CN118146999A - Autotrophic Pseudonocardia and its application - Google Patents
Autotrophic Pseudonocardia and its application Download PDFInfo
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- CN118146999A CN118146999A CN202410319196.4A CN202410319196A CN118146999A CN 118146999 A CN118146999 A CN 118146999A CN 202410319196 A CN202410319196 A CN 202410319196A CN 118146999 A CN118146999 A CN 118146999A
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- Prior art keywords
- dehydrocholesterol
- hydroxy
- culture
- fermentation
- autotrophic
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the field of biocatalysis and organic synthesis, in particular to autotrophic Pseudonocardia and application thereof. The invention provides an autotrophic Pseudonocardia Pseudonocardia autotrophica capable of tolerating high-concentration 7-dehydrocholesterol, and the preservation number is CGMCC No.26831. The preparation method for the chemical synthesis of the 25-hydroxy vitamin D3 is improved in that a novel bioconversion method is adopted to prepare the key intermediate product of the 25-hydroxy-7-dehydrocholesterol, the feeding amount of the 7-dehydrocholesterol is obviously improved, and the conversion rate is improved to about 85 percent; the 25-hydroxy-7-dehydrocholesterol has simple purification, less crystal impurities and high purity. The process is simple and feasible, mild in condition, safe and controllable, and good in process stability.
Description
Technical Field
The invention relates to the field of biocatalysis and organic synthesis, in particular to autotrophic Pseudonocardia and application thereof.
Background
Osteoporosis (Osteoporosis, OP) is a systemic bone disease that is susceptible to fracture due to a decrease in bone density and bone mass, destruction of bone microstructure, and increased bone fragility, due to a variety of causes, and is increasingly a chronic disease that severely threatens the health of the elderly. The 25-hydroxy vitamin D3 is an active metabolite of the vitamin D3, has activity far higher than that of the vitamin D3, is mainly used for treating various chronic bone disorders such as senile osteoporosis and the like by regulating calcium and phosphorus metabolism and immune function in vivo, is widely used in the field of animal nutrition in recent years, and has wide market prospect as a feed additive for promoting bone development of livestock and poultry animals.
At present, a chemical synthesis technical route is mainly adopted in the industrial production of 25-hydroxy vitamin D3, wherein the key intermediate 25-hydroxy-7-dehydrocholesterol is obtained through chemical synthesis, 25-hydroxy vitamin D3 is further obtained through photochemical reaction, 25-hydroxy-7-ketocholesterol derivatives are used as raw materials in the patent CN104910231, 25-hydroxy-7-dehydrocholesterol is prepared through chemical synthesis, 5,7, 24-triene cholesterol is used as raw materials in the patent CN106831921, 25-hydroxy-7-dehydrocholesterol is prepared through chemical synthesis, the two chemical synthesis reaction routes have the defects of difficult obtainment of reaction raw materials, more reaction steps, lower reaction selectivity and the like, and simultaneously, more organic solvents are used, so that the environment cannot be polluted greatly. Liu Gongjuan et al explored a bioconversion method for preparing 25-hydroxy-7-dehydrocholesterol (journal Hua Xiyao, 2014,29 (5): 507-509), which uses readily available 7-dehydrocholesterol as a raw material, and utilizes a microbial enzyme one-step reaction to obtain 25-hydroxy-7-dehydrocholesterol, which has good selectivity, mild conditions, low cost and environmental friendliness, and the bioconversion method has the potential to replace chemical synthesis methods, but has the disadvantages of lower conversion rate, only 25%, low dosage and final concentration of only 0.6mg/mL in a catalytic system.
Therefore, it is of great importance to provide an industrial scale production process capable of significantly increasing the conversion of 25-hydroxy-7-dehydrocholesterol and further obtaining 25-hydroxyvitamin D3 by photochemical reaction.
Disclosure of Invention
In view of this, the present invention provides autotrophic Pseudonocardia and uses thereof.
The invention provides autotrophic Pseudonocardia and application thereof. The invention provides an autotrophic Pseudonocardia Pseudonocardia autotrophica capable of tolerating high-concentration 7-dehydrocholesterol, and the preservation number is CGMCC No.26831. The improvement of the preparation method of the 25-hydroxy vitamin D3 provided by the invention is that the key intermediate product of the 25-hydroxy-7-dehydrocholesterol is prepared by adopting a novel bioconversion method, the feeding amount of the 7-dehydrocholesterol is obviously improved, and the conversion rate is improved to about 85 percent; the 25-hydroxy-7-dehydrocholesterol has simple purification, less crystal impurities and high purity. The process is simple and feasible, mild in condition, safe and controllable, and good in process stability.
In order to achieve the above object, the present invention provides the following technical solutions:
The invention provides autotrophic Pseudonocardia (Pseudonocardia autotrophica) with a preservation number of CGMCC No.26831.
The invention also provides a microbial preparation comprising the autotrophic nocardia (Pseudonocardia autotrophica) and acceptable auxiliary materials or auxiliary agents.
On the basis of the above study, the present invention also provides a composition comprising any of the following:
(1) -said autotrophic nocardia (Pseudonocardia autotrophica) and polyethylene glycol 600; and/or
(2) The microbial formulation and polyethylene glycol 600.
The invention also provides the use of said autotrophic nocardia (Pseudonocardia autotrophica), said microbial preparation or said composition in any of the following:
(1) Improving the conversion rate of 25-hydroxy-7-dehydrocholesterol; and/or
(2) Preparing 25-hydroxy-7-dehydrocholesterol; and/or
(3) Preparing 25-hydroxy vitamin D3;
(4) 7-dehydrocholesterol resistant.
In some embodiments of the invention, the 7-dehydrocholesterol resistant comprises 7-dehydrocholesterol resistant to 1-5 mg/mL.
The invention also provides a process for the preparation of 25-hydroxy-7-dehydrocholesterol, based on 7-dehydrocholesterol and any of the following:
(1) -said autotrophic nocardia (Pseudonocardia autotrophica); and/or
(2) The microbial preparation; and/or
(3) The composition.
In some embodiments of the present invention, the method for preparing 25-hydroxy-7-dehydrocholesterol specifically comprises the steps of:
Step 1: culturing the autotrophic Pseudonocardia (Pseudonocardia autotrophica), the microbial agent, the autotrophic Pseudonocardia (Pseudonocardia autotrophica) in the composition or the microbial agent in the composition to obtain a fermentation broth;
Step 2: mixing the fermentation liquor with polyethylene glycol 600 to obtain fermentation liquor containing polyethylene glycol 600;
Step 3: mixing the fermentation liquor containing polyethylene glycol 600 with 7-dehydrocholesterol substrate liquor, and converting to obtain bioconversion liquor;
Step 4: and extracting, concentrating, performing column chromatography and crystallizing the bioconversion liquid to obtain the 25-hydroxy-7-dehydrocholesterol.
In some embodiments of the invention, the culturing in step 1 comprises one or more of slant culture, seed culture, or fermentation culture;
The seed culture comprises the step of inoculating spore suspension obtained by the slant culture into a seed culture medium;
the fermentation culture comprises the step of inoculating seed liquid obtained by seed culture to a fermentation culture medium;
the inoculation amount of the seed liquid is 10-30%.
In some embodiments of the invention, the number of spores in the spore suspension is 1X 10 8 spores/mL.
In some embodiments of the invention, the seed solution is inoculated at 20%.
In some embodiments of the invention, the conditions of the slant culture are: culturing for 5-10 d at 20-35 ℃;
The culture medium used for slant culture comprises the following components in percentage by weight and volume: glucose 1-10%, peptone 1-10%, corn steep liquor 0.1-2%, sodium chloride 0.1-1% and/or agar 1-2.5%, pH 6.5-7.5;
The seed culture conditions are as follows: the rotation speed is 100-300 r/min, the temperature is 20-35 ℃, and the culture is carried out for 2-5 d;
The culture medium for seed culture comprises the following components in percentage by weight and volume: peptone 0.2-2%, corn steep liquor 0.2-2%, glucose 0.5-5%, soybean meal 0.1-1.5%, sodium chloride 0.1-1.5% and/or potassium dihydrogen phosphate 0.1-1.5%, pH 7.0-8.0;
The conditions of the fermentation culture are as follows: the rotation speed is 100-300 r/min, the temperature is 20-35 ℃, and the culture is carried out for 2-5 d;
The culture medium used for fermentation culture comprises, by weight and volume percentage, 0.2-2% of peptone, 0.2-2% of corn steep liquor, 0.5-5% of glucose, 0.1-1.5% of soybean meal, 0.1-1.5% of sodium chloride, 0.1-1.5% of potassium dihydrogen phosphate, 0.2-1.0% of glutathione and/or cyclodextrin;
The mass of the cyclodextrin is 4-10 times of the mass of the 7-dehydrocholesterol in the 7-dehydrocholesterol substrate liquid in the step 3.
In some embodiments of the invention, the conditions of the slant culture are: culturing at 28 ℃ for 7d;
the culture medium used for slant culture comprises the following components in percentage by weight and volume: glucose 1.5%, peptone 1.0%, corn steep liquor 0.3%, sodium chloride 0.4% and/or agar 1.5%, pH7.0.
In some embodiments of the invention, the seed culture conditions are: culturing for 3d at 28 ℃ at 220 r/min;
The culture medium for seed culture comprises the following components in percentage by weight and volume: 1.5% of peptone, 1.5% of corn steep liquor, 1.5% of glucose, 0.4% of soybean meal, 0.5% of sodium chloride and/or 0.2% of potassium dihydrogen phosphate.
In some embodiments of the invention, the conditions of the fermentation culture are: culturing for 3d at 28 ℃ at 220 r/min;
The culture medium used for fermentation culture comprises the following components in percentage by weight and volume: 1.5% of peptone, 1.5% of corn steep liquor, 1.5% of glucose, 0.4% of soybean meal, 0.5% of sodium chloride, 0.2% of monopotassium phosphate, and 0.2-1.0% of glutathione and/or cyclodextrin.
In some embodiments of the invention, the cyclodextrin is 6 times the mass of 7-dehydrocholesterol in the 7-dehydrocholesterol substrate solution of step 3.
In some embodiments of the invention, the cyclodextrin comprises one or more of α -cyclodextrin, β -cyclodextrin, γ -cyclodextrin, carboxymethyl β -cyclodextrin, hydroxyethyl β -cyclodextrin, hydroxypropyl β -cyclodextrin, methyl β -cyclodextrin, sulfobutyl β -cyclodextrin.
In some embodiments of the invention, the cyclodextrin is sulfobutylβ cyclodextrin.
In some embodiments of the present invention, the concentration of polyethylene glycol 600 in the fermentation broth containing polyethylene glycol 600 in step 2 is 0.2-1.0%;
The final concentration of 7-dehydrocholesterol in the 7-dehydrocholesterol substrate solution in the step 3 is 1-5 mg/mL;
the conversion time in the step 3 is 1-5 d.
In some embodiments of the invention, the concentration of polyethylene glycol 600 in the fermentation broth comprising polyethylene glycol 600 in step 2 is 0.5%.
In some embodiments of the invention, the final concentration of 7-dehydrocholesterol in the 7-dehydrocholesterol substrate solution in step 3 is 1mg/mL or 3mg/mL.
In some embodiments of the invention, the conversion time is 4d.
The 7-dehydrocholesterol substrate solution in step 3 comprises 7-dehydrocholesterol, tween and isopropanol.
In some embodiments of the invention, the solvent extracted in step 4 is ethyl acetate.
In some embodiments of the invention, the crystallization is performed using an ethanol-water system.
In some embodiments of the invention, the column used for column chromatography is a silica gel column.
The invention also provides a method for preparing 25-hydroxy vitamin D3, which is based on 7-dehydrocholesterol and any of the following to prepare 25-hydroxy vitamin D3:
(1) -said autotrophic nocardia (Pseudonocardia autotrophica); and/or
(2) The microbial preparation; and/or
(3) The composition.
In some embodiments of the present invention, the method for preparing 25-hydroxyvitamin D3 comprises the steps of:
Step A: preparing 25-hydroxy-7-dehydrocholesterol based on 7-dehydrocholesterol and any of:
(1) -said autotrophic nocardia (Pseudonocardia autotrophica); and/or
(2) The microbial preparation; and/or
(3) The composition;
and (B) step (B): taking the 25-hydroxy-7-dehydrocholesterol, and carrying out illumination ring opening and thermal isomerization to obtain the 25-hydroxy vitamin D3.
The invention provides autotrophic Pseudonocardia and application thereof. The invention provides an autotrophic Pseudonocardia Pseudonocardia autotrophica capable of tolerating high-concentration 7-dehydrocholesterol, and the preservation number is CGMCC No.26831. The industrial scale production method for obtaining the 25-hydroxy vitamin D3 by photochemical reaction has important significance, and can obviously improve the conversion rate of the 25-hydroxy-7-dehydrocholesterol.
Description of biological preservation
Biological material: UV-288, class designation: the autotrophic Pseudonocardia (Pseudonocardia autotrophica) is preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) at 3 and 17 days of 2023, and has an address of North Star, west Lu No. 1, 3 of the Korean area of Beijing, and a preservation number of CGMCC No.26831.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the mycelium status of the mutagenized strains Pseudonocardia autotrophica UV-288 of the present invention when grown in a fermentation medium containing 1mg/mL 7-dehydrocholesterol;
FIG. 2 shows the mycelium status of the control strain Pseudonocardia autotrophica CGMCC No.7935 according to the present invention when grown in a fermentation medium containing 1mg/mL of 7-dehydrocholesterol.
Detailed Description
The invention discloses autotrophic Pseudonocardia and application thereof, and a person skilled in the art can properly improve the process parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.
The invention aims to solve the problems of low conversion rate and difficult industrialization existing in the biological conversion process of preparing 25-hydroxy-7-dehydrocholesterol, thereby providing an industrial scale production method capable of remarkably improving the conversion rate of 25-hydroxy-7-dehydrocholesterol and further obtaining 25-hydroxy vitamin D3 through photochemical reaction.
In order to screen and obtain strains capable of converting 7-dehydrocholesterol into 25-hydroxy-7-dehydrocholesterol, a total of 1200 strains are subjected to shake flask experiment screening, 7-dehydrocholesterol is taken as a substrate, after shake flask bioconversion is finished, whether 25-hydroxy-7-dehydrocholesterol exists in a conversion solution is detected by HPLC, and as a result, 9 strains have the capability of converting 7-dehydrocholesterol into 25-hydroxy-7-dehydrocholesterol, wherein the strains comprise 4 strains of mould and 5 strains of actinomycetes; referring to the journal of western medicine, 2014,29 (5): the biological conversion rate of the 9 strains is measured by the method described in 507-509, the conversion rate is found to be in the range of 11% -45%, four strains, two strains of mould and two strains of actinomycetes are obtained, the strain with the best conversion result is autotrophic Nocardia fungus Pseudonocardia autotrophica CGMCC No.7935 (the preservation number in China general microbiological culture Collection center is CGMCC No. 7935) in the two strains of actinomycetes, the conversion rate of Pseudonocardia autotrophica CGMCC No.7935 is found to be 45%, and the invention finds that Pseudonocardia autotrophica CGMCC No.7935 has better capability of converting 7-dehydrocholesterol into 25-hydroxy-7-dehydrocholesterol through a large amount of basic screening work.
To be suitable for commercial production, it is desirable to further increase the ability of Pseudonocardia autotrophica CGMCC No.7935 to convert 7-dehydrocholesterol to 25-hydroxy-7-dehydrocholesterol. In general, after screening for the transformed strain, optimization of the transformation conditions is critical for improving the transformation rate, and in the transformation system, the optimization of the transformation conditions must take into consideration the characteristics of the transformed substrate, such as the stability of the substrate to oxygen, light and heat, the solubility of the substrate, the toxicity of the substrate to the transformed strain, and the like.
The conversion conditions are correspondingly adjusted according to the characteristics of the substrates to obtain the optimal conversion process, different conversion substrates are converted, the structures of the compounds are different, the solubility of the substrates in a conversion system is different due to the difference, so that the number of substrate molecules contacted by the strain is different, the conversion efficiency is obviously different, an experimenter needs to adopt different solubilization modes, the different solubilization modes have influence on the strain and are unpredictable factors, and a great amount of creative research work is needed to determine that the solubility of the substrates is improved on the premise of not influencing the performance of the strain, so that the conversion performance is improved; the polarity of the compound is different due to different conversion substrates, and the substrate has different transmembrane permeabilities when the substrate is contacted with bacterial strain cells, so that the conversion efficiency is affected; in addition, toxicity of different compounds to the bacterial cells is also different, which is shown by that the tolerance concentration of the bacterial cells to different substrates is obviously different, the feeding concentration of the substrates needs to be adjusted, otherwise, the transformation process cannot be carried out due to intolerance of the bacterial strains, the valuable bioconversion process usually needs high feeding concentration of the substrates, so that the improvement of the tolerance of the transformed bacterial strains to the substrates is very important, but for different substrates, different transformed bacterial strains often need to develop personalized or specific process parameters, and in addition, when the specific condition optimization to the substrates cannot be carried out or the optimization effect is not good, research and development personnel often need to modify the transformed bacterial strains or select the creative work, so that the tolerance of the bacterial strains to the substrates can be improved. As previously mentioned, the optimization of transformation conditions takes into account the structural and chemical specificity of the substrate, optimizes the performance of the transformed strain, and adjusts and optimizes the transformation conditions so that a valuable bioconversion process is possible. In the process of converting 7-dehydrocholesterol into 25-hydroxy-7-dehydrocholesterol using Pseudonocardia autotrophica CGMCC No.7935, the conversion process design should be performed with respect to the characteristics of 7-dehydrocholesterol, such as solubility in bioconversion systems and the degree of damage of 7-dehydrocholesterol to mycelium.
According to the invention, after intensive researches, a large number of comparison experiments show that when the final concentration of 7-dehydrocholesterol in a conversion system is lower than 0.8mg/mL, the mycelium of Pseudonocardia autotrophica CGMCC No.7935 is less influenced, the concentration change of mycelium is small, the length of mycelium is basically unchanged, but when the final concentration of 7-dehydrocholesterol in the conversion system is higher than 0.8mg/mL, the influence of mycelium is gradually increased along with the increase of the final concentration, the mycelium becomes shorter during microscopic examination, the edge is slightly blurred, and the like, meanwhile, the concentration of mycelium is gradually reduced, the appearance is shown to be diluted by fermentation broth, the conversion process is correspondingly obviously slowed down, the consumption of substrate 7-dehydrocholesterol is reduced, the increase of product 25-hydroxy-7-dehydrocholesterol is also reduced, and the conversion rate is low. However, in the previous research work, the applicant found that when the substrate is vitamin D3 and the final concentration reaches 5mg/mL, pseudonocardia autotrophica CGMCC No.7935 mycelium is not affected, the conversion process can still be smoothly carried out, which indicates that the effect of different substrates on Pseudonocardia autotrophica CGMCC No.7935 is different, and when the substrate is 7-dehydrocholesterol, the final concentration is preferably controlled within 0.8mg/mL, so that the mycelium can be ensured to be affected little, the conversion process can be carried out, but the feeding amount of the substrate 7-dehydrocholesterol is low, so that the bioconversion cost is high, and industrialization is difficult to realize;
A great deal of mutation breeding work is carried out on Pseudonocardia autotrophica CGMCC No.7935, the breeding work needs to be specially designed aiming at the growth characteristics of Pseudonocardia autotrophica CGMCC No.7935 and the structural characteristics of the substrate 7-dehydrocholesterol, but even so, whether the strain which can tolerate high-concentration 7-dehydrocholesterol and has better conversion rate can be bred is uncertain.
Performing ultraviolet mutagenesis on Pseudonocardia autotrophica CGMCC No.7935 monospore suspension, preparing a mature inclined plane of an original strain into the monospore suspension by using normal saline, oscillating, dispersing glass beads, collecting the monospore suspension by sterile filtration, and irradiating ultraviolet light in a sterile culture dish for 30s with the power of an ultraviolet lamp of 30W and the lamp distance of 25-30cm;
The isolation medium 2L was prepared according to the following formulation: 15g/L of glucose, 10g/L of peptone, 3g/L of corn steep liquor, 4g/L of sodium chloride, 15g/L of agar and 1g/L of 7-dehydrocholesterol, and pH7.0. 100 sterile culture dishes were prepared, and after sterilization of the medium, the culture dishes were poured into a plate for separation. 1mL of the monospore suspension after mutagenesis treatment was diluted and spread on a plate, and cultured in an incubator at 28℃for 10d. Strains with good growth and spore production were selected from plates containing high concentrations of 7-dehydrocholesterol (1 g/L) and inoculated onto slant medium of the same formulation as the isolation medium.
Preparing a seed culture medium and a fermentation culture medium for screening, and preparing 7-dehydrocholesterol solution at the same time: 1g of 7-dehydrocholesterol, 2mL of Tween 80 were dissolved in isopropanol in a total volume of 40mL; preparing a 7-dehydrocholesterol-free control solution: 2mL of Tween 80 was dissolved in isopropanol, and the total volume was 40mL; after the strain obtained by ultraviolet mutagenesis is cultivated and matured, the spore suspension with the spore number of about 1X 10 8/mL is prepared on a sterile water inclined plane, 5mL is added into 25mL of seed culture medium (the formula is the same as in example 1), meanwhile, the inclined plane of the original strain Pseudonocardia autotrophica CGMCC No.7935 used for mutagenesis treatment is also treated and is added into the seed culture medium to serve as a control, the rotating speed of a shaking table is 280r/min, after the strain is cultivated for 72h at 28 ℃, the strain is added into a fermentation culture medium (the formula is the same as in example 1) according to the inoculation amount of 10%, after the strain is cultivated for 72h at 28 ℃, the mutagenized strain and the control strain are respectively divided into two groups, one group is supplemented with 7-dehydrocholesterol liquid so that the final concentration of 7-dehydrocholesterol in the fermentation liquid is 1mg/mL, and the other group is supplemented with the same amount of 7-dehydrocholesterol-free control liquid, and the influence of tween 80 and isopropanol on the strain growth is examined. Culturing for 96 hr, stopping fermentation, performing microscopic examination on mycelium, and measuring 7-dehydrocholesterol conversion rate, and selecting strain with tolerance of 7-dehydrocholesterol concentration above 0.8mg/mL and conversion rate not lower than control strain Pseudonocardia autotrophica CGMCC No. 7935.
Through a large number of strain mutagenesis and screening works, the invention finally breeds the autotrophic Pseudonocardia Pseudonocardia autotrophica UV-288 which can tolerate 1mg/mL 7-dehydrocholesterol, the strain grows well in a fermentation medium containing 1mg/mL 7-dehydrocholesterol, mycelium microscopic examination is normal (see figure 1), the conversion rate can be kept at about 45%, the growth of a control strain is abnormal, the mycelium microscopic examination is obviously shortened, and the edge is slightly blurred (see figure 2); the invention discovers that the selected autotrophic Pseudonocardia Pseudonocardia autotrophica UV-288 not only can tolerate 1mg/mL of 7-dehydrocholesterol, but also can tolerate 2-5 mg/mL of 7-dehydrocholesterol, but when the concentration of 7-dehydrocholesterol exceeds 5mg/mL, mycelium grows slowly, and the conversion efficiency is affected. The selected autotrophic Pseudonocardia Pseudonocardia autotrophica UV-288 is preserved in China general microbiological culture Collection center (China Committee for culture Collection), and has an address of North Star Xiyu No.1, 3 in the Korean region of Beijing, a collection number of CGMCC No.26831, and a collection date of 2023, 3 and 17.
The selected autotrophic Pseudonocardia Pseudonocardia autotrophica UV-288 can solve the problem of low dosage of substrate 7-dehydrocholesterol, in the deep research for further improving the conversion rate of Pseudonocardia autotrophica UV-288, the invention discovers that the conversion rate is improved to about 85% after the conversion is finished by adding substrate liquid containing 7-dehydrocholesterol into grown mycelium, the conversion rate is basically 40-45%, and a plurality of substrates 7-dehydrocholesterol in the conversion liquid are not converted, which is unfavorable for cost control, and the invention discovers that the conversion rate is not improved by adding non-ionic surfactants such as triton X-100, triton X-114, tween 20, tween 60 into a system through further deep research on factors such as a culture medium formula, conversion conditions, and the like, but the conversion rate is improved to about 85% after the conversion is finished by adding polyethylene glycol 600, but the common polyethylene glycol 200 and polyethylene glycol 400 are not good in improving effect, and the conversion rate is surprisingly improved by adding polyethylene glycol 600 without improving 7-dehydrocholesterol in the biological conversion by adopting a contrast strain Pseudonocardia autotrophica CGMCC No.7935, thus the invention has the unique conversion rate of Pseudonocardia autotrophica UV-25; in addition, it was found that the inoculum size of the seed liquid introduced into the fermentation broth had a significant effect on the improvement of the conversion rate of 7-dehydrocholesterol; based on the above intensive studies, the present invention has been completed.
The invention provides a strain of autotrophic Pseudonocardia Pseudonocardia autotrophica CGMCC No.26831 capable of tolerating high concentration of 7-dehydrocholesterol, and also provides a method for preparing 25-hydroxy-7-dehydrocholesterol by bioconversion, and further carrying out photochemical reaction on the obtained 25-hydroxy-7-dehydrocholesterol to obtain 25-hydroxy vitamin D3, wherein the bioconversion preparation of 25-hydroxy-7-dehydrocholesterol comprises the following steps:
(1) The strain Pseudonocardia autotrophica UV-288 slant culture is sequentially cultured in a seed bottle and a fermentation bottle to amplify mycelium,
(2) And (3) adding substrate solution containing 7-dehydrocholesterol and polyethylene glycol 600 into the fermentation broth of the strain Pseudonocardia autotrophica UV-288, and performing bioconversion to obtain bioconversion solution.
(3) Extracting the bioconversion liquid obtained in the step (2) by using an organic solvent, concentrating, performing column chromatography and crystallizing to obtain the 25-hydroxy-7-dehydrocholesterol.
The seed of the strain Pseudonocardia autotrophica UV-288 in the step (1) and the fermentation medium contain peptone, corn steep liquor, glucose, soybean meal, sodium chloride, potassium dihydrogen phosphate, cyclodextrin and glutathione.
After the fermentation broth of the strain Pseudonocardia autotrophica UV-288 in the step (2) is supplemented with the substrate solution containing 7-dehydrocholesterol and polyethylene glycol 600, the bioconversion is started, and the conversion time is 1-5 d.
The medium for column chromatography in step (3) is silica gel.
The method for preparing 25-hydroxy-7-dehydrocholesterol by bioconversion of strain Pseudonocardia autotrophica UV-288 is operated as follows:
(1) The strain Pseudonocardia autotrophica UV-288 slant culture is sequentially cultured in a seed bottle and a fermentation bottle to amplify mycelium, and the contents of the following components are weight volume percent, namely g/l00mL.
Slant culture medium and culture:
Preparing culture medium containing 1-10% of glucose, 1-10% of peptone, 0.1-2% of corn steep liquor, 0.1-1% of sodium chloride, 1-2.5% of agar and the balance of water, sterilizing at 121 ℃ for 30min, cooling after sterilization, preparing an inclined plane, inoculating, and culturing at 20-35 ℃ for 5-10 d.
The preferable formula is as follows: glucose 1.5%, peptone 1.0%, corn steep liquor 0.3%, sodium chloride 0.4%, agar 1.5%, and water in balance, pH7.0.
Preferred culture conditions are: culturing at 28℃for 7d.
Seed culture medium and culture:
Preparing seed culture medium containing 0.2-2% of peptone, 0.2-2% of corn steep liquor, 0.5-5% of glucose, 0.1-1.5% of soybean meal, 0.1-1.5% of sodium chloride, 0.1-1.5% of monopotassium phosphate and the balance of water, filling the seed culture medium with pH value of 7.0-8.0 into a 750mL shake flask for sterilization, cooling, inoculating bevel strain, washing the cultured bevel strain with sterile water to prepare spore suspension with the spore number of about 1X 10 8/mL, taking 5mL, inoculating into the shake flask filled with the seed culture medium, and culturing for 2-5 d at the rotation speed of 100-300 r/min and the temperature of 20-35 ℃ to obtain shake flask seed liquid.
The preferable formula is as follows: 1.5% of peptone, 1.5% of corn steep liquor, 1.5% of glucose, 0.4% of soybean meal, 0.5% of sodium chloride, 0.2% of monopotassium phosphate and the balance of water, and pH7.5.
Preferred culture conditions are: the flask was incubated at a rotation speed of 220r/min at 28℃for 3d.
Fermentation medium and culture:
Preparing a fermentation culture medium containing 0.2-2% of peptone, 0.2-2% of corn steep liquor, 0.5-5% of glucose, 0.1-1.5% of soybean meal, 0.1-1.5% of sodium chloride, 0.1-1.5% of monopotassium phosphate and 0.2-1.0% of glutathione, wherein the adding mass of cyclodextrin is 4-10 times of the adding mass of 7-dehydrocholesterol to be supplemented later, the balance is water, and pH 7.0-8.0, filling the fermentation culture medium into a 750mL shake flask for sterilization, cooling, and then filling the cultured seed liquid into the shake flask filled with the fermentation culture medium, wherein the inoculum size is 10-30%, the shake flask rotating speed is 100-300 r/min, the temperature is 20-35 ℃, and culturing is carried out for 2-5 days to obtain a shake flask fermentation liquid.
The cyclodextrin is one or more of alpha-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, carboxymethyl beta-cyclodextrin, hydroxyethyl beta-cyclodextrin, hydroxypropyl beta-cyclodextrin, methyl beta-cyclodextrin and sulfobutyl beta-cyclodextrin.
Sulfobutylβ cyclodextrin is preferred.
The preferable formula is as follows: 1.5% of peptone, 1.5% of corn steep liquor, 1.5% of glucose, 0.4% of soybean meal, 0.5% of sodium chloride, 0.2% of monopotassium phosphate, 0.5% of glutathione, 6 times of sulfobutyl beta cyclodextrin and the balance of water, wherein the added mass is 7-dehydrocholesterol, and the pH is 7.5.
Preferred culture conditions are: the seed liquid inoculation amount of the shake flask is 20%, the rotational speed of the shake flask is 220r/min, and the culture is carried out for 3d at 28 ℃.
(2) Adding substrate solution containing 7-dehydrocholesterol and polyethylene glycol 600 into fermentation broth of strain Pseudonocardia autotrophica UV-288, and performing bioconversion;
the preparation method of the substrate solution comprises the following steps: 1g of 7-dehydrocholesterol, 2mL of Tween 80 was dissolved in isopropanol in a total volume of 40mL.
Adding polyethylene glycol 600 into the fermentation broth Pseudonocardia autotrophica UV-288 to make the concentration of the fermentation broth reach 0.2-1.0%, then adding substrate solution containing 7-dehydrocholesterol to make the final concentration of 7-dehydrocholesterol in the conversion solution be 1-5 mg/mL, starting bioconversion, and making the conversion time be 1-5 d so as to obtain bioconversion solution.
Preferred conversion conditions are: the concentration of polyethylene glycol 600 in the conversion solution was 0.5%, and the conversion rate was close to 1mg/mL even when the final concentration of 7-dehydrocholesterol in the conversion solution was 3mg/mL, and the conversion time was 4d.
(3) Extracting the bioconversion liquid obtained in the step (2) by using an organic solvent, concentrating, performing column chromatography and crystallizing to obtain the 25-hydroxy-7-dehydrocholesterol.
The method for separating the 25-hydroxy-7-dehydrocholesterol is to extract bioconversion liquid by using ethyl acetate firstly by adopting a conventional method in the field, concentrate the ethyl acetate and then separate the bioconversion liquid by using a silica gel column to obtain target components, and then crystallize and recrystallize to obtain the 25-hydroxy-7-dehydrocholesterol.
The preparation method of the 25-hydroxy vitamin D3 provided by the invention comprises the steps of preparing 25-hydroxy-7-dehydrocholesterol by adopting the method, and then carrying out illumination ring opening and thermal isomerism on the obtained 25-hydroxy-7-dehydrocholesterol to obtain the 25-hydroxy vitamin D3.
The improvement of the preparation method of the 25-hydroxy vitamin D3 provided by the invention is that a new method is adopted to prepare the intermediate product of the 25-hydroxy-7-dehydrocholesterol, and the process and conditions of the 25-hydroxy-7-dehydrocholesterol for obtaining the 25-hydroxy vitamin D3 through illumination ring opening and thermal isomerization are the same as those of the prior art, and the process and conditions can be known to those skilled in the art, and are not repeated herein.
The sterilization conditions in this example were 121℃for 30 minutes, unless otherwise specified. % is mass percent. The preparation method of the substrate solution comprises the following steps: 1g of 7-dehydrocholesterol, 2mL of Tween 80 was dissolved in isopropanol in a total volume of 40mL. 25-hydroxy-7-dehydrocholesterol HPLC detection conditions: the chromatographic column is KromasilC columns (250×4.6mm,5 um), the mobile phase is methanol/water (95:5), the flow rate is 1.0mL/min, the column temperature is 40 ℃, and the detection wavelength is 265nm. The method for calculating the 7-dehydrocholesterol conversion rate comprises the following steps: (mass of 25-hydroxy-7-dehydrocholesterol obtained by bioconversion/(mass of 7-dehydrocholesterol dosed). Times.100%).
The autotrophic Pseudonocardia provided by the invention and the raw materials and the reagents used in the application of the autotrophic Pseudonocardia can be purchased from the market.
The invention is further illustrated by the following examples:
example 1
1. Medium configuration:
slant culture medium: glucose 1.5%, peptone 1.0%, corn steep liquor 0.3%, sodium chloride 0.4%, agar 1.5%, and water in balance, pH7.0.
Seed culture medium: 1.5% of peptone, 1.5% of corn steep liquor, 1.5% of glucose, 0.4% of soybean meal, 0.5% of sodium chloride, 0.2% of monopotassium phosphate and the balance of water, and pH7.5.
Fermentation medium: 1.5% of peptone, 1.5% of corn steep liquor, 1.5% of glucose, 0.4% of soybean meal, 0.5% of sodium chloride, 0.2% of monopotassium phosphate, 0.5% of glutathione, 6 times of sulfobutyl beta cyclodextrin and the balance of water, wherein the added mass is 7-dehydrocholesterol, and the pH is 7.5.
2. Fermentation
Washing the cultured Pseudonocardia autotrophica UV-288 slant strain with sterile water to obtain spore suspension with spore number of about 1× 8/mL, taking 5mL, inoculating into 250mL shake flask containing 25mL seed culture medium, culturing at rotation speed of 220r/min at 28deg.C for 3d, and co-culturing for 10 bottles; and (3) inoculating the cultured seed liquid into 750mL shake flasks filled with 100mL of fermentation medium, wherein the seed liquid inoculation amount of each flask is 20%, the shake flask rotation speed is 220r/min, and culturing is carried out at 28 ℃ for 3d, and 10 flasks are co-cultured.
3. Bioconversion, separation and purification
(1) Polyethylene glycol 600 was fed into the fermented broth after 3d cultivation so that the concentration of polyethylene glycol in the fermented broth reached 0.5%.
(2) Adding substrate liquid containing 7-dehydrocholesterol into the fermentation liquid, wherein the total amount of the fermentation liquid in each bottle is 120mL, so that the final concentration of the 7-dehydrocholesterol in the fermentation liquid is 3mg/mL, adding 3600mg of 7-dehydrocholesterol in total, starting bioconversion, the conversion time is 4d, stopping bioconversion, and the combined conversion liquid is about 1120mL in total due to partial liquid volatilization, wherein the concentration of 25-hydroxy-7-dehydrocholesterol is about 2.7mg/mL through HPLC detection, and the conversion rate is 84%;
(3) Extracting the bioconversion solution with ethyl acetate for three times, concentrating the ethyl acetate solution, separating with a silica gel column to obtain a target component, crystallizing in an ethanol-water system, and vacuum drying at 30 ℃ overnight to obtain 25-hydroxy-7-dehydrocholesterol white flaky crystals. The melting point of the crystal is 191-193 ℃;
EI-MS(m/z):423(M++Na)。HNMR(CDCl3,400MHz):δ
5.57(1H,d,7-H),5.40(1H,d,6-H),3.63(1H,m,3-H),2.28(2H,m,4-H),2.03(1H,m,3-OH),1.98(1H,m,25-OH),1.24(3H×2,s,26-H,27-H).
The above data indicate that the target product is 25-hydroxy-7-dehydrocholesterol.
4. Preparation of 25-hydroxy vitamin D3
The 25-hydroxy vitamin D3 is prepared by carrying out illumination ring opening and thermal isomerization on the 25-hydroxy-7-dehydrocholesterol by adopting a conventional method in the field.
Example 2
This example differs from example 1 in (different bioconversion and isolation and purification step (1)):
(1) Polyethylene glycol 600 was fed into the fermented broth after 3d cultivation so that the concentration of polyethylene glycol in the fermented broth reached 0.2%.
All other conditions are consistent with example 1, with a 7-dehydrocholesterol conversion of 62%.
Example 3
This example differs from example 1 in (different bioconversion and isolation and purification step (1)):
(1) Polyethylene glycol 600 was fed into the fermented broth after 3d cultivation so that the concentration of polyethylene glycol in the fermented broth reached 1.0%.
All other conditions are consistent with example 1, with a 7-dehydrocholesterol conversion of 75%.
Comparative example 1
The difference between this comparative example and example 1 is (the bioconversion and isolation and purification step (1) is different):
(1) And adding Tween 20 into the fermented liquid after 3d culture so that the concentration of the Tween 20 in the fermented liquid reaches 0.5%.
All other conditions are consistent with example 1, with a 7-dehydrocholesterol conversion of 43%.
Comparative example 2
The difference between this comparative example and example 1 is (the bioconversion and isolation and purification step (1) is different):
(1) And adding tween 60 into the fermented liquid after 3d culture so that the concentration of tween 60 in the fermented liquid reaches 0.5 percent.
All other conditions are consistent with example 1, with a 7-dehydrocholesterol conversion of 45%.
Comparative example 3
The difference between this comparative example and example 1 is (the bioconversion and isolation and purification step (1) is different):
(1) And supplementing the fermentation liquor after 3d culture with the triton X-100 so that the concentration of the triton X-100 in the fermentation liquor reaches 0.5%.
All other conditions are consistent with example 1, with a 7-dehydrocholesterol conversion of 40%.
Comparative example 4
The difference between this comparative example and example 1 is (the bioconversion and isolation and purification step (1) is different):
(1) And supplementing the fermentation broth after 3d culture with the triton X-114 to ensure that the concentration of the triton X-114 in the fermentation broth reaches 0.5 percent.
All other conditions are consistent with example 1, with a 7-dehydrocholesterol conversion of 37%.
Comparative example 5
The difference between this comparative example and example 1 is (the bioconversion and isolation and purification step (1) is different):
(1) Polyethylene glycol 200 was fed into the fermented broth after 3d cultivation so that the concentration of polyethylene glycol in the fermented broth reached 0.5%.
All other conditions are consistent with example 1, with a 7-dehydrocholesterol conversion of 52%.
Comparative example 6
The difference between this comparative example and example 1 is (the bioconversion and isolation and purification step (1) is different):
(1) Polyethylene glycol 400 was fed into the fermented broth after 3d cultivation so that the concentration of polyethylene glycol in the fermented broth reached 0.5%.
All other conditions are consistent with example 1, with a 7-dehydrocholesterol conversion of 55%.
Example 4
This example differs from example 1 in (different bioconversion and isolation and purification step (2)):
(2) Adding substrate liquid containing 7-dehydrocholesterol into the fermentation liquid, wherein the total amount of each bottle of the fermentation liquid is 120mL, so that the final concentration of the 7-dehydrocholesterol in the fermentation liquid is 1mg/mL;
All other conditions are consistent with example 1, with a 7-dehydrocholesterol conversion of 85%.
Example 5
This example differs from example 1 in (different bioconversion and isolation and purification step (2)):
(2) Adding substrate liquid containing 7-dehydrocholesterol into the fermentation liquid, wherein the total amount of the fermentation liquid in each bottle is 120mL, so that the final concentration of the 7-dehydrocholesterol in the fermentation liquid is 4mg/mL;
all other conditions are consistent with example 1, with a 7-dehydrocholesterol conversion of 73%.
Example 6
This example differs from example 1 in (different bioconversion and isolation and purification step (2)):
(2) Adding substrate liquid containing 7-dehydrocholesterol into the fermentation liquid, wherein the total amount of the fermentation liquid in each bottle is 120mL, so that the final concentration of the 7-dehydrocholesterol in the fermentation liquid is 5mg/mL;
all other conditions are consistent with example 1, with a 7-dehydrocholesterol conversion of 51%.
Example 7
The difference between this example and example 1 is (the inoculum size in the fermentation step is different):
2. Fermentation
Washing the cultured Pseudonocardia autotrophica UV-288 slant strain with sterile water to obtain spore suspension with spore number of about 1× 8/mL, taking 5mL, inoculating into 250mL shake flask containing 25mL seed culture medium, culturing at rotation speed of 220r/min at 28deg.C for 3d, and co-culturing for 10 bottles; and (3) inoculating the cultured seed liquid into 750mL shake flasks filled with 100mL of fermentation medium, wherein the seed liquid inoculation amount of each flask is 10%, the shake flask rotation speed is 220r/min, and culturing is carried out at 28 ℃ for 3d, and 10 flasks are co-cultured.
All other conditions are consistent with example 1, with a 7-dehydrocholesterol conversion of 67%.
Example 8
The difference between this example and example 1 is (the inoculum size in the fermentation step is different):
2. Fermentation
Washing the cultured Pseudonocardia autotrophica UV-288 slant strain with sterile water to obtain spore suspension with spore number of about 1× 8/mL, taking 5mL, inoculating into 250mL shake flask containing 25mL seed culture medium, culturing at rotation speed of 220r/min at 28deg.C for 3d, and co-culturing for 10 bottles; and (3) inoculating the cultured seed liquid into 750mL shake flasks filled with 100mL of fermentation medium, wherein the seed liquid inoculation amount of each flask is 30%, the shake flask rotation speed is 220r/min, and culturing is carried out at 28 ℃ for 3d, and 10 flasks are cultivated altogether.
All other conditions are consistent with example 1, with a 7-dehydrocholesterol conversion of 73%.
Comparative example 7
2. Fermentation
Preparing a spore suspension with the spore number of about 1 multiplied by 10 8/mL by using a control strain Pseudonocardia autotrophica CGMCC No.7935 inclined plane strain cultured by sterile water, taking 5mL, inoculating into a 250mL shake flask filled with 25mL of seed culture medium, culturing for 3d at the speed of 220r/min and the temperature of 28 ℃, and culturing for 10 bottles altogether; and (3) inoculating the cultured seed liquid into 750mL shake flasks filled with 100mL of fermentation medium, wherein the seed liquid inoculation amount of each flask is 20%, the shake flask rotation speed is 220r/min, and culturing is carried out at 28 ℃ for 3d, and 10 flasks are co-cultured.
All other conditions are consistent with example 1, with a 7-dehydrocholesterol conversion of 46%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The autotrophic Pseudonocardia (Pseudonocardia autotrophica) is characterized by having a preservation number of CGMCC No.26831.
2. A microbial preparation comprising the autotrophic nocardia (Pseudonocardia autotrophica) of claim 1 and an acceptable adjuvant or adjuvant.
3. A composition comprising any of the following:
(1) The autotrophic nocardia bacterium (Pseudonocardia autotrophica) of claim 1 and polyethylene glycol 600; and/or
(2) The microbial preparation of claim 2 and polyethylene glycol 600.
4. Use of the autotrophic nocardia (Pseudonocardia autotrophica) of claim 1, the microbial preparation of claim 2, or the composition of claim 3 in any of the following:
(1) Improving the conversion rate of 25-hydroxy-7-dehydrocholesterol; and/or
(2) Preparing 25-hydroxy-7-dehydrocholesterol; and/or
(3) Preparing 25-hydroxy vitamin D3;
(4) 7-dehydrocholesterol resistant.
A process for the preparation of 25-hydroxy-7-dehydrocholesterol, characterized in that 25-hydroxy-7-dehydrocholesterol is prepared based on 7-dehydrocholesterol and any of the following:
(1) The autotrophic nocardia bacterium (Pseudonocardia autotrophica) of claim 1; and/or
(2) The microbial preparation of claim 2; and/or
(3) A composition according to claim 3.
6. The preparation method according to claim 5, comprising the following steps:
Step 1: taking the autotrophic nocardia (Pseudonocardia autotrophica) according to claim 1, the microbial preparation according to claim 2, the autotrophic nocardia (Pseudonocardia autotrophica) in the composition according to claim 3 or the microbial preparation in the composition according to claim 3, and culturing to obtain a fermentation broth;
Step 2: mixing the fermentation liquor with polyethylene glycol 600 to obtain fermentation liquor containing polyethylene glycol 600;
Step 3: mixing the fermentation liquor containing polyethylene glycol 600 with 7-dehydrocholesterol substrate liquor, and converting to obtain bioconversion liquor;
Step 4: and extracting, concentrating, performing column chromatography and crystallizing the bioconversion liquid to obtain the 25-hydroxy-7-dehydrocholesterol.
7. The method of claim 6, wherein the culturing in step 1 comprises one or more of slant culture, seed culture, or fermentation culture;
The seed culture comprises the step of inoculating spore suspension obtained by the slant culture into a seed culture medium;
the fermentation culture comprises the step of inoculating seed liquid obtained by seed culture to a fermentation culture medium;
the inoculation amount of the seed liquid is 10-30%.
8. The method of claim 7, wherein the conditions of the slant culture are: culturing for 5-10 d at 20-35 ℃;
The culture medium used for slant culture comprises the following components in percentage by weight and volume: glucose 1-10%, peptone 1-10%, corn steep liquor 0.1-2%, sodium chloride 0.1-1% and/or agar 1-2.5%, pH 6.5-7.5;
The seed culture conditions are as follows: the rotation speed is 100-300 r/min, the temperature is 20-35 ℃, and the culture is carried out for 2-5 d;
The culture medium for seed culture comprises the following components in percentage by weight and volume: peptone 0.2-2%, corn steep liquor 0.2-2%, glucose 0.5-5%, soybean meal 0.1-1.5%, sodium chloride 0.1-1.5% and/or potassium dihydrogen phosphate 0.1-1.5%, pH 7.0-8.0;
The conditions of the fermentation culture are as follows: the rotation speed is 100-300 r/min, the temperature is 20-35 ℃, and the culture is carried out for 2-5 d;
The culture medium used for fermentation culture comprises, by weight and volume percentage, 0.2-2% of peptone, 0.2-2% of corn steep liquor, 0.5-5% of glucose, 0.1-1.5% of soybean meal, 0.1-1.5% of sodium chloride, 0.1-1.5% of potassium dihydrogen phosphate, 0.2-1.0% of glutathione and/or cyclodextrin;
The mass of the cyclodextrin is 4-10 times of the mass of the 7-dehydrocholesterol in the 7-dehydrocholesterol substrate liquid in the step 3.
9. The method according to claim 6, wherein the concentration of polyethylene glycol 600 in the fermentation broth containing polyethylene glycol 600 in step 2 is 0.2 to 1.0%;
The final concentration of 7-dehydrocholesterol in the 7-dehydrocholesterol substrate solution in the step 3 is 1-5 mg/mL;
the conversion time in the step 3 is 1-5 d.
The preparation method of the 25-hydroxy vitamin D3 is characterized by comprising the following steps:
Step A: preparing 25-hydroxy-7-dehydrocholesterol based on 7-dehydrocholesterol and any of:
(1) The autotrophic nocardia bacterium (Pseudonocardia autotrophica) of claim 1; and/or
(2) The microbial preparation of claim 2; and/or
(3) A composition according to claim 3;
and (B) step (B): taking the 25-hydroxy-7-dehydrocholesterol, and carrying out illumination ring opening and thermal isomerization to obtain the 25-hydroxy vitamin D3.
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