CN116410871A - Fusarium equisetum and method for hydroxylating cholic acid by using same - Google Patents

Fusarium equisetum and method for hydroxylating cholic acid by using same Download PDF

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CN116410871A
CN116410871A CN202310241202.4A CN202310241202A CN116410871A CN 116410871 A CN116410871 A CN 116410871A CN 202310241202 A CN202310241202 A CN 202310241202A CN 116410871 A CN116410871 A CN 116410871A
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李杉
刘芬
张雷
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South China University of Technology SCUT
Zhongshan Institute of Modern Industrial Technology of South China University of Technology
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Zhongshan Institute of Modern Industrial Technology of South China University of Technology
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Abstract

The invention belongs to the technical field of biology, and relates to a Fusarium equisetum (Fusarium equiseti) and a method for hydroxylating cholic acid by using the same. The Fusarium equisetum is Fusarium equisetum (Fusarium equiseti) HG18, and the preservation number is CCTCC M2023160. By adopting the microbial fermentation method, the conversion rate of ursodeoxycholic acid reaches 42% at the substrate concentration of 2g/L of lithocholic acid.

Description

Fusarium equisetum and method for hydroxylating cholic acid by using same
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for preparing hydroxyl cholic acid from Fusarium equisetum (Fusarium equiseti).
Background
Ursodeoxycholic acid (Ursodeoxycholic Acid, UDCA), also known as 3 alpha, 7 beta-dihydroxyl-5 beta-cholestane-24-acid, is a hydrophilic bile acid with various biological activities, is also a main active ingredient of traditional rare traditional Chinese medicine bear gall powder, can increase secretion of bile, inhibit cytotoxicity of hydrophobic cholic acid, protect bile duct cells and liver cells from toxic bile injury, has extremely strong function of promoting fat and fatty acid hydrolysis, reduces cholesterol and cholesterol ester in bile, is favorable for gradually dissolving cholesterol in gall stone, and is mainly used for treating various liver and gall diseases and digestive tract diseases clinically, such as treating gall stone, cholestatic liver disease, fatty liver, various hepatitis, toxic liver disorder, cholecystitis, cholangitis, bile dyspepsia, bile reflux gastritis, ocular disease and the like.
The existing ursodeoxycholic acid preparation methods generally comprise the following steps, namely, the method is obtained through bear bile extraction, and the method is gradually eliminated due to limited sources or animal protection laws; secondly, animal cholic acid substances such as cholic acid, chenodeoxycholic acid and the like are used as raw materials to be prepared by chemical or biological synthesis methods, and the methods have the problems of limited raw material sources, long reaction steps, low yield and the like (X.L.He, L.T.Wang, X.Z.Gu, J.X.Xiao, W.W.Qiu, steroids,2018,140:173-178;M.M.Zheng,R.F.Wang,C.X.Li,J.H.Xu,Process Biochemistry,2015,50:598-604); thirdly, a synthetic method which takes plant-derived dehydroandrosterone as a raw material prepares ursodeoxycholic acid (W.Chen, D.Hu, Z.Feng, Z.Liu, steroids,2021, 172:10887.) through eight steps of reactions, and the method belongs to a method for preparing the raw material of non-animal cholic acid, but the separation, purification and separation amount of plants are very large problems; fourth, it is prepared by an enzymatic method, and chinese patent CN109154016a reports that lithocholic acid undergoes hydroxylation reaction under the catalytic action of hydroxylase and coenzyme to obtain ursodeoxycholic acid. The coenzyme is needed in the reaction, the cost is high, and in addition, the hydroxylase of the technology is obtained by separating from thalli, the steps are complex, the cost is high, and the technology is only suitable for experimental stage research and is difficult to realize industrialized application.
Disclosure of Invention
One of the technical problems to be solved by the invention is to provide the fusarium equiseti (Fusarium equiseti) which has the function of catalyzing and converting the lithocholic acid into ursodeoxycholic acid.
One of the technical problems to be solved by the invention is to provide a method for preparing ursodeoxycholic acid by microbial fermentation, which has higher reaction yield.
In order to achieve the above purpose, the invention obtains a microorganism strain HG18 through screening, and the microorganism strain is identified as Fusarium equisetum (Fusarium equiseti) (see figure 1), and is preserved in the China center for type culture collection (preservation number CCTCC M2023160) of No. 299 in Wuchang district of Wuhan, hubei province for 20 days in 2023. The strain can take lithocholic acid as a substrate, introduce beta-configuration hydroxyl into the 7-position of the lithocholic acid by a microbial transformation method, and obtain ursodeoxycholic acid after simple treatment. The method has the advantages of low cost, simple process, mild reaction conditions, simple and easily obtained reagent and suitability for industrialized mass production.
The equation for the above reaction is as follows:
Figure SMS_1
the aim of the invention is realized by the following technical scheme:
the first aim of the invention is to provide Fusarium equisetum (Fusarium equiseti) HG18 with a preservation number of CCTCC M2023160.
Another object of the present invention is to provide a method for hydroxylating cholic acid using the above fusarium equiseti, comprising the steps of:
adding a liquid culture containing Fusarium equisetosum into a fermentation medium, adding lithocholic acid, and culturing to obtain a fermentation broth containing ursodeoxycholic acid;
the Fusarium equisetum is Fusarium equisetum (Fusarium equiseti) HG18 CCTCC M2023160.
Preferably, the fermentation medium is composed of: 20-30g/L of carbon source, 10-30g/L of nitrogen source, 1-5g/L of yeast powder and KH 2 PO 4 1-5g/L，MgSO 4 ·7H 2 O 0.1-1g/L，FeSO 4 ·7H 2 0.01-0.5g/L O, 0-0.1M metal ion compound; adjusting pH of the culture medium to 5.5-10 before sterilization, and sterilizing at 121+ -5deg.C under high pressure steam for 20+ -5 min.
More preferably, the composition of the fermentation medium is as follows: 20g/L of carbon source, 15g/L of nitrogen source, 1g/L of yeast powder and KH 2 PO 4 3g/L，MgSO 4 ·7H 2 O 0.5g/L，FeSO 4 ·7H 2 O0.02 g/L; the pH of the medium was adjusted to 7.5 before sterilization.
Preferably, the carbon source is one or more than two of glucose, lactose, sucrose, soluble starch and dextrin;
the nitrogen source is NH 4 One or more of Cl, corn steep liquor dry powder, wall-broken dry yeast, peptone, yeast powder and L-asparagine;
the metal ion compound is NaCl, caCl 2 、MgCl 2 、CuCl 2 、ZnCl 2 、NH 4 One or more of Cl and KCl.
Preferably, the inoculum size of Fusarium equisetum is 10-30% (v/v), and the final concentration of lithocholic acid in the fermentation medium is 2g/L; the culture conditions were as follows: the culture temperature is 25-30 ℃, and the fermentation culture is carried out for 96-144h at the rotating speed of 120-220 rpm.
Preferably, the liquid culture containing fusarium equiseti is obtained by the following steps: 100 mu L of fusarium equiseti spore frozen stock solution is coated on a PDA solid culture medium, cultured for 5-7 days at 25-40 ℃, resuspended by 0.2% Tween 80 water to obtain spore suspension, and inoculated into a seed culture medium for culture, thus obtaining the fusarium equiseti-containing liquid culture.
Preferably, the concentration of spores in the spore suspension is (1-1.7). Times.10 6 The spore/mL is inoculated into 50mL of seed culture medium at the inoculation proportion of 1mL spore suspension, and the culture condition is that the temperature is 25-30 ℃ and the culture is 120-220rpm for 12-72h.
More preferably, the seed culture medium comprises the following components in percentage by weight: 20g/L of carbon source, 15g/L of nitrogen source, 1g/L of yeast powder and KH 2 PO 4 3g/L，MgSO 4 ·7H 2 O0.5 g/L; natural pH, sterilizing at 121deg.C under high pressure steam for 20min;
the PDA culture medium comprises the following components in percentage by weight: 200g/L potato; glucose 20g/L; 20g/L of agar powder; naturally pH, and sterilizing at 121deg.C under high pressure steam for 20min.
Preferably, the carbon source of the seed culture medium is one or more than two of glucose, lactose, sucrose, soluble starch and dextrin;
the nitrogen source of the seed culture medium is NH 4 One or more of Cl, corn steep liquor dry powder, wall-broken dry yeast, peptone, yeast powder and L-asparagine;
the incubation time of the spore suspension in the seed medium was 36 hours.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) The strain can take lithocholic acid as a substrate, introduce beta-configuration hydroxyl into the 7-position of the lithocholic acid by a microbial transformation method, and obtain ursodeoxycholic acid after simple treatment. The method has the advantages of low cost, simple process, mild reaction conditions, simple and easily obtained reagent and suitability for industrialized mass production.
(2) The method provided by the invention has higher reaction yield, and the conversion rate of ursodeoxycholic acid reaches 42% under the condition that the concentration of lithocholic acid is 2 g/L.
Drawings
FIG. 1 is a graph showing the relationship between the TEF-1 alpha region of Fusarium equisetum HG18 (Fusarium equiseti) of the present invention.
FIG. 2 is a liquid chromatogram of the reaction substrates and products of the present invention.
FIG. 3 is an optimized graph of the liquid culture of Fusarium equisetum HG18 (Fusarium equiseti) of the present invention, FIG. 3A is the effect of carbon source on Fusarium equisetum growth; FIG. 3B is an effect of nitrogen source on Fusarium equisetum growth.
FIG. 4 is a graph showing the growth of Fusarium equisetum HG18 (Fusarium equiseti) of the present invention.
FIG. 5 is an optimized diagram of fermentation medium of Fusarium equisetum HG18 (Fusarium equiseti) of the present invention, FIG. 5A is the effect of carbon source on Fusarium equisetum conversion; FIG. 5B is the effect of nitrogen source on Fusarium equisetum conversion.
FIG. 6 is a graph showing the effect of pH of the fermentation medium of the present invention on biocatalysis.
FIG. 7 is a graph showing the effect of metal ions on biocatalysis in the fermentation medium of the present invention.
FIG. 8 is a biocatalytic schedule of Fusarium equisetum HG18 (Fusarium equiseti) of the present invention.
Detailed Description
The invention is described in further detail below with reference to the drawings and examples.
Example 1 identification of Fusarium equisetum HG18 species
200mg of fresh mycelium with transformation capability obtained by screening is taken and ground into powder by liquid nitrogen, and then genomic DNA thereof is extracted by referring to a kit. After the extraction, TEF-1. Alpha. Sequence amplification was performed and the PCR amplified product was sent to Shanghai Biotechnology Co., ltd for sequencing. Sequencing results were aligned by BLAST program. And the relationship of Fusarium equisetum HG18 was mapped using MEGA11 software to determine its genus.
As shown in FIG. 1, the genus HG18 was determined to be Fusarium equisetum (Fusarium equiseti).
Example 2 detection method of reaction product (UDCA)
The substrate and the product of the reaction are detected by adopting a high performance liquid chromatography, and the detection conditions are as follows:
chromatographic column: a C18 reverse-phase chromatographic column,
mobile phase: methanol-acetonitrile-water (5:4:3, v/v) and 0.03% NaH 2 PO 4 ·2H 2 O and 0.08% H 3 PO 4 ，
Flow rate: the concentration of the solution is 0.8mL/min,
column temperature: 30 ℃,
a detector: differential refractive light detector.
The liquid chromatogram under this condition is shown in FIG. 2, with LCA retention time of 5.25 minutes and UDCA retention time of 25.75 minutes.
Example 3 preparation of Fusarium equisetum HG18 cultures
1) Preparation of Fusarium equisetum HG18 solid cultures
100. Mu.L of Fusarium equisetum spore frozen stock stored in a-80℃freezer was spread on PDA solid medium. Incubated for 5-7 days at 28℃in a biochemical incubator.
2) Optimized preparation of Fusarium equisetum HG18 liquid culture
Washing Fusarium equisetum HG18 on PDA solid medium with 10ml of 0.2% Tween 80 sterile water, gently blowing with a pipetting gun, collecting spore suspension for use, controlling the density of spore suspension to be (1-1.7). Times.10 6 spores mL -1 . 1mL of the spore suspension was then inoculated into a 250mL conical flask containing 50mL of seed medium and incubated at 28℃and 200 rpm. Different kinds of carbon sources and nitrogen sources were added to the seed medium to examine the effect of the different carbon sources and nitrogen sources on the growth of Fusarium equisetum HG 18. The optimal carbon and nitrogen source for the growth of Fusarium equisetum HG18 is determined based on the dry weight of the mycelium. As a result, as shown in FIGS. 3A and 3B, the most suitable carbon source and nitrogen source were dextrin and peptone, respectively.
The seed culture medium comprises the following components in percentage by weight: 20g/L of carbon source, 15g/L of nitrogen source, 1g/L of yeast powder and KH 2 PO 4 3g/L，MgSO 4 ·7H 2 O0.5 g/L; naturally pH, and sterilizing at 121deg.C under high pressure steam for 20min.
3) Exploration of Fusarium equisetum HG18 growth kinetics
The liquid culture is put into a centrifuge tube for repeated centrifugation and washing with clear water for three times by adopting a cell dry weight method, and then is dried in vacuum at 105 ℃ and then weighed. The growth curve of Fusarium equisetum HG18 was monitored as the dry weight of the cells. The results are shown in FIG. 4. Fusarium equisetum HG18 grew logarithmically at 24h, entered stationary phase after 72h, and then gradually declined.
4) Connection of growth period of fusarium equiseti HG18 microorganism and biocatalysis
According to the growth curve in 3), the mycelia with different growth periods are adopted for microbial catalysis, and mycelia with the optimal period are selected for the next experiment.
Mycelia grown to 12, 24, 36, 48, 60 and 72 hours were centrifuged at 6000rpm for 5min, respectively, discarded and then washed 3 times with sterile water and centrifuged at 6000rpm for 5min. Then, the sterilized fermentation medium was inoculated with an inoculum size of 10% (v/v), and an appropriate amount of lithocholic acid, which was dissolved in 1% (v/v) DMSO, was weighed, and put into the above fermentation medium so that the final concentration of lithocholic acid was 2g/L, and fermented at 28℃and 200rpm for 96 hours. The results are shown in Table 1. Therefore, mycelia grown to 36h in the early logarithmic growth phase were selected as seed cells and transferred to a transformation medium for transformation study.
TABLE 1 relationship between microbial growth phase and biocatalysis of Fusarium equisetum HG18 (Fusarium equiseti)
Figure SMS_2
Example 4 Fusarium equisetum conversion of lithocholic acid (LCA) to ursodeoxycholic acid (UDCA)
1) Optimized fusarium equiseti HG18 fermentation medium
The above-mentioned liquid culture of Fusarium equisetum HG18, which had grown to a log phase of 36 hours, was centrifuged at 6000rpm for 5 minutes, then washed 3 times with sterile water and centrifuged at 6000rpm for 5 minutes, and then inoculated into a sterilized fermentation medium at an inoculum size of 10% (v/v). Different kinds of carbon sources and nitrogen sources were added to the fermentation medium to examine the effect of the different carbon sources and nitrogen sources on conversion of Fusarium equisetum HG18 into lithocholic acid. The optimal carbon source and nitrogen source for the biocatalysis of Fusarium equisetum HG18 are determined based on the yield of ursodeoxycholic acid. As a result, as shown in FIGS. 5A and 5B, the most suitable carbon source and nitrogen source were dextrin and peptone, respectively.
The pH of the fermentation medium was adjusted to 5.5, 6.5, 7.5, 8.5, 9.5, 10 at the time of formulation to examine the effect of different pH on conversion of Fusarium equisetum HG18 to lithocholic acid. The results are shown in FIG. 6, with an optimum pH of 7.5.
The components and the proportion of the fermentation medium are as follows: 20g/L of carbon source, 15g/L of nitrogen source, 1g/L of yeast powder and KH 2 PO 4 3g/L，MgSO 4 ·7H 2 O 0.5g/L，FeSO 4 ·7H 2 O0.02 g/L; the pH of the medium was adjusted to 7.5 before sterilization, and the medium was sterilized at 121℃under high pressure steam for 20min.
2) Exploration of metal ion for biocatalysis of Fusarium equisetum HG18
During the preparation of the fermentation medium, 0.1M NaCl and CaCl are added 2 、MgCl 2 、CuCl 2 、ZnCl 2 、NH 4 Cl, KCl and other metal ion compounds to examine microelementsThe effect of the pigments and inorganic salts on the biocatalysis of Fusarium equisetum. As a result, potassium ions have a promoting effect on biocatalysis as shown in FIG. 7.
3) Biocatalysis
In the optimized fermentation medium, 10% (v/v) of fusarium equiseti HG18 is inoculated, a proper amount of lithocholic acid is precisely weighed, 1% (v/v) of DMSO is used for dissolving, the lithocholic acid is added into the optimized fermentation medium to make the final concentration of the lithocholic acid be 2g/L, and the fermentation medium is cultivated for 144 hours under the conditions of 28 ℃ and 200rpm, so as to obtain a fermentation broth.
The pH of the fermentation broth obtained above was adjusted to 12 with NaOH aqueous alkali to dissolve cholic acid compounds in the fermentation broth, and then centrifuged at 12000rpm for 5min. Separating the supernatant from thallus, regulating pH of the supernatant to 2 with HCl strongly acidic aqueous solution, standing for 10min, and centrifuging at 12000rpm for 5min to obtain white precipitate as cholic acid compound. After centrifugation, most of the supernatant was discarded, the lower precipitate and part of the supernatant were resuspended, the resuspension was extracted 3 times with an equal volume of ethyl acetate, the extracts were combined, the mixture was spun on a rotary evaporator until crystals appeared, the crystals were reconstituted with methanol and removed by filtration through an organic membrane of 0.22 μm, the ursodeoxycholic acid, lithocholic acid content was analyzed by high performance liquid chromatography of example 2, and the conversion of LCA to UDCA was 42%.

Claims (10)

1. The fusarium equiseti is characterized in that the fusarium equiseti is fusarium equiseti (Fusarium equiseti) HG18, and the preservation number is CCTCC M2023160.
2. A method of using the fusarium equiseti hydroxy fossil cholic acid of claim 1, comprising the steps of:
adding a liquid culture containing Fusarium equisetosum into a fermentation medium, adding lithocholic acid, and culturing to obtain a fermentation broth containing ursodeoxycholic acid;
the Fusarium equisetum is Fusarium equisetum (Fusarium equiseti) HG18 CCTCC M2023160.
3. The method of claim 2, wherein the fermentation medium is composed of: 20-30g/L of carbon source, 10-30g/L of nitrogen source, 1-5g/L of yeast powder and KH 2 PO 4 1-5g/L，MgSO 4 ·7H 2 O 0.1-1g/L，FeSO 4 ·7H 2 0.01-0.5g/L O, 0-0.1M metal ion compound; adjusting pH of the culture medium to 5.5-10 before sterilization, and sterilizing at 121+ -5deg.C under high pressure steam for 20+ -5 min.
4. The method according to claim 2, characterized in that the composition of the fermentation medium is as follows: 20g/L of carbon source, 15g/L of nitrogen source, 1g/L of yeast powder and KH 2 PO 4 3g/L，MgSO 4 ·7H 2 O 0.5g/L，FeSO 4 ·7H 2 O0.02 g/L; the pH of the medium was adjusted to 7.5 before sterilization.
5. The method according to claim 2, wherein the carbon source is one or more of glucose, lactose, sucrose, soluble starch, dextrin;
the nitrogen source is NH 4 One or more of Cl, corn steep liquor dry powder, wall-broken dry yeast, peptone, yeast powder and L-asparagine;
the metal ion compound is NaCl, caCl 2 、MgCl 2 、CuCl 2 、ZnCl 2 、NH 4 One or more of Cl and KCl.
6. The method according to claim 2, wherein the inoculum size of fusarium equiseti is 10-30% (v/v), and the final concentration of lithocholic acid in the fermentation medium is 2g/L; the culture conditions were as follows: the culture temperature is 25-30 ℃, and the fermentation culture is carried out for 96-144h at the rotating speed of 120-220 rpm.
7. The method according to claim 2, wherein the liquid culture containing fusarium equiseti is obtained by: 100 mu L of fusarium equiseti spore frozen stock solution is coated on a PDA solid culture medium, cultured for 5-7 days at 25-40 ℃, resuspended by 0.2% Tween 80 water to obtain spore suspension, and inoculated into a seed culture medium for culture, thus obtaining the fusarium equiseti-containing liquid culture.
8. The method of claim 7, wherein the concentration of spores in the spore suspension is (1-1.7). Times.10 6 The spore/mL is inoculated into 50mL of seed culture medium at the inoculation proportion of 1mL spore suspension, and the culture condition is that the temperature is 25-30 ℃ and the culture is 120-220rpm for 12-72h.
9. The method of claim 7, wherein the seed culture medium comprises the following components in percentage: 20g/L of carbon source, 15g/L of nitrogen source, 1g/L of yeast powder and KH 2 PO 4 3g/L，MgSO 4 ·7H 2 O0.5 g/L; natural pH, sterilizing at 121deg.C under high pressure steam for 20min;
the PDA culture medium comprises the following components in percentage by weight: 200g/L potato; glucose 20g/L; 20g/L of agar powder; naturally pH, and sterilizing at 121deg.C under high pressure steam for 20min.
10. The method of claim 9, wherein the carbon source of the seed medium is one or more of glucose, lactose, sucrose, soluble starch, dextrin;
the nitrogen source of the seed culture medium is NH 4 One or more of Cl, corn steep liquor dry powder, wall-broken dry yeast, peptone, yeast powder and L-asparagine;
the incubation time of the spore suspension in the seed medium was 36 hours.
CN202310241202.4A 2023-03-13 2023-03-13 Fusarium equisetum and method for hydroxylating cholic acid by using same Pending CN116410871A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117025709A (en) * 2023-07-31 2023-11-10 华南理工大学 Application of cytochrome P450 enzyme and cytochrome P450 reductase combined in synthesis of ursodeoxycholic acid

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117025709A (en) * 2023-07-31 2023-11-10 华南理工大学 Application of cytochrome P450 enzyme and cytochrome P450 reductase combined in synthesis of ursodeoxycholic acid

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