CN111500498A

CN111500498A - Novel mycobacterium aurum and application thereof

Info

Publication number: CN111500498A
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: 2020-08-07
Anticipated expiration: 2040-04-30
Also published as: CN111500498B

Abstract

The invention provides a new mycobacterium aureum MN HI L-4 for converting phytosterol into HI L, wherein the preservation number of the new mycobacterium aureum is CGMCC No. 17948. the invention also provides a seed culture of the new mycobacterium aureum MN HI L-4. the invention provides a method for preparing HI L, the method comprises the step of converting the phytosterol into HI L by using the new mycobacterium aureum MN HI L-4 or the seed culture thereof, the strain takes the phytosterol as a substrate, the weight yield of the converted HI L is 53.83% (g/g%) phytosterol (pure), the yield of HI L is 7.88 times of the yield of the disclosed strain HI L, the HI L accounts for 99.29% in fermentation broth, the products contain no common byproducts HIP and HK, and the yield of the byproducts is greatly lower than that of the disclosed strain.

Description

Novel mycobacterium aurum and application thereof

Technical Field

The invention belongs to the field of bioengineering, and relates to a new mycobacterium aureofaciens and application thereof in preparing a steroid compound intermediate HI L.

Background

Steroid hormone drugs are important in the field of Chinese medicine and are widely used clinically. However, the steroid compound has complex structure, many synthesis steps, complex production process, high pollution and low yield, so that the cost is high and the economic benefit is weak. In recent years, some microorganisms such as nocardia, pseudomonas, mycobacterium, arthrobacter and the like can grow by taking sterol as a unique carbon source, and then certain strains in bacteria, actinomycetes, yeast and mould can transform specific sites of steroid compounds, and the reaction specificity shows great advantages. Therefore, the production of steroid drug intermediates by microbial degradation of sterols has gradually become the mainstream of the production of steroid hormone drugs in the modern pharmaceutical industry, and occupies an extremely important position.

The degradation of steroid substances in the bacteria is aerobic degradation, taking mycobacteria to degrade cholesterol, the degradation process is that cholesterol (I) is converted into cholest-4-en-3-one (cholest-4-en-3-one), the side chain degradation is carried out simultaneously with the first oxidation, the process is similar to fatty acid β -oxidation, under the catalysis of a series of enzymes, an important steroid hormone precursor androst-4-ene-3,17-dione (II, androst-4-ene-3,17-dione (AD) is generated, and the degradation of the central ring is that AD is split into 2-hydroxy-2, 4-dienoic acid (III, 2-hydroxyoxa-2, 4-dienoic acid) and 9,17-dioxo-1,2,3,4,10, 19-hexa-oxohexanoic acid (III, 2-hydroxyoxa-2-563-2, 4-dienoic acid) under the action of a series of enzymes, and the energy of AD is further degraded into 2,3,4, 19-hexa-hexahydro-5-ene-3-one (III, 19-2-hydroxy-2, 3-oxo-2, 19-2-diol (III, 2-hydroxy-2, 3-2-oxo-5-one) and further degraded into hydroxy-2-hydroxy-2, 3-oxo-2-hydroxy-2, 3-one (HIP-2-one), and finally, 3-hydroxy-2-hydroxy-2-hydroxy-2-hydroxy-2-5-4-hydroxy-lactone (III, 4-hydroxy₂And H₂O。

Wherein HI L is lactone, also called A-ring degradation product and the like, CAS number 64053-02-7, which is important medicine or intermediate for synthesizing estrone, estradiol and derivatives thereof and the like.

In the prior art, reports exist for directly preparing HI L by degrading phytosterol by using microorganisms, and particularly, although HI L can be accumulated by degrading phytosterol in NRR L B-8128 disclosed in US4,042,459 and NK-XHX-103 disclosed in CN103756940A, a large amount of byproducts are generated at the same time, the HI L yield is low, the weight yield is 8.05-39%, the production cost is high, and the industrialization is difficult.

Therefore, there is a need for a low cost, simple HI L production process.

Disclosure of Invention

The invention also provides a method for preparing HI L based on the novel mycobacterium aurum, compared with the prior method, the method for preparing HI L by using the novel mycobacterium aurum has the advantages of high HI L yield, less byproducts and no environmental pollution, and in addition, the strain MN HI L-4 has the characteristics of high yield and stable properties, and the performance of the strain is kept stable after 6 months of passage.

According to the novel mycobacterium aurum mutant strain provided by the invention, phytosterol can be converted into HI L, and byproducts such as HIP, HK and the like can be reduced.

Herein, HIP, HI L and HK are, unless otherwise specified, compounds shown below, respectively:

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

HI L, also known as lactone, is 3a α -H-4 α - (3' -propionic acid) -5 α -hydroxy-7a β -methylhexahydro-1-methanone-lactone, represented by formula V below:

HK, 3a α -H-4 α - (3' -propanol) -7a β -methylhexahydro-1, 5-imprinted dione hemiketal, represented by the following formula VI:

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

in one aspect, the present invention provides a Mycobacterium neogold (m) MN HI L-4 with a accession number of CGMCC No.17948 for converting phytosterols to HI L.

The strain is preserved in China general microbiological culture Collection center (address: West Lu No.1 of the North Chen West Lu of the sunward region, Beijing, China academy of sciences, microbiological research institute, postal code 100101) in 2019, and is classified and named as Mycobacterium neoaurum MN HI L-4 with the preservation number of CGMCC No. 17948.

The strain has the following properties:

1. morphological characteristics of colonies:

the bacterial strain is cultured and grown on a nutrient solid culture medium at the temperature of 32 ℃, and after 3-5 days, a golden yellow bacterial colony with the diameter of about 3-10 mm is obtained, and the bacterial colony is circular and has a smooth surface.

2. The morphological characteristics of the strain are as follows:

the strain of the invention presents a round rod shape under a microscope and is consistent with the shape under a microscope of mycobacteria.

3. Physiological and biochemical properties:

the culture temperature of the strain is 28-32 ℃, the optimal growth temperature is 32 ℃, and the strain can grow well under the condition that the pH value is 7.0-7.6.

4. The nutrition characteristics are as follows:

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

In another aspect, the present invention provides a method for preparing the strain, the method comprising mutagenizing M.neoformans using nitrosoguanidine.

Specifically, the method comprises the step of mutagenizing a new mycobacterium aurum strain MNPJ-1 (the preservation number is CGMCC No.14181) by using nitrosoguanidine to obtain the strain.

In a further aspect, the present invention provides a seed culture of M.aureofaciens MN HI L-4 as described above.

Wherein the seed culture is prepared by culturing Mycobacterium vaccae MN HI L-4 and is a seed culture of Mycobacterium vaccae MN HI L-4.

In a further aspect, the present invention provides a method for preparing a seed culture of M.aureofaciens MN HI L-4, said method comprising the steps of:

inoculating the new mycobacterium aureum MN HI L-4 into a seed culture medium, carrying out shake culture for 36-60 hours, and collecting fermentation liquor to obtain a seed culture;

wherein the seed culture medium comprises the following components:

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

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

Preferably, the seed culture medium is prepared by mixing the components with water, adjusting pH, sterilizing with high pressure steam at 121 deg.C for 30min, and cooling.

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

inoculating Mycobacterium vaccae MN HI L-4 (such as strain preserved in 1m L frozen glycerol tube) into seed culture medium, culturing at 28-32 deg.C, preferably 32 deg.C, under shaking at 200-250 rpm, preferably 220 rpm for 36-60 hr, preferably 48 hr, and collecting fermentation broth to obtain seed culture.

The novel mycobacterium aureofaciens MN HI L-4 or the seed culture provided by the invention can be used for preparing HI L.

In a further aspect, the invention provides the use of new mycobacterium aurum MN HI L-4 or a seed culture thereof in the preparation of HI L.

In a further aspect, the present invention provides a method for preparing HI L, the method comprising converting phytosterols into HI L using mycobacterium aureofaciens MN HI L-4 or a seed culture thereof of the present invention.

Wherein, the phytosterol is a mixture of a plurality of sterols extracted from various plants, mainly comprises sitosterol, sitostanol, stigmasterol, campesterol, brassicasterol, etc., and can be directly purchased.

Preferably, the method comprises the steps of:

1) inoculating Mycobacterium aureum MN HI L-4 or its seed culture into transformation medium containing phytosterol, and fermenting;

2) extracting HI L from the fermentation broth obtained in step 1).

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

soybean peptone 1-5 g/L, yeast extract 0.5-2.0 g/L, glucose 0.5-2.0 g/L, citric acid 0.1-0.3 g/L, ferric ammonium citrate 0.01-0.05 g/L, K₂HPO₄0.5～1.5g/L，MgSO₄·7H₂O0.01～0.1g/L，NH₄NO₃0.1-0.5 g/L, 5.0-8.0 g/L (pure) phytosterol, 801-5 g/L tween and 7.0-7.6 of pH;

preferably, the transformation medium comprises the following components:

3 g/L of soybean peptone, 1.0 g/L of yeast extract, 1.0 g/L of glucose, 0.2 g/L of citric acid, 0.01 g/L of ferric ammonium citrate and K₂HPO₄1.0g/L，MgSO₄·7H₂O 0.05g/L，NH₄NO₃0.3 g/L, phytosterol 6.0 g/L (pure form), Tween 802 g/L, pH 7.2.

As will be understood by those skilled in the art, the phytosterol in the transformation medium of the present invention may be replaced by a phytosterol extract comprising phytosterol, and accordingly, the phytosterol content in the transformation medium after purification is 5.0-8.0 g/L.

In the preparation of the transformation medium, water with the temperature of 50 ℃ is firstly used for preparing Tween 80 solution, 5.0-8.0 g/L phytosterol (purified) or corresponding phytosterol extract is added while stirring, other components are added, the pH is adjusted, high-pressure steam sterilization is carried out, the shake flask sterilization condition is that sterilization is carried out for 30 minutes at the temperature of 121 ℃, and the ingredients are cooled and shaken up for use.

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

The method of the invention, wherein in the step 1), the fermentation culture is shake flask shaking culture;

preferably, in step 1), the phytosterol is 6 g/L (pure) in the transformation medium for shaking flask shaking culture;

more preferably, in the step 1), the shaking culture is carried out at 28-32 ℃, preferably 32 ℃ for 5-13 days at 200-250 rpm, preferably 220 rpm.

The method according to the invention, wherein the step 2) is realized by a method comprising the following steps:

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

More preferably, the method further comprises:

filtering the collected organic phase, distilling and concentrating under normal pressure to 1/10-1/6, preferably 1/8 of the original volume of the organic phase, adding n-hexane with the volume being 0.2-0.6 times, preferably 0.4 times, cooling to 12-14 ℃, growing crystals, filtering, washing and drying to obtain HI L.

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

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

taking the fermentation liquid fermented and cultured in the step 1), and analyzing by gas chromatography to obtain the product.

Preferably, a 200 μ L sample of the fermentation liquid obtained in step 1) is taken, acidified to pH 2 with HCl, extracted with 4 times volume of ethyl acetate, vortexed and shaken for 2min, 12000rpm, centrifuged for 10min, the supernatant is collected, dried with nitrogen, the obtained product is dissolved with ethyl acetate to prepare a solution of 1mg/m L, and the solution is filtered through an organic membrane of 0.22 μ L to remove impurities, and the filtrate is detected by gas chromatography.

The gas chromatography conditions include:

the chromatographic column is Agilent HP-5; the detector is an FID detector; the sample introduction amount is 1 mu l, and the sample introduction port temperature is 240 ℃; the temperature of the detector is 280 ℃;

adopting temperature programming: initial temperature of 180 ℃, 7min, heating rate of 30 ℃ and min^-1The temperature is raised to 240 ℃ for 6 min.

The preservation number of the strain is CGMCC No.17948, the strain uses phytosterol as a substrate, the mutant strain is utilized to realize high-efficiency transformation of HI L for the first time, the yield of HI L in a shake flask fermentation liquid is 53.83 percent (g/g percent) phytosterol (pure), the yield is 14.83-45.78 percent higher than that of the existing strain (CN, 103756940B, NK-XHX-103) HI L, the proportion of HI L in the fermentation liquid product accounts for 99.29 percent of the total product, the product has no common HIP byproducts and HK, and the yield of the byproducts is greatly lower than that of the disclosed strain.

The beneficial effects of the invention include:

1. the new mycobacterium aureofaciens capable of highly producing HI L by taking phytosterol as a substrate is obtained by screening, the high-efficiency conversion of HI L is realized for the first time by utilizing the new mycobacterium aureofaciens, the HI L weight yield of shake flask fermentation liquor is 53.83 percent (g/g percent) phytosterol (pure), the HI L yield is 7.88 times of the HI L yield of the disclosed strain (NRR L B-8128), and the HI L in the fermentation liquor product accounts for 99.29 percent of the total products;

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

3. the new mycobacterium aurum has the characteristic of stable high-yield character, and the strain is passaged for 6 months, so that the performance is kept stable;

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

Drawings

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

FIG. 1A is a gas phase diagram of a fermentation product of shake flask fermentation production of HI L using control strain NRR L B-8128;

FIG. 1B is a gas phase diagram of the product of the shake flask fermentation production of HI L using M.aureofaciens MN HI L-4 of the present invention;

FIG. 2 is a gas phase spectrum of HIP, HI L and HK standards, wherein FIG. 2A is a detection result of HIP standard with retention time of 5.205min, FIG. 2B is a detection result of HI L standard with retention time of 6.440min, and FIG. 2C is a detection result of HK standard with retention time of 3.321 min.

FIG. 3 shows the metabolic pathway of cholesterol in mycobacteria.

Preservation of biological materials

The new Mycobacterium aurum MN HI L-4 is classified and named as new Mycobacterium aurum (Mycobacterium neoaurum) which is preserved in China general microbiological culture Collection center (CGMCC) in 2019 at 17.06.7.A preservation unit address is No. 3 of Beijing university Chen Xilu No.1 of the sunward district, and the preservation number is CGMCC No.17948 at the institute of microbiology of China academy of sciences.

Detailed Description

The invention is illustrated below with reference to specific examples. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

The experimental procedures in the following examples are conventional unless otherwise specified. The strains, plasmids, kits and the like used in the following examples are all commercially available products unless otherwise specified.

The Mycobacterium neo-gold MN HI L-4 used in the following examples was deposited in the general microbiological center of China Committee for culture Collection of microorganisms (address: Beijing, West Luo No.1, North Chen West Lu, institute of microbiology, China academy of sciences, postal code 100101) at 17.06.2019, and was classified and named as Mycobacterium neo-aurum MNHI L-4, CGMCC No. 17948.

The novel Mycobacterium aurum NRR L B-8128 used in the examples described below was obtained from the American Agricultural Research Culture Collection.

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

taking a 200 mu L fermentation liquid sample, acidifying with HCl to pH 2, extracting with 4 times volume of ethyl acetate, vortexing and shaking for 2min at 12000rpm, centrifuging for 10min, collecting supernatant, drying with nitrogen, dissolving the obtained product with ethyl acetate to prepare a solution of 1mg/m L, filtering through an organic membrane of 0.22 mu L to remove impurities, and detecting the filtrate by gas chromatography.

The preparation method of solid sample comprises dissolving the obtained product with ethyl acetate, preparing 1mg/m L solution, filtering with 0.22 μm organic membrane to remove impurities, and analyzing the content of the product with gas chromatography;

gas chromatography assay:

taking a sample of 1 mu L, injecting the sample into a gas chromatograph, recording a chromatogram, taking 25mg of HIP, HI L and HK samples, precisely weighing, placing the samples in a 25ml volumetric flask, adding ethyl acetate to dissolve and dilute the samples to scale, shaking up the samples to be used as reference solution, measuring by the same method, and calculating the concentrations of HIP, HI L and HK in the test sample by peak areas according to an external standard method.

Calculating the formula:

ax is the area of the peak of HI L in the test sample

Ar is the peak area of HI L in the control

Cr is the concentration of HI L in the control (mg/m L)

HIP and HK were calculated in the same manner as HI L.

Sample introduction requirements are as follows: the samples for content determination, single sample double needle, parallel determination.

Gas chromatography measurement conditions:

the chromatographic column is Agilent HP-5;

the detector is an FID detector;

the sample introduction amount is 1 mu L, and the sample introduction port temperature is 240 ℃;

the temperature of the detector is 280 ℃;

the split ratio is as follows: 20: 1;

carrier gas: high purity N₂The flow rate is 3.0m L/min;

adopting temperature programming: the initial temperature is 180 ℃, the time is 7min,the heating rate is 30 ℃ and min^-1The temperature is raised to 240 ℃ for 6 min.

The peak time of each substance was compared with that of HIP, HI L and HK standards to detect the production of the product, HI L was purchased from baodingjifu biotechnology limited, and HIP and HK were obtained by laboratory separation, HI L retention time T5.205 min, HI L retention time T6.440 min, and HK retention time T3.321 min, as shown in fig. 2A, 2B and 2C.

In the following examples, a phytosterol extract having a phytosterol content of 85% was used.

Example 1: preparation of the strains of the invention

The new mycobacterium aureofaciens MNPJ-1 (the preservation number is CGMCC No.14181, obtained from the common microorganism center of China Committee for culture Collection of microorganisms in the preservation organization) is used as an initial strain, and the new mycobacterium aureofaciens is prepared by adopting the following method:

1) weighing 6mg nitrosoguanidine (NTG for short, purchased from Sigma) in a sterile centrifuge tube, adding 0.05m L acetone for dissolution assistance, and adding 0.2mM phosphate buffer solution with pH 6.0, 1m L for complete dissolution;

the new Mycobacterium aureofaciens MNPJ-1 is prepared to have a concentration of 10^8-9Cell/m L bacterial suspension, 5m L bacterial suspension and the nitrosoguanidine solution;

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

3) collecting thallus by centrifugation, washing thallus twice with 5m L above phosphate buffer solution to terminate mutagenesis of NTG, adding 5m L sterile physiological saline into centrifuge tube, and shaking;

4) diluting the mutagenized bacterial suspension by 10 times, coating the diluted bacterial suspension on a nutrient agar culture medium plate, and culturing for 4-6 days to obtain a single colony;

5) a single colony is picked to carry out a transformation test by taking phytosterol as a substrate, and the mutant strain of the invention, namely the new mycobacterium aureum MN HI L-4 is screened from the single colony.

Example 2: new mycobacterium aurum MN HI L-4 seed culture and passage

The seed culture medium comprises beef extract 0.3 g/L, peptone 1.0 g/L, yeast powder 0.3 g/L, glycerol 1.5 g/L and tween 2.0 g/L, wherein after the components are prepared with water, the pH is adjusted to 7.2, and the beef extract is sterilized for 30 minutes at 121 ℃ by high-pressure steam and used after being cooled.

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

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

1. morphological characteristics of colonies:

the bacterial strain is cultured and grown on a nutrient solid culture medium at 28 ℃, and a golden yellow bacterial colony with the diameter of about 3-10 mm is obtained after 3-5 days, and the bacterial colony is circular and has a smooth surface.

2. The morphological characteristics of the strain are as follows:

3. Physiological and biochemical properties:

Example 3: shaking flask shaking culture of new mycobacterium aurum MN HI L-4 preparation of HI L

Firstly, seed culture:

seed cultures of the novel Mycobacterium aureum MN HI L-4 of the invention and of the control strain NRR L B-8128, respectively, were prepared as described in example 2.

(II) transformation culture:

the transformation medium comprises soybean peptone 3 g/L, yeast extract 1 g/L, glucose 1.0 g/L, citric acid 0.2 g/L, ferric ammonium citrate 0.01 g/L, and K₂HPO₄1.0g/L，MgSO₄·7H₂O 0.05g/L，NH₄NO₃0.3 g/L, Tween 802.0 g/L, phytosterol 6.0 g/L (after 85% of external standard content is converted to pure), pH 7.2, when prepared, the composition is prepared byPreparing Tween 80 solution with 50 deg.C water, adding phytosterol extract under stirring, adding the above components, adding water to make up volume, adjusting pH to 7.2, subpackaging and shaking to 100ml/500ml, sterilizing with high pressure steam at 121 deg.C for 30min, and cooling.

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

In addition, the product accumulation in the fermentation broths of the two strains was compared and the analytical results are shown in Table 1.

TABLE 1 comparison of the accumulation of the major products of the strains of the invention with those of the control strains

Note: 1. the yield is the product g/g phytosterol (pure); the numerical values are rounded off.

2. The table only calculates the yield ratio of the common products HI L, HIP and HK.

As a result, the fermentation broth of the control strain NRR L B-8128 can produce HI L.41 g/L, and can accumulate a large amount of byproducts HIP and HK in addition to HI L, wherein the proportion of HI L in the product is only 18.64%.

The results prove that the HI L produced by fermenting the new mycobacterium aurum MN HI L-4 strain has obvious advantages compared with the method of the U.S. patent US4,062,729.

The results of gas chromatography for the flask fermentation of NRR L B-8128 are shown in FIG. 1A, and the results of the flask fermentation of MN HI L-4 are shown in FIG. 1B.

Example 4 extraction of HI L

And (4) harvesting fermentation liquor after transformation culture, centrifuging at 8000rpm for 10min, and separating to obtain supernatant. Then, the mixture is filtered by using a 5-micron filter, and the filtrate is put into an extraction tank. And adjusting the pH of the filtrate to 2-3 by using sulfuric acid, adding 0.4 volume of dichloromethane, fully mixing, standing for layering, and collecting a lower organic phase. Filtering the organic phase by using a 5-micron filter, distilling the organic phase to 1/8 of the original volume of the organic phase under normal pressure, and concentrating the organic phase until the feed liquid is viscous and the wall built-up is obvious.

Adding 0.4 times volume of n-hexane into the concentrated solution, gradually cooling to 12-14 deg.C, and growing crystal for 1 hr. The crystals were filtered and washed with n-hexane. Drying the crystal, conveying the sample for detection, and recrystallizing the unqualified product.

The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various changes and modifications according to the present invention without departing from the spirit of the present invention, which is defined by the scope of the appended claims.

Claims

1. A Mycobacterium neogold (MNHI L-4) for converting phytosterol into HI L, having a accession number of CGMCC No. 17948.

2. A seed culture of Mycobacterium aurum MN HI L-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 new mycobacterium aureum MN HI L-4 according to claim 1 to a seed culture medium, performing shake culture for 36-60 hours, and collecting fermentation liquor to obtain a seed culture;

wherein the seed culture medium comprises the following components:

4. The method of claim 3, wherein the seed culture is obtained by a method comprising:

inoculating the new mycobacterium aureum MN HI L-4 into a seed culture medium, culturing at 28-32 ℃, preferably at 32 ℃ under shaking at 200-250 rpm, preferably 220 rpm for 36-60 hours, preferably 48 hours, and collecting fermentation liquor to obtain a seed culture.

5. Use of mycobacterium aureofaciens MN HI L-4 according to claim 1 or a seed culture of mycobacterium aureofaciens MN HI L-4 according to claim 2 for the preparation of HI L.

6. A method for preparing HI L, the method comprising converting phytosterols into HI L using a seed culture of mycobacterium neoaurum MNHI L-4 according to claim 1 or mycobacterium neoaurum MN HI L-4 according to claim 2.

7. The method according to claim 6, wherein the method comprises the steps of:

2) extracting HI L from the fermentation broth obtained in step 1).

8. The method of claim 7, wherein in step 1), the transformation medium comprises the following components:

soybean peptone 1-5 g/L, yeast extract 0.5-2 g/L, glucose 0.5-2 g/L, citric acid 0.1-0.3 g/L, ferric ammonium citrate 0.01-0.05 g/L, K₂HPO₄0.5～1.5g/L，MgSO₄·7H₂O 0.01～0.1g/L，NH₄NO₃0.1-0.5 g/L, 5.0-8.0 g/L (pure) phytosterol, 801-5 g/L tween and 7.0-7.6 of pH;

preferably, the transformation medium comprises the following components:

3 g/L of soybean peptone, 1 g/L of yeast extract, 1.0 g/L of glucose, 0.2 g/L of citric acid, 0.01 g/L of ferric ammonium citrate and K₂HPO₄1.0g/L，MgSO₄·7H₂O 0.05g/L，NH₄NO₃0.3 g/L, phytosterol 6.0 g/L (pure form), Tween 802 g/L, pH 7.2.

9. The method according to claim 7, wherein in step 1), the seed culture is inoculated in the transformation medium in an inoculum size of 3-10%, preferably 4-6%, further preferably 5%, relative to the volume of the transformation medium;

preferably, in step 1), the fermentation culture is shake flask shaking culture;

10. The method of claim 7, wherein the step 2) is achieved by a method comprising the steps of:

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

preferably, the method further comprises:

filtering the collected organic phase, distilling and concentrating the organic phase at normal pressure until the volume of the organic phase is 1/10-1/6, preferably 1/8, adding n-hexane with the volume of 0.2-0.6 times, preferably 0.4 times, cooling to 12-14 ℃, growing crystals, filtering, washing and drying to obtain HI L;

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