
Example 3
Fermentation medium components: glucose: 30g/L, soy peptone: 5g/L, corn steep liquor: 30g/L, potassium dihydrogen phosphate: 5g/L, yeast extract: 4g/L, manganese chloride: 0.003g/L, 0.03g/L ferrous sulfate, 0.12g/L tween, 0.2g/L natural sodium chloride, initial pH of fermentation 7.0, and water analysis pretreatment AD substrate as a substrate.
The fermentation process is as follows: first-order seed culture, adding a culture medium into a jacketed 50L fermentation tank, wherein the charging coefficient is 0.6, sterilizing, cooling to 33 ℃, inoculating Arthrobacter simplex, inoculating 10 percent of inoculum size, and fermenting in two stages: firstly, a thallus growth stage: after inoculation, the air flow was adjusted to 4.0m3H, stirring speed 300r/min, temperatureControlling the temperature at 33 ℃, culturing for 7h, monitoring the bacterial concentration to be 40%, and feeding. ② microbial transformation stage: the feeding concentration is 10g/L, and the air flow is adjusted to be 2.0m3H, stirring speed of 400r/min, temperature of 33 ℃, conversion time of 72h and conversion rate of 93.62 percent.
Extraction and refining: centrifuging the fermentation liquor, extracting the fermentation filter cake by using 85% acetone-water mixed solvent, drying the extracted crude product, and refining by using 85% acetone-water mixed solvent again to obtain the fine product ADD, wherein the yield of the fine product is 79%, and the content of the fine product is more than 99%.
Example 4
Fermentation medium components: glucose: 20g/L, soy peptone: 5g/L, corn steep liquor: 20g/L, potassium dihydrogen phosphate: 3g/L, yeast extract: 2g/L, manganese chloride: 0.003g/L, 0.03g/L ferrous sulfate, 0.2g/L tween, 0.4g/L natural sodium chloride, initial pH of fermentation 7.0, and water analysis pretreatment AD substrate as a substrate.
The fermentation process is as follows: first-order seed culture, adding a culture medium into a jacketed 50L fermentation tank, wherein the charging coefficient is 0.6, sterilizing, cooling to 33 ℃, inoculating Arthrobacter simplex, inoculating 10 percent of inoculum size, and fermenting in two stages: firstly, a thallus growth stage: after inoculation, the air flow was adjusted to 3.0m3And h, stirring at the speed of 400r/min, controlling the temperature at 33 ℃, culturing for 6h, monitoring the bacterial concentration to be 50%, and feeding. ② microbial transformation stage: the feeding concentration is 10g/L, and the air flow is adjusted to be 3.0m3The stirring speed is 500r/min, the temperature is 33 ℃, the conversion time is 72h, and the conversion rate is 94.23%.
Extraction and refining: centrifuging the fermentation liquor, extracting the fermentation filter cake by using 85% acetone-water mixed solvent, drying the extracted crude product, and refining by using 85% acetone-water mixed solvent again to obtain the fine product ADD, wherein the yield of the fine product is 83%, and the content of the fine product is more than 99%.
Example 5
Fermentation medium components: glucose: 20g/L, soy peptone: 5g/L, corn steep liquor: 20g/L, potassium dihydrogen phosphate: 3g/L, yeast extract: 2g/L, manganese chloride: 0.003g/L, 0.03g/L ferrous sulfate, 0.12g/L Tween, 0.2g/L natural sodium chloride, initial pH of fermentation 7.0, and methanol-pretreated AD substrate as substrate.
The fermentation process is as follows: first-order seed culture, adding a culture medium into a jacketed 50L fermentation tank, wherein the charging coefficient is 0.6, sterilizing, cooling to 33 ℃, inoculating Arthrobacter simplex, inoculating 10 percent of inoculum size, and fermenting in two stages: firstly, a thallus growth stage: after inoculation, the air flow was adjusted to 3.0m3And h, stirring at the speed of 300r/min, controlling the temperature at 33 ℃, culturing for 7h, monitoring the bacterial concentration to be 40%, and feeding. ② microbial transformation stage: the feeding concentration is 30g/L, the stirring speed is 400r/min, and the air flow is adjusted to be 2.0m3The reaction temperature is 33 ℃, the conversion time is 72h, and the conversion rate is 98.95%.
Extraction and refining: centrifuging the fermentation liquor, extracting the fermentation filter cake by using 85% acetone-water mixed solvent, drying the extracted crude product, and refining by using 85% acetone-water mixed solvent again to obtain the fine product ADD, wherein the yield of the fine product is 92%, and the content of the fine product is more than 99%.
Example 6
Fermentation medium components: glucose: 20g/L, soy peptone: 5g/L, corn steep liquor: 20g/L, potassium dihydrogen phosphate: 3g/L, yeast extract: 2g/L, manganese chloride: 0.003g/L, 0.03g/L ferrous sulfate, 0.12g/L tween, 0.2g/L natural sodium chloride, initial pH of fermentation 7.0, and acetone pretreatment AD substrate as a substrate.
The fermentation process is as follows: first-order seed culture, adding a culture medium into a jacketed 50L fermentation tank, wherein the charging coefficient is 0.6, sterilizing, cooling to 33 ℃, inoculating Arthrobacter simplex, inoculating 10 percent of inoculum size, and fermenting in two stages: firstly, a thallus growth stage: after inoculation, the air flow was adjusted to 3.0m3And h, stirring at the speed of 300r/min, controlling the temperature at 33 ℃, culturing for 7h, monitoring the bacterial concentration to be 40%, and feeding. ② microbial transformation stage: the feeding concentration is 30g/L, the stirring speed is 400r/min, and the air flow is adjusted to be 2.0m3The temperature is 34 ℃, the conversion time is 85h, and the conversion rate is 95.66%.
Extraction and refining: centrifuging the fermentation liquor, extracting the fermentation filter cake by using 85% acetone-water mixed solvent, drying the extracted crude product, and refining by using 85% acetone-water mixed solvent again to obtain the fine product ADD, wherein the yield of the fine product is 88%, and the content of the fine product is more than 99%.
Example 7
Fermentation medium components: glucose: 20g/L, soy peptone: 5g/L, corn steep liquor: 20g/L, potassium dihydrogen phosphate: 3g/L, yeast extract: 2g/L, manganese chloride: 0.003g/L, 0.03g/L ferrous sulfate, 0.12g/L tween, 0.2g/L natural sodium chloride, initial pH of fermentation 7.0, and ethyl acetate as a substrate for pretreatment of AD substrate.
The fermentation process is as follows: first-order seed culture, adding a culture medium into a jacketed 50L fermentation tank, wherein the charging coefficient is 0.6, sterilizing, cooling to 33 ℃, inoculating Arthrobacter simplex, inoculating 10 percent of inoculum size, and fermenting in two stages: firstly, a thallus growth stage: after inoculation, the air flow was adjusted to 3.0m3And h, stirring at the speed of 300r/min, controlling the temperature at 33 ℃, culturing for 7h, monitoring the bacterial concentration to be 40%, and feeding. ② microbial transformation stage: the feeding concentration is 30g/L, and the air flow is adjusted to be 2.0m3The stirring speed is 400r/min, the temperature is 33 ℃, the conversion time is 96h, and the conversion rate is 95.27%.
Extraction and refining: centrifuging the fermentation liquor, extracting the fermentation filter cake by using 85% acetone-water mixed solvent, drying the extracted crude product, and refining by using 85% acetone-water mixed solvent again to obtain the fine product ADD, wherein the yield of the fine product is 83%, and the content of the fine product is more than 99%.
Example 8
Fermentation medium components: glucose: 20g/L, soy peptone: 5g/L, corn steep liquor: 20g/L, potassium dihydrogen phosphate: 3g/L, yeast extract: 2g/L, manganese chloride: 0.003g/L, 0.03g/L ferrous sulfate, 0.12g/L tween, 0.2g/L natural sodium chloride, initial pH of fermentation 7.0, and methanol-pretreated AD substrate as substrate.
The fermentation process is as follows: first-order seed culture, adding a culture medium into a jacketed 50L fermentation tank, wherein the charging coefficient is 0.6, sterilizing, cooling to 33 ℃, inoculating Arthrobacter simplex, inoculating 10 percent of inoculum size, and fermenting in two stages: firstly, a thallus growth stage: after inoculation, the air flow was adjusted to 3.0m3And h, stirring at the speed of 300r/min, controlling the temperature at 33 ℃, culturing for 7h, monitoring the bacterial concentration to be 40%, and feeding. ② microbial transformation stage: the feeding concentration is 60g/L, the stirring speed is 400r/min, and the air flow is adjusted to be 2.0m3H, temperatureThe conversion time was 72h at 33 ℃ and the conversion was 97.18%.
Extraction and refining: centrifuging the fermentation liquor, extracting the fermentation filter cake by using 85% acetone-water mixed solvent, drying the extracted crude product, and refining by using 85% acetone-water mixed solvent again to obtain the fine product ADD, wherein the yield of the fine product is 91%, and the content of the fine product is more than 99%.
Example 9
Fermentation medium components: glucose: 20g/L, soy peptone: 5g/L, corn steep liquor: 20g/L, potassium dihydrogen phosphate: 3g/L, yeast extract: 2g/L, manganese chloride: 0.003g/L, 0.03g/L ferrous sulfate, 0.12g/L tween, 0.2g/L natural sodium chloride, initial pH of fermentation 7.0, and methanol-pretreated AD substrate as substrate.
The fermentation process is as follows: first-order seed culture, adding a culture medium into a jacketed 50L fermentation tank, wherein the charging coefficient is 0.6, sterilizing, cooling to 33 ℃, inoculating Arthrobacter simplex, inoculating 10 percent of inoculum size, and fermenting in two stages: firstly, a thallus growth stage: after inoculation, the air flow was adjusted to 3.0m3And h, stirring at the speed of 300r/min, controlling the temperature at 33 ℃, culturing for 7h, monitoring the bacterial concentration to be 40%, and feeding. ② microbial transformation stage: the feeding concentration is 80g/L, the stirring speed is 400r/min, and the air flow is adjusted to be 2.0m3H, temperature 33 ℃, conversion time 85h and conversion rate 96.03%.
Extraction and refining: centrifuging the fermentation liquor, extracting the fermentation filter cake by using 85% acetone-water mixed solvent, drying the extracted crude product, and refining by using 85% acetone-water mixed solvent again to obtain the fine product ADD, wherein the yield of the fine product is 87%, and the content of the fine product is more than 99%.
Comparative example 1
Fermentation medium components: glucose: 20g/L, soy peptone: 5g/L, corn steep liquor: 20g/L, potassium dihydrogen phosphate: 3g/L, yeast extract: 2g/L, manganese chloride: 0.003g/L, 0.03g/L ferrous sulfate, 0.12g/L tween, 0.2g/L natural sodium chloride, initial pH of fermentation 7.0, and direct feeding of AD substrate.
The fermentation process is as follows: first-stage seed culture, adding culture medium into jacketed 50L fermentation tank, with a charging coefficient of 0.6, sterilizing, cooling to 33 deg.C, inoculating Arthrobacter simplexThe inoculation amount is 10 percent, and the fermentation is divided into two stages: firstly, a thallus growth stage: after inoculation, the air flow was adjusted to 3.0m3And h, stirring at the speed of 300r/min, controlling the temperature at 33 ℃, culturing for 8h, monitoring the bacterial concentration at 30 percent, and feeding. ② microbial transformation stage: the feeding concentration is 10g/L, and the air flow is adjusted to be 2.0m3The stirring speed is 400r/min, the temperature is 33 ℃, the conversion time is 80h, and the conversion rate is 87%.
Extraction and refining: centrifuging the fermentation liquor, extracting the fermentation filter cake by using 85% acetone-water mixed solvent, drying the extracted crude product, and refining by using 85% acetone-water mixed solvent again to obtain the fine product ADD, wherein the yield of the fine product is 70%, and the content of the fine product is more than 98.5%.
Claims (8)
1. A method for producing androstadienedione by Arthrobacter simplex fermentation is characterized in that Arthrobacter simplex CPCC 140451 is used as a fermentation strain, AD is converted into ADD by optimizing a Arthrobacter simplex whole-cell fermentation system and optimizing substrate AD pretreatment, wherein the optimizing of the whole-cell fermentation system comprises optimizing fermentation medium components and feeding point bacterial concentration.
2. The method of claim 1, wherein the fermentation medium comprises glucose: 10-30g/L, soy peptone: 3-7g/L, corn steep liquor: 10-30g/L, potassium dihydrogen phosphate: 1-5g/L, yeast extract: 2-4g/L, manganese chloride: 0.003g/L, ferrous sulfate: 0.03g/L, Tween-80: 0.04-0.2% and molinate: 0.04-0.4%, and the pH of the fermentation medium is 7.0.
3. The method for producing androstadienedione from Arthrobacter simplex by fermentation as claimed in claim 1, wherein the feed point concentration is 30-50%.
4. The method for producing androstadienedione from Arthrobacter simplex fermentation as claimed in claim 1, wherein the pretreatment of the substrate AD comprises pretreatment of the substrate AD with methanol, acetone, ethyl acetate or water-out.
5. The method for producing androstadienedione by fermentation of Arthrobacter simplex according to claim 1 or 4, wherein the pretreatment of the substrate AD is performed by: the methanol pretreatment method comprises dissolving substrate AD at 65-67 deg.C by volume of 10-12v, concentrating under reduced pressure at 45 deg.C or below and vacuum degree of 0.06-0.08Mpa to obtain methanol pretreated substrate; the acetone pretreatment method comprises dissolving substrate AD at 55-57 deg.C by volume of 10-12v, concentrating under reduced pressure at 45 deg.C or below and vacuum degree of 0.06-0.08Mpa to obtain acetone pretreated substrate; dissolving substrate AD at 74-76 deg.C by volume of 10-12v, concentrating under reduced pressure at 45 deg.C or below and vacuum degree of 0.06-0.08Mpa to obtain pretreated substrate; the water separation pretreatment method comprises the steps of dissolving a substrate AD clearly by adopting a DMF solvent with the volume of 1-1.5v, slowly dripping the substrate AD into a water phase with the volume of 5-6v, starting stirring, cooling to below 10 ℃ after dripping is finished, and filtering to obtain the water separation pretreatment substrate.
6. The method for producing androstadienedione from Arthrobacter simplex fermentation according to claim 1 or 4, wherein methanol is used for pretreatment of the substrate AD, and the feed concentration is 30-60 g/L.
7. The method for producing androstadienedione from Arthrobacter simplex by fermentation as claimed in claim 1, further comprising a step of providing a microorganism growth stage air flow of 3.0-5.0m3The stirring speed is 200-400r/min, and the temperature is 31-34 ℃; the air flow rate of the microbial conversion stage is 1.0-3.0m3The stirring speed is 300-500r/min, the temperature is 33-35 ℃, and the conversion time is 72-96 h.
8. The method for producing androstadienedione by Arthrobacter simplex fermentation as claimed in claim 1, further comprising crude ADD extraction and purification, by the following steps: extracting and concentrating the crude fermentation product by using a 10-12v volume of 85% acetone-water mixed solvent, wherein the yield of the crude product is more than 97%; refining the crude product by using 5-6v of 85% acetone-water mixed solvent to obtain an ADD refined product.
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