CN114317372B - Arthrobacter urate and application thereof - Google Patents
Arthrobacter urate and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial steroid transformation, and particularly relates to a Arthrobacter urate strain and application thereof in C1 and 2 dehydrogenation. The Arthrobacter urate strain is specifically Arthrobacter urate (Arthrobacter uratoxydans) AAR-47, and has the preservation number: CGMCC No.23650. The Arthrobacter urate AAR-47 is obtained by physical mutagenesis, the strain after mutagenesis uses hydrocortisone acetate as a substrate, fermentation is carried out for 30-96h under the condition of adding the hydrocortisone acetate with the concentration of 10-60g/L, the substrate conversion rate can reach 90.81-100%, the strain after mutagenesis uses hydrocortisone as the substrate, the strain after mutagenesis carries out fermentation conversion for 60-96h under the condition of 5-40g/L, and the substrate conversion rate can reach 90.56-94.58%.
Description
Technical field:
the invention belongs to the technical field of microbial steroid transformation, and particularly relates to a Arthrobacter urate strain and application thereof in C1 and 2 dehydrogenation.
The background technology is as follows:
steroid drugs have important physiological activities, and are second most important drugs next to antibiotics. Prednisolone is an adrenocortical hormone drug, and is mainly used for treating various acute serious bacterial infections, serious allergic diseases, rheumatism, rheumatoid arthritis, serious bronchial asthma and the like. Meanwhile, the prednisolone can also be used as a raw material of other corticosteroids, is an important intermediate for synthesizing triamcinolone acetonide, 16 alpha-hydroxy prednisolone and other drugs, and has wide application value and very important. The preparation research of the product is continuous at home and abroad.
At present, the preparation method of prednisolone mainly comprises the following steps:
1. preparing prednisolone by taking a steroid mother nucleus as a raw material through multi-step chemical reaction; for example, CN101397324a discloses that prednisolone is obtained by a multi-step chemical synthesis process using 17α -hydroxy-1, 4, 9-triesterone-3, 20 as a raw material; CN200710061258.2 discloses a method for synthesizing prednisolone, which synthesizes prednisolone through bromination, debromination, iodine adding, replacement and hydrolysis. In addition, a similar multi-step chemical synthesis process for preparing prednisolone is disclosed in CN1361108A, CN105384790a et al. However, the method has the defects of complicated process route, complex process flow, longer production period, lower total yield, unfriendly environment and the like, and the short plates make the production cost of the prednisolone high.
2. The prednisone is taken as a raw material, dissolved by methanol, added with semicarbazide hydrochloride solution for condensation reaction, reduced by lithium aluminum hydride, and hydrolyzed to obtain the prednisolone. However, the prednisone has high raw material cost, which is unfavorable for reducing the production cost.
3. Prednisolone is prepared from hydrocortisone serving as a starting material through a C1 and 2-position dehydrogenation process, and the synthetic route is as follows:
methods for dehydrogenating steroid at the C1,2 position generally include chemical methods, biological fermentation methods, and the like. Chemical dehydrogenation generally requires the use of SeO 2 The yield is relatively low, and SeO 2 Is a serious poison and has great environmental pollution and has been gradually replaced by biological methods at present. Regarding the biological fermentation methods, CN102206696a and CN101760495A disclose microbial fermentation and cell transformation for producing products, and these methods, although avoiding the above-mentioned drawbacks of the chemical methods, generally involve multiple steps of strain selection, fermentation, transformation, purification, etc., and have the disadvantages of low substrate concentration, strong substrate inhibition effect with high concentration, long transformation time, lower transformation rate, difficult separation and purification, etc.
4. The preparation method for directly producing the prednisolone by taking hydrocortisone acetate as a starting material and adopting simple arthrobacterium through biological fermentation in one step comprises the following synthetic route:
CN106893753a discloses a preparation method for directly producing prednisolone from hydrocortisone acetate serving as a starting material through one-step biological fermentation by using simple arthrobacter, wherein the prednisolone has a structure that a plurality of groups including 1-position double bond, 4-position double bond, 11-position hydroxyl group, 21-position hydroxyl group and 17-position hydroxyl group are arranged on a steroidal mother nucleus, and compared with the structure of hydrocortisone acetate, the hydrocortisone acetate can be found to react through two units of 1, 2-position dehydrogenation and 21-position hydrolysis to form 1, 2-position double bond and 21-position hydroxyl group. Although the patent takes the cheaper hydrocortisone acetate as the starting material to generate the prednisolone in one step, the conversion time is long and is generally longer than 60 hours, the conversion rate is about 80 percent, the substrate concentration of the converted hydrocortisone acetate is not very high, and the highest substrate concentration is only 3 percent.
The invention comprises the following steps:
the invention aims to solve the technical problems of complex preparation process, long time consumption and low conversion rate of the existing preparation method of the prednisolone, and further provides a preparation method of the prednisolone, which is low in cost, easy to prepare and high in conversion rate. In order to overcome the defects of the prior art, the invention provides a one-step preparation method of prednisolone by taking hydrocortisone acetate or hydrocortisone as a raw material and using Arthrobacter urate as a biocatalyst, and the method has the advantages of high yield, good selectivity, simple process, economy, practicability and safety to environment and operators.
In order to achieve the above purpose, one of the technical schemes provided by the invention is a strain of Arthrobacter urate, which is a strain obtained by ARTP mutagenesis and breeding and can improve the conversion rate of hydrocortisone acetate and hydrocortisone at high feeding concentration.
The Arthrobacter urate strain, in particular Arthrobacter urate (Arthrobacter uratoxydans) AAR-47, has been deposited in China general microbiological culture Collection center (address: national institute of microbiology, national academy of sciences, 3, university of Chinese, p.m. 100101, of the North Xiya, 1, G.Chen) of the Beijing city at day 25 of 2021, deposit number: CGMCC No.23650.
The Arthrobacter urate AAR-47 has the following microbiological characteristics:
morphological characteristics are:
(1) The cell morphology was short-rod and gram-positive (as shown in FIG. 3).
(2) The colonies were smooth and full, the thalli were sticky, the edges were clean, opaque, and the colonies appeared beige (as shown in fig. 2).
The physiological and biochemical characteristics are as follows:
(1) Culturing for 1-2 days; aerobic; the optimal growth pH value is 6.8-7.0; the optimal growth temperature is 30 ℃; (2) conversion of the major product: prednisolone.
The second technical scheme provided by the invention is the application of the Arthrobacter urate AAR-47, in particular the application in the production of prednisolone;
further, the method for producing the prednisolone by fermenting the Arthrobacter urate AAR-47 comprises the following steps:
hydrocortisone acetate is used as a substrate: inoculating the strain into fermentation medium containing hydrocortisone acetate with final concentration of 10-60g/L according to inoculum size of 3-20% after seed culture, and fermenting at 28-34 deg.C and 160-180r/min for 30-96 hr; the substrate conversion rate can reach 90.81-100%;
hydrocortisone is used as a substrate: after the strain is cultured by seeds, inoculating fermentation medium containing hydrocortisone with a final concentration of 5-40g/L according to the inoculum size of 3% -20%, fermenting for 60-96h at 28-34 ℃ under 160-180r/min, and the substrate conversion rate can reach 90.56-94.58% after fermentation.
Preferably, the fermentation medium is as follows: 10-15g/L of glucose, 12-20g/L of corn steep liquor, 4-10g/L of peptone, 1-4g/L of yeast extract, 2.5-5g/L of monopotassium phosphate, the balance of water, 10-80g/L of HP-beta-CD and pH of 6.5-7.5; sterilizing at 121deg.C under 0.1-0.15Mpa for 20min.
The beneficial effects are that:
the Arthrobacter urate AAR-47 provided by the invention is obtained through physical mutagenesis, the strain after mutagenesis uses hydrocortisone acetate as a substrate, and the substrate conversion rate can reach 90.81-100% after fermentation for 30-96h under the condition of adding 10-60g/L of concentration. The strain after mutagenesis takes hydrocortisone as a substrate, and the fermentation conversion is carried out for 60-96 hours under the concentration of 5-40g/L, and the substrate conversion rate can reach 90.56-94.58%. The transformation rate of the mutagenesis strain for transforming 30g/L hydrocortisone acetate can reach 96.19% in 72 hours, and the transformation rate of the original strain is only 84.77%. Effectively solves the problem of inhibiting the dehydrogenation conversion of C1 and 2 by high feeding concentration in the prior art.
Description of the drawings:
FIG. 1 is a graph showing the lethality of ARTP mutagenesis;
FIG. 2 shows colony morphology of mutagens on a plate;
FIG. 3 is a photograph of a mutagenized strain.
The specific embodiment is as follows:
in order to make the objects, technical solutions and advantages of the present patent more apparent, the present patent will be described in further detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the present invention.
Example 1: mutagenesis screening of Arthrobacter urate AAR-47
ARTP mutagenesis of Arthrobacter urate:
(1) The initial strain is a strain of Arthrobacter urate deposited in applicant laboratory, and the initial strain Arthrobacter urate is scraped into a loop and inoculated into 50mL of seed culture medium, the temperature is 30 ℃ and the culture is carried out at 160r/min until the logarithmic phase (18-20 h).
The seed medium composition was as follows: 15g/L glucose, 20g/L corn steep liquor, 10g/L peptone, 6g/L potassium dihydrogen phosphate and the balance of water, wherein the pH value is 6.8-7.0.
(2) ARTP mutagenesis
And (3) carrying out mutagenesis on the seed solution of the Arthrobacter urate by utilizing ARTP, centrifugally collecting the bacterial solution cultured to the logarithmic phase, washing 2-3 times by using sterile physiological saline, and diluting with a proper amount of sterile physiological saline to prepare the OD600 value of 0.6-0.8 or the concentration of bacteria is 10 6 Bacterial suspension of about one/mL. mu.L of the bacterial suspension was dropped onto a slide glass. After drawing a mortality curve (shown in figure 1), selecting 20, 30 and 40 as mutagenesis time, taking 100 mu L of mutagenized bacterial suspension to be coated on a flat plate, picking single bacteria for culturing, reversely culturing for 2d, screening by a method of developing colors of 9 alpha-OH-AD and 2, 4-dinitrophenylhydrazine, and breeding a production strain capable of efficiently converting hydrocortisone acetate.
Preliminary screening of mutant strains of Arthrobacter urate:
selecting 2018 single colonies growing in an agar culture medium after mutagenesis, taking out an initial strain as a control, performing primary screening on a 24-micropore culture medium based on a 2, 4-dinitrophenylhydrazine color development technology, breeding 150 strains capable of efficiently converting hydrocortisone acetate, performing 24-micropore plate secondary screening on the 150 strains obtained by the primary screening to obtain 17 mutagenized strains capable of efficiently converting hydrocortisone acetate, performing shake flask fermentation secondary screening on the mutagenized strains obtained by the 17 secondary screening, and screening to obtain the high-yield prednisolone uric acid Arthrobacter AAR-47 by taking the content of prednisolone as an index at the end point of the conversion process. The results of culturing and microscopic examination of Arthrobacter urate AAR-47 on a plate (1.5% agar added to seed medium) are shown in FIGS. 2 and 3.
Example 2: production of prednisolone by Arthrobacter urate oxidase
Experimental strains: AAR-47 and starting strain;
(1) Seed culture
Taking one fresh AAR-47 strain and one inclined surface of a starting strain which are cultured for 1-2 days respectively, taking a full loop by an inoculating loop respectively, inoculating the full loop into a 250mL triangular flask filled with 30mL of seed culture medium, and culturing at 30 ℃ for 160r/min until the logarithmic phase (18-20 h).
The seed culture medium comprises the following components: 15g/L glucose, 20g/L corn steep liquor, 10g/L peptone, 6g/L potassium dihydrogen phosphate and the balance of water, wherein the pH value is 6.8-7.0.
(2) Fermentation culture
Inoculating 5% seed solution into fermentation medium, adding hydrocortisone acetate with final concentration of 10g/L, and fermenting at 30deg.C and 160r/min for 30 hr.
The method adopts a fermentation culture medium with the following proportion: 12g/L glucose, 15g/L corn steep liquor, 6g/L peptone, 1.5g/L yeast extract, 3.5g/L potassium dihydrogen phosphate and the balance of water, wherein the pH is 6.8-7.0, the HP-beta-CD is 10g/L, and the mixture is sterilized for 20min at 121 ℃ under the pressure of 0.1-0.15 Mpa.
(3) In the conversion process, 300 mu L of fermentation liquor is taken, diploid volume of ethyl acetate is added, ultrasonic treatment is carried out for 30min, centrifugation is carried out for 10min at 12000r/min, and HPLC detection is carried out on the content of prednisolone from the supernatant.
HPLC determination is adopted, the separation column is a silica gel column, the detection wavelength is 240nm, and the mobile phase is dichloromethane: diethyl ether: methanol=82: 12:6 was 1.0mL/min. Substrate conversion was calculated using the area normalization method (substrate conversion = substrate molar mass converted to product/initial substrate molar mass x 100%).
The conversion rate of the mutant strain AAR-47 conversion substrate can reach 100%, the time is 30 hours, and the starting strain is 36 hours after the conversion of the substrate with the same concentration.
Example 3: production of prednisolone by Arthrobacter urate oxidase
Experimental strains: AAR-47 and starting strain;
(1) Seed culture
Taking one fresh AAR-47 strain and one inclined surface of a starting strain which are cultured for 1-2 days respectively, taking a full loop by an inoculating loop respectively, inoculating the full loop into a 250mL triangular flask filled with 30mL of seed culture medium, and culturing at 30 ℃ for 160r/min until the logarithmic phase (18-20 h).
The seed culture medium comprises the following components: 15g/L glucose, 20g/L corn steep liquor, 10g/L peptone, 6g/L potassium dihydrogen phosphate and the balance of water, wherein the pH value is 6.8-7.0.
(2) Fermentation culture
Inoculating 8% seed solution into fermentation medium, adding hydrocortisone acetate with final concentration of 30g/L, and fermenting at 30deg.C and 160r/min for 96 hr.
The method adopts a fermentation culture medium with the following proportion: 12g/L glucose, 15g/L corn steep liquor, 6g/L peptone, 2g/L yeast extract, 3.5g/L potassium dihydrogen phosphate and the balance of water, wherein the pH is 6.8-7.0, the HP-beta-CD is 50g/L, and the mixture is sterilized for 20min at 121 ℃ under the pressure of 0.1-0.15 Mpa.
The conversion rate of the mutant strain AAR-47 conversion substrate can reach 96.19%, the time is 72 hours, and the conversion rate of the original strain conversion 72 hours is 84.7%.
EXAMPLE 4 AAR-47 fermentative production of prednisolone
(1) The production strain is AAR-47, and the seed culture is the same as in example 2;
(2) Fermentation culture: inoculating the seed solution into a fermentation culture medium according to 15% of inoculum size, adding hydrocortisone acetate with substrate concentration of 60g/L into the fermentation culture medium, fermenting at 32 ℃ under 180r/min for 96 hours, and enabling the substrate conversion rate to reach 90.81% after fermentation.
The method adopts a fermentation culture medium with the following proportion: 15g/L glucose, 20g/L corn steep liquor, 10g/L peptone, 4g/L yeast extract, 5g/L potassium dihydrogen phosphate and the balance of water, wherein the pH is 6.8-7.0, the HP-beta-CD is 50g/L, and the sterilization is carried out for 20min at 121 ℃ under the pressure of 0.1-0.15 Mpa.
EXAMPLE 5 AAR-47 fermentation of hydrocortisone to prednisolone
(1) The production strain is AAR-47, and the seed culture is the same as in example 2;
(2) Fermentation culture: inoculating the seed solution into a fermentation medium according to the inoculum size of 5%, adding hydrocortisone with substrate concentration of 10g/L into the fermentation medium, fermenting at 30 ℃ under 160r/min for 60h, and the conversion rate can reach 94.58% after the fermentation is finished.
The method adopts a fermentation culture medium with the following proportion: 15g/L glucose, 20g/L corn steep liquor, 10g/L peptone, 2g/L yeast extract, 5g/L potassium dihydrogen phosphate, the balance of water, 15g/L HP-beta-CD, pH6.8-7.0, and sterilizing at 121 ℃ for 20min under 0.1-0.15 Mpa.
EXAMPLE 6 AAR-47 fermentation of hydrocortisone to prednisolone
(1) The production strain is AAR-47, and the seed culture is the same as in example 2;
(2) Fermentation culture: inoculating the seed solution into a fermentation culture medium according to the inoculation amount of 8%, adding hydrocortisone with the substrate concentration of 15g/L into the fermentation culture medium, fermenting for 72h at 30 ℃ under 180r/min, and the conversion rate can reach 93.91% after the fermentation is finished.
The method adopts a fermentation culture medium with the following proportion: 12g/L glucose, 15g/L corn steep liquor, 6g/L peptone, 3g/L yeast extract, 3.5g/L potassium dihydrogen phosphate and the balance of water, wherein the pH is 6.8-7.0, the HP-beta-CD is 35g/L, and the mixture is sterilized for 20min at 121 ℃ under the pressure of 0.1-0.15 Mpa.
EXAMPLE 7 AAR-47 fermentation of hydrocortisone to prednisolone
(1) The production strain is AAR-47, and the seed culture is the same as in example 2;
(2) Fermentation culture: inoculating the seed solution into a fermentation culture medium according to 10% of inoculum size, adding hydrocortisone with substrate concentration of 30g/L into the fermentation culture medium, fermenting at 32 ℃ under 180r/min for 84 hours, and after fermentation, the conversion rate can reach 91.89%.
The method adopts a fermentation culture medium with the following proportion: 12g/L glucose, 15g/L corn steep liquor, 6g/L peptone, 3.5g/L yeast extract, 3.5g/L potassium dihydrogen phosphate, and the balance water, wherein the pH is 6.8-7.0, the HP-beta-CD is 45g/L, and the sterilization is carried out for 20min at 120-122 ℃ under the pressure of 0.1-0.15 Mpa.
The substrate conversion effect of the strain AAR-47 on the hydrocortisone acetate is superior to that of the hydrocortisone acetate, the feeding concentration of the hydrocortisone acetate serving as a substrate is higher than that of the hydrocortisone acetate, the time of the hydrocortisone acetate is short when the hydrocortisone acetate is converted by adopting the substrate with the same concentration, and the final conversion rate is higher than that of the hydrocortisone acetate. The main reason is that the substrate inhibition effect of hydrocortisone exists, and the hydrocortisone acetate needs to be hydrolyzed into hydrocortisone before dehydrogenation reaction during conversion, so that the substrate inhibition effect is relieved. Meanwhile, most strains have difficulty in simultaneously transforming hydrocortisone acetate and hydrocortisone with high efficiency due to the substrate inhibition.
The above embodiments represent only a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the patent. It should be noted that, for a person skilled in the art, the above embodiments may also make several variations, combinations and improvements, without departing from the scope of the present patent. Therefore, the protection scope of the patent is subject to the claims.
Claims (4)
1. A strain of Arthrobacter urate, characterized in that it is specifically Arthrobacter urate (Arthrobacter uratoxydans) AAR-47, accession No.: CGMCC No.23650.
2. Use of Arthrobacter urate (Arthrobacter uratoxydans) AAR-47 according to claim 1 for the production of prednisolone.
3. The use according to claim 2, wherein the method for producing prednisolone by fermentation with the use of the bacteria Arthrobacter urate AAR-47 is as follows:
after the strain is cultured by seeds, inoculating fermentation medium containing hydrocortisone acetate with the final concentration of 10-60g/L according to the inoculum size of 3-20%, and fermenting for 30-96h under the conditions of 28-34 ℃ and 160-180 r/min.
4. The use according to claim 2, wherein the method for producing prednisolone by fermentation with the use of the bacteria Arthrobacter urate AAR-47 is as follows: after the strain is cultured by seeds, inoculating fermentation medium containing hydrocortisone with a final concentration of 5-40g/L according to the inoculum size of 3% -20%, and fermenting for 60-96h under the conditions of 28-34 ℃ and 160-180 r/min.
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