CN111500498B

CN111500498B - Mycobacterium neogold and application thereof

Info

Publication number: CN111500498B
Application number: CN202010360558.6A
Authority: CN
Inventors: 常慧
Original assignee: Shenyang Botai Biopharmaceutical Co ltd
Current assignee: Shenyang Botai Biopharmaceutical Co ltd
Priority date: 2020-04-30
Filing date: 2020-04-30
Publication date: 2023-10-20
Anticipated expiration: 2040-04-30
Also published as: CN111500498A

Abstract

The application provides a novel Mycobacterium aurum and application thereof. The application provides a novel Mycobacterium aurum MN HIL-4 for converting phytosterol into HIL, wherein the preservation number of the novel Mycobacterium aurum is CGMCC No.17948. The application also provides a seed culture of the novel Mycobacterium aurum MN HIL-4. The present application provides a method for preparing HIL, which comprises converting phytosterols into HIL using the novel Mycobacterium aurum MN HIL-4 or seed culture thereof of the present application. The strain takes the phytosterol as a substrate, the weight yield of converted HIL is 53.83% (g/g%) of the phytosterol (fold purity), and the yield of the HIL is 7.88 times that of the disclosed strain. The plant sterol is taken as a substrate, the HIL product in the fermentation broth accounts for 99.29 percent, the product has no common byproducts HIP and HK, and the yield of the byproducts is greatly lower than that of the disclosed strain.

Description

Mycobacterium neogold and application thereof

Technical Field

The application belongs to the field of bioengineering, and relates to a novel Mycobacterium aurum and application thereof in preparation of steroid intermediate HIL.

Background

Steroid hormone drugs are important categories in the field of Chinese medicine and are widely used clinically. However, because the steroid compound has complex structure, more synthesis steps, complex production process, large pollution and low yield, the cost is very high, and the economic benefit is weak. In recent years, it has been found that some microorganisms such as nocardia, pseudomonas, mycobacterium, arthrobacter and the like can grow on sterols as the only carbon source, and then it has been found that certain strains of bacteria, actinomycetes, yeasts and molds can transform specific sites of steroid compounds, and the reaction specificity of the microorganisms shows great advantages. Therefore, the microbial degradation of sterols to produce steroid drug intermediates has become the mainstream of the modern pharmaceutical industry to produce steroid hormone drugs, and takes up an extremely important position.

The degradation of steroid substances in the bacteria is aerobic degradation, taking mycobacteria for degrading cholesterol as an example, the degradation process is as follows: 1. conversion of cholesterol (I) to cholest-4-en-3-one (cholest-4-en-3-one); 2. side chain degradation: simultaneously with the oxidation of the first step, the process is similar to beta-oxidation of fatty acid, and under the catalysis of a series of enzymes, an important steroid hormone precursor namely androstane-4-alkene-3, 17-dione (II, android-4-ene-3, 17-dione, abbreviated as AD) is generated; 3.degradation of the central ring: AD is split into 2-hydroxy-2, 4-dienoic acid (III, 2-hydroxyhexa-2,4-dienoic acid) and 9,17-dioxo-1,2,3,4,10,19-hexa-androstane-5-carboxylic acid (IV, 9, 17-diox-1,2,3,4,10,19-hexanordrostan-5-oid acid, HIP) under the action of a series of enzymes; 4. downstream degradation pathway: the 2-hydroxy-2, 4-dienoic acid is gradually decomposed by a series of enzymes, the energy enters into tricarboxylic acid cycle, and HIP is converted into 3a alpha-H-4 alpha- (3 '-propionic acid) -5 alpha-hydroxy-7 a beta-methyl hexahydro-1-seal ketone-delta-lactone (V, 3a alpha-H-4 alpha- (3' -propionic acid) -5 alpha-hydroxy-7 a beta-methyl hexahydro-1-indanone-delta-lactone, short for HIL) which is further completely degraded into CO ₂ And H ₂ O。

Wherein HIL is delta-lactone of the application, also called A ring degradation product and the like, CAS number: 64053-02-7 is an important drug or intermediate for synthesizing estrone, estradiol, derivatives thereof and the like.

In the prior art, there is a report of directly preparing HIL by degrading phytosterol by utilizing microorganism, specifically, NRRL B-8128 disclosed in US4,042,459 and NK-XHX-103 disclosed in CN103756940A, while the phytosterol can be degraded to accumulate HIL, a large amount of byproducts are produced at the same time, the HIL yield is lower, the weight yield is 8.05-39%, the production cost is high, and industrialization is difficult.

Therefore, there is a need for a production method of HIL that is low in production cost and simple in process.

Disclosure of Invention

It is therefore an object of the present application to address the deficiencies of the prior art and to provide a novel Mycobacterium aurum mutant. The novel Mycobacterium aurum mutant strain provided by the application can be used for converting a large amount of phytosterol into HIL. The application also provides a method for preparing HIL based on the novel Mycobacterium aurum. Compared with the existing method, the method for preparing the HIL by using the novel mycobacterium aurum has the advantages of high HIL yield, less byproducts and no pollution to the environment. In addition, the strain MN HIL-4 has the characteristics of high yield and stable properties, and the performance of the strain is kept stable after passage for 6 months.

According to the mutant strain of the novel Mycobacterium aurum provided by the application, the phytosterol can be converted into HIL, and byproducts such as HIP, HK and the like can be reduced.

In the present application, HIP, HIL and HK are each the following compounds unless otherwise specified:

HIP is 9,17 dioxo-1,2,3,4,10,19-hexanorandrostane-5-carboxylic acid, represented by the following formula IV:

HIL, also known as delta-lactone, is 3aα -H-4α - (3' -propionic acid) -5α -hydroxy-7aβ -methylhexahydro-1-printed keto-delta-lactone represented by formula V:

HK, 3aα -H-4α - (3' -propanol) -7aβ -methylhexahydro-1, 5-seal dione hemiketal, represented by formula VI:

specifically, the application is realized by the following technical scheme:

in one aspect, the application provides a novel Mycobacterium aurum (Mycobacterium neoaurum) MN HIL-4 for use in converting phytosterols to HIL, the novel Mycobacterium aurum having a accession number of CGMCC No.17948.

The strain has been deposited in 2019, 06 and 17 days with China general microbiological culture Collection center (address: north West Lu No.1, 3, china academy of sciences microbiological study, post code 100101), and is named as Mycobacterium neogold: (Mycobacterium neoaurum MN HIL-4) with a preservation number of CGMCC No.17948.

The strain has the following properties:

1. colony morphology characterization:

the strain of the application is cultivated and grown on a nutrient solid culture medium at 32 ℃ for 3-5 days to obtain golden yellow bacterial colonies with the diameter of about 3-10 mm, and the bacterial colonies are round and have smooth surfaces.

2. Morphological characteristics of the strain:

the strain of the application has a round rod shape under a microscope and is consistent with the form under the mycobacteria.

3. Physiological and biochemical characteristics:

the strain of the application has a culture temperature of 28-32 ℃, an optimal growth temperature of 32 ℃, and better growth under the condition of pH of 7.0-7.6.

4. The nutrition characteristics are as follows:

the strain of the present application is cultured without special nutrients using a basal medium such as a nutrient broth, and is obligately aerobic.

In another aspect, the application provides a method of preparing the strain, the method comprising mutagenizing a novel Mycobacterium aurum using nitrosoguanidine.

Specifically, the method comprises the step of mutagenesis of a new Mycobacterium aurum strain MNPJ-1 (with the preservation number of CGMCC No. 14181) by using nitrosoguanidine.

In a further aspect, the present application provides a seed culture of Mycobacterium vaccae MN HIL-4 as described above.

Wherein the seed culture is prepared by culturing Mycobacterium neogold MN HIL-4, and is the seed culture of Mycobacterium neogold MN HIL-4.

In yet another aspect, the present application provides a method for preparing a seed culture of Mycobacterium neogold MN HIL-4, said method comprising the steps of:

inoculating Mycobacterium neogold MN HIL-4 into a seed culture medium, carrying out shaking culture for 36-60 hours, and collecting fermentation liquor to obtain a seed culture;

wherein the seed medium comprises the following components:

beef extract 0.1-0.5 g/L, peptone 0.5-1.5 g/L, yeast powder 0.2-0.4 g/L, glycerol 1.0-2.0 g/L, tween 1.5-2.5 g/L, and pH 7.0-7.4;

preferably, the seed medium comprises: beef extract 0.3g/L, peptone 1.0g/L, yeast powder 0.3g/L, glycerol 1.5g/L, tween 2.0g/L, and pH 7.2.

Preferably, in the preparation of the seed culture medium, after each component is mixed with water, the pH is adjusted, high-pressure steam is used for sterilization at 121 ℃ for 30 minutes, and the mixture is cooled for use.

More preferably, the seed culture is obtained by a method comprising the steps of:

mycobacterium neogold MN HIL-4 (e.g., a strain stored in 1mL of a cryoglycerol tube) is inoculated into a seed medium, and the fermentation broth is collected by shaking at 28 to 32℃for 36 to 60 hours, preferably 48 hours, at 200 to 250 rpm, preferably 220 rpm, preferably 32℃to obtain a seed culture.

The novel Mycobacterium aurum MN HIL-4 or seed culture provided by the application can be used for preparing HIL.

In a further aspect, the application provides the use of Mycobacterium vaccae MN HIL-4 or seed culture thereof in the preparation of HIL.

In a further aspect, the application provides a method of preparing HIL, the method comprising converting phytosterols to HIL using the novel Mycobacterium aurum MN HIL-4 or seed culture thereof of the application.

Wherein the plant sterol is a mixture of various sterols extracted from various plants, mainly including sitosterol, stigmasterol, campesterol, brassicasterol, etc., and can be directly purchased.

Preferably, the method comprises the steps of:

1) Inoculating Mycobacterium neogold MN HIL-4 or seed culture thereof into a transformation medium containing phytosterol for fermentation culture;

2) Extracting HIL from the fermentation broth obtained in step 1).

The method according to the application, wherein, in step 1), the transformation medium comprises the following components:

1 to 5g/L of soybean peptone, 0.5 to 2.0g/L of yeast extract, 0.5 to 2.0g/L of glucose, 0.1 to 0.3g/L of citric acid, 0.01 to 0.05g/L of ferric ammonium citrate and K ₂ HPO ₄ 0.5～1.5g/L，MgSO ₄ ·7H ₂ O0.01～0.1g/L，NH ₄ NO ₃ 0.1-0.5 g/L, 5.0-8.0 g/L (fold purity) of phytosterol, 1-5 g/L of Tween 80 and 7.0-7.6 of pH;

preferably, the transformation medium comprises the following components:

3g/L soybean peptone, 1.0g/L yeast extract, 1.0g/L glucose, 0.2g/L citric acid, 0.01g/L ferric ammonium citrate, K ₂ HPO ₄ 1.0g/L，MgSO ₄ ·7H ₂ O 0.05g/L，NH ₄ NO ₃ 0.3g/L, 6.0g/L (pure), 2g/L Tween 80 and pH 7.2.

It will be appreciated by those skilled in the art that the phytosterols in the transformation media of the present application may be replaced by a phytosterol extract comprising phytosterols and that the phytosterols may be present in the transformation media in an amount of from 5.0 to 8.0g/L after being purified accordingly.

In the preparation of the transformation culture medium, tween 80 solution is prepared by using water at 50 ℃, 5.0-8.0 g/L of phytosterol (folded purity) or corresponding phytosterol extract is added while stirring, then other components are added, the pH is regulated, and high-pressure steam sterilization is carried out. The shake flask sterilization condition is that the sterilization is carried out for 30 minutes at 121 ℃. Shaking up after cooling.

The method according to the application, wherein, in step 1), the seed culture is inoculated into the transformation medium in an inoculum size of 3 to 10%, preferably 4 to 6%, further preferably 5% by volume relative to the transformation medium.

The method according to the present application, wherein in step 1), the fermentation culture is shake flask shake culture;

preferably, in step 1), 6g/L (reduced purity) of phytosterol is used in the transformation medium for shake flask shaking culture;

more preferably, in step 1), the shake flask shaking culture is performed at 28 to 32℃and preferably at 32℃for 5 to 13 days at 200 to 250 rpm and preferably 220 rpm.

The method according to the application, wherein said step 2) is carried out by a method comprising the steps of:

harvesting the fermentation broth obtained in the fermentation culture in the step 1), centrifuging, filtering the supernatant, regulating the pH value of the filtrate to 2-3, adding 0.2-0.6 times of volume, preferably 0.4 times of volume of dichloromethane, mixing, layering and collecting an organic phase.

More preferably, the method further comprises:

filtering the collected organic phase, concentrating to 1/10-1/6, preferably 1/8 of the original volume of the organic phase by normal pressure distillation, adding 0.2-0.6 times of n-hexane, preferably 0.4 times of n-hexane, cooling to 12-14 ℃, crystallizing, filtering, washing and drying to obtain HIL.

More preferably, the filtration is performed using a 5 μm filter.

The method according to the application further comprises a step of detecting the accumulation of products in the fermentation broth of step 1), in particular comprising the following steps:

and (3) taking the fermentation broth obtained in the fermentation culture in the step (1), and performing gas chromatographic analysis to obtain the product.

Preferably, 200 μl of the fermentation broth sample in step 1) is taken, acidified to ph=2 using HCl, extracted with 4 volumes of ethyl acetate, vortexed for 2min, centrifuged at 12000rpm for 10min, the supernatant collected, dried with nitrogen, the resulting product is dissolved in ethyl acetate to prepare a 1mg/mL solution, and filtered through 0.22 μl of organic membrane to remove impurities, and the filtrate is subjected to gas chromatography for detection.

The gas chromatography conditions include:

the chromatographic column is Agilent HP-5; the detector is an FID detector; the sample injection amount is 1 μl, and the temperature of the sample injection port is 240 ℃; the detector temperature was 280 ℃;

the temperature programming is adopted: initial temperature of 180 ℃,7min and heating rate of 30 ℃ min ^-1 Heating to 240 deg.C for 6min.

According to the application, a new Mycobacterium aurum MN HIL-4 and application thereof are provided, and the preservation number of the strain is CGMCC No.17948. The strain takes the phytosterol as a substrate, the mutant strain is utilized to realize the high-efficiency conversion of HIL for the first time, the weight yield of the shake flask fermentation broth HIL is 53.83% (g/g%) of the phytosterol (folded purity), and the weight yield of the HIL is improved by 14.83-45.78% compared with the prior strain (CN, 103756940B,NK-XHX-103). The ratio of HIL in the fermentation liquor product to the whole product reaches 99.29 percent, the product has no common byproducts HIP and HK, and the yield of the byproducts is greatly lower than that of the disclosed strain.

The beneficial effects of the application include:

1. according to the application, the novel Mycobacterium aurum which can be used for high-yield HIL by taking phytosterol as a substrate is obtained by screening, the high-efficiency conversion of the HIL is realized for the first time by utilizing the novel Mycobacterium aurum, the weight yield of the shake flask fermentation broth HIL is 53.83% (g/g%) of phytosterol (fold purity), the yield of the HIL is 7.88 times of that of the published strain (NRRL B-8128), and the HIL in the fermentation broth product accounts for 99.29% of the total product;

2. the method is a microbial transformation method, has mild reaction conditions and has no pollution to the environment;

3. the novel mycobacterium aurum has the characteristics of stable high-yield character, and the performance of the strain is kept stable after passage for 6 months;

4. the application only needs to use the strain of the application for transformation, and has low production cost, simple process and considerable economic benefit.

Drawings

Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1A is a gas phase diagram of a fermentation product for shake flask fermentation to produce HIL using a control strain NRRL B-8128;

FIG. 1B is a gas phase diagram of a product of shake flask fermentation to produce HIL using Mycobacterium neogold MN HIL-4 of the present application;

FIG. 2 is a gas phase diagram of HIP, HIL and HK standards, wherein FIG. 2A is the detection result of HIP standard with retention time of 5.205min; FIG. 2B shows the detection result of the standard HIL, wherein the retention time is 6.440min; FIG. 2C shows the results of detection of standard HK with a retention time of 3.321min.

FIG. 3 shows the metabolic pathways of cholesterol in Mycobacteria.

Preservation of biological materials

The new Mycobacterium aurum MN HIL-4, classified and named as new Mycobacterium aurum (Mycobacterium neoaurum) is preserved in China general microbiological culture Collection center (CGMCC) of China, and the preservation unit address: the collection number of the microbiological institute of China academy of sciences is CGMCC No.17948, and the North Chen Xili No.1, 3 of the Chaoyang area of Beijing city.

Detailed Description

The application is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are for illustration of the application only and are not intended to limit the scope of the application in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The strains, plasmids, kits, etc., used in the examples described below, unless otherwise specified, were commercially available products.

The Mycobacterium neoformans MN HIL-4 used in the following examples was deposited in the China general microbiological culture Collection center (address: north Chen West Lu No.1, 3, institute of microorganisms, academy of sciences of China, post code 100101) of the China general microbiological culture Collection center (China) at 2019, month 06, and was classified and named as Mycobacterium neoformans (Mycobacterium neoaurum) MN HIL-4, CGMCC No.17948.

The novel Mycobacterium aurum NRRL B-8128 used in the examples described below was obtained from the American type agricultural research collection of bacteria (Agricultural Research Service Culture Collection).

In the examples below, the product accumulation in the fermentation broth was analyzed as follows:

200. Mu.L of a fermentation broth sample was taken, HCl was acidified to pH=2 and extracted with 4 volumes of ethyl acetate, vortexed for 2min, centrifuged at 12000rpm for 10min, the supernatant was collected, dried with nitrogen, the resulting product was dissolved with ethyl acetate to prepare a 1mg/mL solution, and the impurities were removed by filtration through a 0.22. Mu.L organic membrane, and the filtrate was detected by gas chromatography.

The preparation method of the solid sample comprises the following steps: dissolving the obtained product with ethyl acetate to prepare a solution of 1mg/mL, filtering the solution through an organic film of 0.22 mu m to remove impurities, and analyzing the content of the product in the filtrate by using a gas chromatography;

gas chromatography assay:

taking 1 mu L of a sample, injecting the sample into a gas chromatograph, and recording a chromatogram; and (3) taking 25mg of HIP, HIL and HK samples, precisely weighing, placing the HIP, HIL and HK samples into a 25ml volumetric flask, adding ethyl acetate for dissolving and diluting to a scale, shaking uniformly, taking the mixture as a reference substance solution, measuring by the same method, and calculating the concentrations of HIP, HIL and HK in the sample according to an external standard method by using peak areas.

The calculation formula is as follows:

ax is the HIL peak area in the test sample

Ar is HIL peak area in reference substance

Cr is the concentration of HIL in the control (mg/mL)

HIP and HK are calculated in the same manner as HIL.

Sample injection requirements: and (3) measuring the content of the sample, namely, measuring the content of the sample in parallel by a single-sample double-needle.

Gas chromatography measurement conditions:

the chromatographic column is Agilent HP-5;

the detector is an FID detector;

the sample injection amount is 1 mu L, and the temperature of a sample injection port is 240 ℃;

the detector temperature was 280 ℃;

split ratio: 20:1;

carrier gas: high purity N ₂ The flow rate is 3.0mL/min;

The production of the products was tested against HIP, HIL purchased from Baoding Jiufu Biotechnology Co., ltd, and HK standards using different peak times for each material. Peak time of each substance: HIP retention time t= 5.205min; HIL retention time t= 6.440min; HK retention time t= 3.321min, as shown in fig. 2A, 2B and 2C.

In the examples below, a phytosterol extract with a phytosterol content of 85% was used.

Example 1: preparation of the strains of the application

The novel Mycobacterium aurum MNPJ-1 (with the preservation number of CGMCC No.14181 and obtained from the common microorganism center of China Committee for culture Collection of microorganisms) is taken as an initial strain, and the novel Mycobacterium aurum is prepared by the following method:

1) 6mg of nitrosoguanidine (NTG for short, purchased from Sigma) is weighed into a sterile centrifuge tube, added with 0.05mL of acetone for dissolution assistance, and added with 1mL of 0.2mM phosphate buffer solution with pH 6.0 for complete dissolution;

preparing Mycobacterium neogold MNPJ-1 into a concentration of 10 ^8-9 Mixing 5mL of bacterial suspension with the nitrosoguanidine solution;

2) Immediately placing the mixed solution obtained in the step 1) in a water bath at 30 ℃ for oscillating treatment for 10 min-1 h;

3) Collecting thalli by centrifugation, washing thalli twice by using 5mL of phosphate buffer solution to terminate the mutagenesis effect of NTG, and finally adding 5mL of sterile physiological saline into a centrifuge tube and shaking uniformly;

4) Diluting the bacterial suspension subjected to mutagenesis treatment by 10 times, then coating a nutrient agar culture medium plate, and culturing for 4-6 days to obtain single bacterial colonies;

5) And (3) selecting a single colony to perform a transformation test by taking plant sterol as a substrate, and screening the single colony to obtain the mutant strain novel Mycobacterium aurum MN HIL-4.

Example 2: mycobacterium aurum MN Seed culture and passage of HIL-4

The formula of the seed culture medium is as follows: beef extract 0.3g/L, peptone 1.0g/L, yeast powder 0.3g/L, glycerol 1.5g/L, tween 2.0g/L. After each component is prepared with water, the pH is regulated to 7.2, and the mixture is sterilized for 30 minutes by high-pressure steam at 121 ℃ and is used after being cooled.

Inoculating Mycobacterium neogold MN HIL-4 into the seed culture medium, culturing at 32deg.C under shaking at 220 rpm for 48 hr, and collecting fermentation broth to obtain seed culture.

The strain is passaged by a plate culture medium for 6 months, inoculated into a nutrient broth solid culture medium and subjected to activation culture for 48 hours at 32 ℃. The strain morphology after passage is consistent with that of the parent strain, and has the following properties:

1. colony morphology characterization:

the strain of the application is cultivated and grown on a nutrient solid culture medium at 28 ℃ for 3-5 days to obtain golden yellow bacterial colonies with the diameter of about 3-10 mm, and the bacterial colonies are round and have smooth surfaces.

2. Morphological characteristics of the strain:

3. Physiological and biochemical characteristics:

Example 3: shake flask shake culture of Mycobacterium neogold MN Preparation of HIL from HIL-4

Seed culture:

seed cultures of the novel Mycobacterium aurum MN HIL-4 of the present application and the control strain NRRL B-8128 were prepared, respectively, as described in example 2.

(II) transformation culture:

the formula of the transformation medium is as follows: 3g/L soybean peptone, 1g/L yeast extract, 1.0g/L glucose, 0.2g/L citric acid, 0.01g/L ferric ammonium citrate, K ₂ HPO ₄ 1.0g/L，MgSO ₄ ·7H ₂ O 0.05g/L，NH ₄ NO ₃ 0.3g/L Tween 80 2.0g/L, and phytosterol 6.0g/L (after 85% of external standard content is folded), and pH 7.2. When in preparation, tween 80 solution is prepared with 50 ℃ water, the phytosterol extract is added while stirring, the above components are added and the volume is complemented with water, the pH is regulated to 7.2, the mixture is split into 100ml/500ml shake flasks, the high pressure steam is used after sterilization for 30min at 121 ℃, and the mixture is cooled.

The prepared seed culture was inoculated into the above-mentioned transformation medium at an inoculum size of 5%, and shake-cultured at 32℃for 13 days at 220 rpm.

In addition, the results of the analysis of the product accumulation in the fermentation broths of the two strains are shown in Table 1.

TABLE 1 accumulation of major products of the strains of the application compared to the control strain

Note that: 1. yield is g/g phytosterol (reduced purity); the values were rounded off.

2. The table only calculates the yield ratios of the three common products HIL, HIP, HK.

As a result, it was found that the fermentation broth of the control strain NRRL B-8128 produced HIL of 0.41g/L, accumulated a large amount of by-products HIP and HK in addition to HIL, and the proportion of HIL in the product was only 18.64%. The novel Mycobacterium aurum MN HIL-4 can produce HIL 3.23g/L in fermentation liquor, the HIL proportion accounts for 100% of the product, and the proportion of byproducts in the product is far lower than that of the original patent strain. Compared with the original patent strain NRRL B-8128, the new Mycobacterium aurum MN HIL-4 of the strain disclosed by the application has the weight yield of 53.83% (g/g%) of phytosterol (folded purity) which is 7.88 times of the weight yield of a control strain when the strain is used for converting the phytosterol into the HIL as a substrate; the weight yield of HIL is improved by 47.00%, and the proportion of HIL in fermentation liquor is improved by 81.36%.

The results demonstrate that the fermentation production of HIL by using the Mycobacterium neogold MN HIL-4 strain of the present application has significant advantages over the method of U.S. Pat. No. 3,182.

NRRL B-8128 shake flask fermentation gas chromatography results are shown in FIG. 1A, and MN HIL-4 shake flask fermentation results are shown in FIG. 1B.

Example 4: extraction of HIL

And (5) harvesting fermentation liquor after transformation culture, centrifuging at 8000rpm for 10min, and separating to obtain supernatant. Then, the mixture was filtered through a 5 μm filter, and the filtrate was fed into an extraction tank. The pH of the filtrate is regulated to 2-3 by sulfuric acid, dichloromethane with the volume of 0.4 times is added, the mixture is fully mixed, the mixture is kept still for layering, and the lower organic phase is collected. The organic phase is filtered by a 5 mu m filter, distilled to 1/8 of the original volume of the organic phase at normal pressure, and concentrated until the feed liquid is sticky and has obvious wall hanging.

Adding 0.4 times of n-hexane into the concentrated feed liquid, gradually cooling to 12-14 ℃, and growing crystals for 1 hour. The crystals were filtered and washed with n-hexane. And (5) drying the crystals, carrying out sample feeding detection, and recrystallizing the unqualified products.

The above description of the embodiments of the present application is not intended to limit the present application, and those skilled in the art can make various changes or modifications according to the present application without departing from the spirit of the present application, and shall fall within the scope of the appended claims.

Claims

1. A new Mycobacterium aurum (Mycobacterium neoaurum) MN HIL-4 for converting phytosterol into 3a alpha-H-4 alpha- (3' -propionic acid) -5 alpha-hydroxy-7 a beta-methyl hexahydro-1-printed ketone-delta-lactone (HIL for short) has a preservation number of CGMCC No.17948.

2. Seed culture of Mycobacterium neogold MN HIL-4 according to claim 1.

3. A method of preparing a seed culture according to claim 2, the method comprising the steps of:

inoculating the Mycobacterium neogold MN HIL-4 according to claim 1 into a seed culture medium, performing shaking culture for 36-60 hours, and collecting a fermentation broth to obtain a seed culture;

wherein the seed medium comprises the following components:

beef extract 0.1-0.5 g/L, peptone 0.5-1.5 g/L, yeast powder 0.2-0.4 g/L, glycerin 1.0-2.0 g/L, tween 1.5-2.5 g/L, and pH 7.0-7.4.

4. A method according to claim 3, wherein the seed medium comprises: beef extract 0.3g/L, peptone 1.0g/L, yeast powder 0.3g/L, glycerol 1.5g/L, tween 2.0g/L, and pH 7.2.

5. A method according to claim 3, wherein the seed culture is obtained by a method comprising the steps of:

inoculating Mycobacterium neogold MN HIL-4 into a seed culture medium, performing shaking culture at the temperature of 28-32 ℃ for 36-60 hours at the speed of 200-250 rpm, and collecting fermentation liquor to obtain a seed culture.

6. The method according to claim 5, wherein the culture is performed with shaking at 32 ℃.

7. The method of claim 5, wherein the culturing is performed with shaking at 220 rpm.

8. The method according to claim 5, wherein the shaking culture is performed for 48 hours.

9. Use of a mycobacterium neo-gold MN HIL-4 according to claim 1 or a seed culture of a mycobacterium neo-gold MN HIL-4 according to claim 2 in the preparation of HIL.

10. A method of preparing HIL, the method comprising converting phytosterols to HIL using the mycobacterium neogold MN HIL-4 of claim 1 or the seed culture of mycobacterium neogold MN HIL-4 of claim 2.

11. The method according to claim 10, wherein the method comprises the steps of:

2) Extracting HIL from the fermentation broth obtained in step 1).

12. The method of claim 11, wherein in step 1), the transformation medium comprises the following components:

1 to 5g/L of soybean peptone, 0.5 to 2g/L of yeast extract, 0.5 to 2g/L of glucose, 0.1 to 0.3g/L of citric acid, 0.01 to 0.05g/L of ferric ammonium citrate and K ₂ HPO ₄ 0.5～1.5g/L，MgSO ₄ ·7H ₂ O0.01～0.1g/L，NH ₄ NO ₃ 0.1-0.5 g/L, and 5.0-8.0 g of phytosterolL, tween 80 1-5 g/L and pH 7.0-7.6.

13. The method of claim 12, wherein in step 1), the transformation medium comprises the following components:

3g/L soybean peptone, 1g/L yeast extract, 1.0g/L glucose, 0.2g/L citric acid, 0.01g/L ferric ammonium citrate, K ₂ HPO ₄ 1.0g/L，MgSO ₄ ·7H ₂ O 0.05g/L，NH ₄ NO ₃ 0.3g/L, 6.0g/L of phytosterol, 2g/L of Tween 80 and pH 7.2.

14. The method according to claim 11, wherein in step 1), the seed culture is inoculated into the transformation medium in an inoculum size of 3 to 10% by volume relative to the transformation medium.

15. The method according to claim 14, wherein in step 1), the seed culture is inoculated into the transformation medium in an inoculum size of 4 to 6% by volume relative to the transformation medium.

16. The method according to claim 15, wherein in step 1), the seed culture is inoculated in the transformation medium in an inoculum size of 5% by volume relative to the transformation medium.

17. The method according to claim 11, wherein in step 1), the fermentation culture is shake flask shake culture.

18. The method according to claim 17, wherein in step 1), the shake flask cultivation is shake flask cultivation at 28 to 32℃for 5 to 13 days at 200 to 250 rpm.

19. The method according to claim 18, wherein in step 1), shake culture is performed at 32 ℃.

20. The method of claim 18, wherein in step 1), the culture is performed with shaking in a shake flask at 220 rpm.

21. The method of claim 11, wherein said step 2) is accomplished by a method comprising the steps of:

harvesting the fermentation liquor obtained by fermentation culture in the step 1), centrifuging, filtering supernatant, regulating the pH value of filtrate to 2-3, adding 0.2-0.6 times of volume of dichloromethane, mixing, layering and collecting an organic phase.

22. The process of claim 21, wherein 0.4 volumes of dichloromethane are added.

23. The method of claim 21, wherein the method further comprises:

filtering the collected organic phase, concentrating to 1/10-1/6 of the original volume of the organic phase by normal pressure distillation, adding 0.2-0.6 times of n-hexane, cooling to 12-14 ℃, growing crystals, filtering, washing and drying to obtain HIL.

24. The method of claim 23, wherein the atmospheric distillation is concentrated to 1/8 of the original volume of the organic phase.

25. The method of claim 23, wherein 0.4 volumes of n-hexane are added.

26. The method of any one of claims 21 to 25, wherein the filtering is performed using a 5 μm filter.