阅读说明：本技术 一种生物转化法降低夫西地酸副产物含量的方法 (Method for reducing content of fusidic acid by-product by biotransformation method ) 是由 高健 黄巍巍 张丽 徐虹 邓丽娜 刘长青 仇静 刘伟 于 2019-11-22 设计创作，主要内容包括：本发明属于发酵工程和微生物甾体转化技术领域,具体涉及一种生物转化法降低夫西地酸副产物含量的方法。本方法以黑根霉或米根霉为转化菌,将夫西地酸粗提液中M副产物转化为夫西地酸。具体包括以下步骤：将黑根霉孢子悬浮液或米根霉孢子悬浮液接入黑根霉或米根霉发酵培养基中,发酵培养24 h；将发酵获得的夫西地酸发酵液经过浸提、离心处理后得到上清液,将上清液蒸发浓缩得到M杂质乙醇浓缩液,将M杂质乙醇浓缩液投入到经发酵培养24 h后的黑根霉或米根霉发酵培养基中,加入氧载体,继续转化M杂质,即可。本发明利用生物转化减低夫西地酸中M杂质含量的方法,使其含量低于2%,显著提升夫西地酸产品质量。(The invention belongs to the technical field of fermentation engineering and microbial steroid conversion, and particularly relates to a method for reducing the content of a fusidic acid by-product by a biotransformation method. The method takes rhizopus nigricans or rhizopus oryzae as a transformation bacterium to transform M by-products in the crude fusidic acid extraction liquid into fusidic acid. The method specifically comprises the following steps: inoculating the rhizopus nigricans spore suspension or the rhizopus oryzae spore suspension into a rhizopus nigricans or rhizopus oryzae fermentation culture medium, and carrying out fermentation culture for 24 hours; leaching and centrifuging the fusidic acid fermentation liquor obtained by fermentation to obtain supernatant, evaporating and concentrating the supernatant to obtain an M impurity ethanol concentrated solution, putting the M impurity ethanol concentrated solution into a rhizopus nigricans or rhizopus oryzae fermentation culture medium after 24 hours of fermentation culture, adding an oxygen carrier, and continuously converting the M impurity. The invention utilizes biotransformation to reduce the content of M impurity in fusidic acid, so that the content of the M impurity is lower than 2%, and the product quality of fusidic acid is obviously improved.)

1. A method for reducing the content of fusidic acid by-products by a biotransformation method is characterized in that Rhizopus nigricans or Rhizopus oryzae is used as a transformation bacterium to transform M by-products in a crude fusidic acid extracting solution into fusidic acid.

2. The method of bioconversion to reduce the content of fusidic acid by-product according to claim 1, comprising the following steps:

(1) culturing of the transformed bacteria: washing the spores of the rhizopus nigricans or rhizopus oryzae after being cultured for 4-5 days by using a PDA solid culture medium to obtain rhizopus nigricans spore suspension or rhizopus oryzae spore suspension; inoculating the rhizopus nigricans spore suspension or the rhizopus oryzae spore suspension into a rhizopus nigricans or rhizopus oryzae fermentation culture medium, and carrying out fermentation culture for 24 hours;

(2) adjusting the pH of the fusidic acid fermentation liquor obtained by fermentation to 3.5-4.0, leaching and centrifuging to obtain a supernatant, evaporating and concentrating the supernatant to obtain an M impurity ethanol concentrated solution, putting the M impurity ethanol concentrated solution into the rhizopus nigricans or rhizopus oryzae fermentation culture medium fermented and cultured for 24 hours in the step (1), adding an oxygen carrier, and continuously converting the M impurity.

3. A bioconversion process to reduce the by-product content of fusidic acid according to claim 2, characterized in that fusidic acid broth in step (2) is prepared as follows: inoculating a fusidic acid production strain cultured for 5-6 days by a PDA solid culture medium into a primary seed culture medium, culturing at 26 ℃ for 72 +/-5 h, then inoculating into a secondary amplification culture medium according to 1% of inoculation amount, culturing at 26 ℃ for 25 +/-5 h, then inoculating into a fermentation culture medium according to 10% of inoculation amount, and culturing at 26 ℃ for 7-8 days to obtain the fusidic acid fermentation liquor.

4. The biological conversion process for reducing the by-product content of fusidic acid as claimed in claim 2, wherein said oxygen carrier in step (2) is H₂O₂Or oleic acid.

5. The method of claim 2, wherein the conditions of transformation in step (2) are normal pressure, 200 rpm, and 28 ℃ shaking whole cell transformation for 43 hours.

6. A bioconversion process to reduce the content of fusidic acid by-product according to claim 2, wherein the composition of rhizopus nigricans or rhizopus oryzae fermentation medium in step (1) is: glucose 5%, corn steep liquor 4%, (NH)₄)₂SO₄0.3%， KH₂PO₄0.03%，MgSO₄·7H₂O 0.075%，ZnSO₄·7H₂O 0.02%， CaCO₃0.5%, natural pH.

7. A bioconversion process reduction enzyme according to claim 3The method for determining the content of the by-product of the sithydric acid is characterized in that the composition of the primary seed culture medium is as follows: the seed culture medium contains glucose 2.5%, fish meal peptone 1.0%, yeast powder 0.2%, KH₂PO₄0.1%，MgSO₄0.05 percent, 4.0 percent of corn steep liquor, 0.2 percent of light calcium carbonate and pH 5.9 +/-0.1.

8. A bioconversion method according to claim 3, wherein the composition of the secondary expanding seed medium is: 2.5 percent of glucose, 1.0 percent of fish meal peptone, 0.2 percent of yeast powder and KH₂PO₄0.1%，MgSO₄0.05 percent, 4.0 percent of corn steep liquor, 0.2 percent of light calcium carbonate and pH 5.9 +/-0.1.

9. A bioconversion process to reduce the content of fusidic acid by-product according to claim 3, characterized in that the composition of the fermentation medium is: 10.0 percent of sucrose, 0.5 percent of gluten powder, 2.0 percent of yeast powder, 1.0 percent of corn steep liquor, 0.5 percent of soybean cake powder and KH₂PO4 0.1%，MgSO₄0.05 percent, 0.3 percent of light calcium carbonate and pH 5.9 +/-0.1.

10. A bioconversion process to reduce the content of fusidic acid by-product according to claim 2 or 3, wherein said PDA solid medium consists of: potato 20.0%, glucose 2.0%, agar 2.0%, natural pH.

Technical Field

The invention belongs to the technical field of fermentation engineering and microbial steroid conversion, and particularly relates to a method for reducing the content of a fusidic acid by-product by a biotransformation method.

Background

Fusidic acid of formula C₃₁H₄₈O₆Molecular weight of 516.69, chemical name of 16 β -acetoxy

-3 α,11 α -dihydroxy-4 α,8 α,14 β -trimethyl-18-nor-5 α,10 β -cholest- (17)Z) -17(20), 24-diene-21-acid, which has a steroid-like structure, wherein the molecular structure of the acid has higher similarity with the molecular structures of the fumagillolic acid and the cephalosporin P, only a few groups have differences, and the presence of the difference groups endows the acid with a special bacteriostatic action. Studies show that fusidic acid has extremely low water solubility and good fat solubility, while sodium fusidate is easily soluble in water.

Fusidic acid is an important antibiotic for G⁺The bacteria have strong inhibitory activity. The pharmaceutical commodities mainly comprise sodium fusidate ointment, sodium fusidate injection, dry suspension and the like, and different dosage forms can be suitable for diseases caused by infection of various sensitive bacteria, for example, sodium fusidate for injection can be used for: joint infection of limbs, septicemia (caused by bacteria), endocarditis, cystic fibrosis, osteomyelitis, skin tissue infection, pneumonia, and traumatic infection of various parts of the body; sodium fusidate ointment may be useful in the treatment of infections caused by staphylococci, streptococci, corynebacteria infinitesimal and other bacteria sensitive to sodium fusidate. Fusidic acid belongs to steroid compounds, and due to the existence of an asymmetric center in molecules, chemical synthesis steps are complex, cost is high, yield is low, a chemical method is difficult to apply to actual industrialized production, and the fusidic acid is mainly produced and prepared by a microbial fermentation method at home and abroad at present.

Fusidic acid is isolated from secondary metabolites of fungal fermentation, and at present, other species have not been found to be capable of synthesizing fusidic acid except for Fusarium globosum. Meanwhile, the secondary metabolite fusidic acid fermented by the fusiform coccospora is often accompanied by the existence of byproducts, particularly M impurities are extremely similar to the main product fusidic acid in structure and only exist in C₁₁One α -OH is missing, the content of M substance in fusidic acid product is less than 2% as specified by European pharmacopoeia and industry standard plaintext, the content of M by-product in crude fusidic acid extract obtained by high-yield preparation of fusidic acid by a fermentation method of fusiform coccobacillus is usually about 7.0-8.0%, at present, the steroid C is transformed by related strain₁₁Therefore, the M impurity in the fusidic acid prepared by a bacterial strain transformation fermentation method with high expression of 11 α -hydroxylase is selected, the content of the M impurity is reduced, the product quality of the fusidic acid is improved, the standard requirement of pharmacopeia is met, and the method has important economic value and social benefit.

Disclosure of Invention

The invention aims to overcome the defect that the content of M by-products in fusidic acid obtained by high-yield preparation through a fermentation method of Alternaria sphaerica in the prior art is higher, about 7.0-8.0%, so as to provide a method for reducing the content of the fusidic acid by-products through a biotransformation method.

In order to achieve the above purpose, the invention provides the following technical scheme:

the invention utilizes rhizopus nigricans (A), (B), (C), (Rhizopus nigricans) Rhizopus oryzae (A) and (B)Rhizopus oryzae) A process for the bioconversion reduction of the content of major by-products in the fermentative preparation of fusidic acid, respectively. The invention relates to three strains, the names of the strains are rhizopus nigricans, rhizopus oryzae and fusiform spore bacterium (A), (B)FusidiumcoccineumThe culture medium is preserved in the China general microbiological culture Collection center, No. 3 Xilu No. 1 of the Chaoyang district, Beijing, the preservation date is 3 months and 21 days in 2018, and the preservation number is CGMCC number 15473).

A method for reducing the content of fusidic acid by-products by a biotransformation method, takes Rhizopus nigricans or Rhizopus oryzae as transformation bacteria, and converts M by-products in crude fusidic acid extract into fusidic acid.

Preferably, the method for reducing the content of fusidic acid by-product by the biotransformation process comprises the following steps:

(1) culturing of the transformed bacteria: washing the spores of the rhizopus nigricans or rhizopus oryzae after being cultured for 4-5 days by using a PDA solid culture medium to obtain rhizopus nigricans spore suspension or rhizopus oryzae spore suspension; inoculating the rhizopus nigricans spore suspension or the rhizopus oryzae spore suspension into a rhizopus nigricans or rhizopus oryzae fermentation culture medium, and carrying out fermentation culture for 24 hours;

(2) adjusting the pH of the fusidic acid fermentation liquor obtained by fermentation to 3.5-4.0 by using dilute hydrochloric acid, leaching and centrifuging to obtain a supernatant, evaporating and concentrating the supernatant to obtain an M impurity ethanol concentrated solution, putting the M impurity ethanol concentrated solution into the rhizopus nigricans or rhizopus oryzae fermentation culture medium fermented and cultured for 24 hours in the step (1), adding an oxygen carrier, and continuously converting the M impurity.

Preferably, the preparation method of the fusidic acid fermentation broth in the step (2) is as follows: inoculating a fusidic acid production strain cultured for 5-6 days by a PDA solid culture medium into a primary seed culture medium, culturing at 26 ℃ for 72 +/-5 h, then inoculating into a secondary amplification culture medium according to 1% of inoculation amount, culturing at 26 ℃ for 25 +/-5 h, then inoculating into a fermentation culture medium according to 10% of inoculation amount, and culturing at 26 ℃ for 7-8 days to obtain the fusidic acid fermentation liquor.

Preferably, the oxygen carrier in the step (2) is H₂O₂Or oleic acid.

Preferably, the transformation conditions in step (2) are normal pressure, 200 rpm, and 28 ℃ shaking whole cell transformation for 43 h.

Preferably, the rhizopus nigricans or rhizopus oryzae fermentation medium in the step (1) has the following composition: glucose 5%, corn steep liquor 4%, (NH)₄)₂SO₄0.3%， KH₂PO₄0.03%，MgSO₄·7H₂O 0.075%，ZnSO₄·7H₂O 0.02%， CaCO₃0.5%, natural pH.

Preferably, theThe first-order seed culture medium comprises the following components: the seed culture medium contains glucose 2.5%, fish meal peptone 1.0%, yeast powder 0.2%, KH₂PO₄0.1%，MgSO₄0.05 percent, 4.0 percent of corn steep liquor, 0.2 percent of light calcium carbonate and pH 5.9 +/-0.1.

Preferably, the composition of the secondary amplification seed culture medium is as follows: 2.5 percent of glucose, 1.0 percent of fish meal peptone, 0.2 percent of yeast powder and KH₂PO₄0.1%，MgSO₄0.05 percent, 4.0 percent of corn steep liquor, 0.2 percent of light calcium carbonate and pH 5.9 +/-0.1.

Preferably, the composition of the fermentation medium is: 10.0 percent of sucrose, 0.5 percent of gluten powder, 2.0 percent of yeast powder, 1.0 percent of corn steep liquor, 0.5 percent of soybean cake powder and KH₂PO4 0.1%，MgSO₄0.05 percent, 0.3 percent of light calcium carbonate and pH 5.9 +/-0.1.

Preferably, the PDA solid medium consists of: potato 20.0% (peeling, cutting, boiling to paste, filtering with six layers of gauze to remove residue), glucose 2.0%, agar 2.0%, and natural pH.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention obviously improves the quality of the fusidic acid product, is fully applied to the production of efficiently preparing the fusidic acid by an actual biological method, and has good economic value and social benefit.

(2) The fermentation yield of the fusidic acid provided by the invention is 3205 mu g/ml, the content of M impurities is 7.72%, after the rhizopus nigricans and the rhizopus oryzae are respectively transformed, the yields of the fusidic acid are 3293 mu g/ml and 3251 mu g/ml, and the yields of the fusidic acid are improved.

(3) The yield of the impurity substance M in the fermentation liquor of the fusidic acid strain provided by the invention is 268 mu g/ml, after the rhizopus nigricans and the rhizopus oryzae are respectively converted, the impurity residues are 38 mu g/ml and 58 mu g/ml, the conversion rates respectively reach 85.72 percent and 78.49 percent, and the impurity contents are both lower than 2 percent.

(4) The method for reducing the content of the M impurity in the fusidic acid by biotransformation has the advantages of mild cultivation conditions, simple operation, high output-input ratio, obvious transformation effect and easy implementation; the crude fusidic acid ethanol extract is subjected to pretreatment such as low-temperature rotary evaporation concentration and the like, and then is fed, so that the influence of a high-concentration organic solvent on a rhizopus nigricans and rhizopus oryzae whole-cell transformation system is reduced.

Drawings

FIG. 1 is a diagram of three strains according to the present invention, and FIG. 1 (a) shows Rhizopus nigricans: (A)Rhizopus nigricans) FIG. 1 (b) shows Rhizopus oryzae (A)Rhizopus oryzae) FIG. 1 (c) shows Alternaria sphaerica NJWW (C)Fusidium coccineum）；

FIG. 2 is a molecular structure diagram of two major steroids in fusidic acid fermentation broth prepared from Fusarium globosum, wherein FIG. 2 (a) is fusidic acid as a main product, and FIG. 2 (b) is impurity of byproduct M;

figure 3 is a standard graph of high performance liquid chromatography for determining fusidic acid concentration.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The rhizopus nigricans strain for transformation grows on a PDA culture medium, can grow into a sheet of colony after being cultured at 27 ℃ for 4-5 days, and has abundant and white aerial hyphae at the early stage of the colony, less hyphae in a basal layer, more hyphae in a basal layer at the later stage, further increased aerial hyphae, deepened color and large amount of black spores (as shown in figure 1 (a)).

The transformation strain Rhizopus oryzae grows on a PDA culture medium, is cultured at 27 ℃ for 4-5 days, can grow into a sheet of colony, is white at the early stage of the colony, is rich in aerial hyphae and less in substrate hyphae, and can generate a plurality of black spores at the later stage (as shown in figure 1 (b)).

The fusidic acid high-yield strain Alternaria sphaerica grows on a PDA culture medium, the culture is carried out for 6 days at 26 ℃, the diameter of a bacterial colony is 4-6 mm, the color of the bacterial colony is white to off-white, light pink is occasionally seen, aerial hyphae are rich, intrabasal hyphae and spores are fewer, small white sample-like protrusions formed by the spores are arranged in the center, slight exudate is generated, soluble pigments are not generated, and the overall state of the bacterial colony is dry and hard (as shown in figure 1 (c)).