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Mycobacterium neoaurum and application thereof

A new technology of Mycobacterium aureus and seeds, applied in the field of bioengineering, can solve the problems of industrialization difficulties, low yield of HIL, high production cost, etc.

Active Publication Date: 2020-08-07
SHENYANG BOTAI PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In the prior art, there are reports of using microorganisms to degrade phytosterols to directly prepare HIL, specifically NRRL B-8128 disclosed in US4,042,459 and NK-XHX-103 disclosed in CN103756940A. Although it can degrade phytosterols and accumulate HIL, it also produces A large number of by-products, the yield of HIL is low, the weight yield is 8.05-39%, the production cost is high, and industrialization is difficult

Method used

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  • Mycobacterium neoaurum and application thereof
  • Mycobacterium neoaurum and application thereof
  • Mycobacterium neoaurum and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0087] The preparation method of the solid sample: dissolve the obtained product in ethyl acetate, prepare a 1 mg / mL solution, filter through a 0.22 μm organic membrane to remove impurities, and analyze the content of the product in the filtrate by gas chromatography;

[0088] Gas Chromatography:

[0089] Take 1 μL of the sample, inject it into the gas chromatograph, and record the chromatogram; take another 25 mg of HIP, HIL and HK samples, weigh them accurately, put them in a 25 ml volumetric flask, add ethyl acetate to dissolve and dilute to the mark, shake well, and use it as a reference solution , determined with the same method, and calculated the concentration of HIP, HIL and HK in the test product by the peak area according to the external standard method.

[0090] Calculation formula:

[0091]

[0092] Ax is the HIL peak area in the test product

[0093] Ar is the HIL peak area in the reference substance

[0094] Cr is the concentration of HIL in the reference s...

Embodiment 1

[0107] Embodiment 1: prepare bacterial strain of the present invention

[0108] Using Mycobacterium aureus MNPJ-1 (preservation number is CGMCC No.14181, obtained from the General Microbiology Center of China Microorganism Culture Collection Management Committee of the depository institution) as the starting strain, the following method is used to prepare Mycobacterium aureus of the present invention :

[0109] 1) Weigh 6 mg of nitrosoguanidine (abbreviated as NTG, purchased from Sigma) in a sterile centrifuge tube, add 0.05 mL of acetone to aid dissolution, and add 1 mL of 0.2 mM pH 6.0 phosphate buffer to dissolve completely;

[0110] The new mycobacterium aureus MNPJ-1 is made into a concentration of 10 8-9 1 cell / mL bacterial suspension, mix 5mL bacterial suspension with the above-mentioned nitrosoguanidine solution;

[0111] 2) Immediately place the mixed solution obtained in step 1) in a 30°C water bath and shake for 10 minutes to 1 hour;

[0112] 3) Collect the cel...

Embodiment 2

[0115] Embodiment 2: new mycobacterium aureus MN Seed culture and subculture of HIL-4

[0116] The formula of the seed medium is: beef extract 0.3g / L, peptone 1.0g / L, yeast powder 0.3g / L, glycerol 1.5g / L, Tween 2.0g / L. After preparing each component with water, adjust the pH to 7.2, sterilize with high-pressure steam at 121° C. for 30 minutes, and use it after cooling.

[0117] Mycobacterium neoaureum MN HIL-4 was inoculated on the above seed culture medium, cultured at 32° C. with shaking at 220 rpm for 48 hours, and the fermentation broth was collected to obtain a seed culture.

[0118] The strain was subcultured on the plate medium for 6 months, inoculated on the solid medium of nutrient broth, and activated for 48 hours at 32°C. The morphology of the subcultured strain is consistent with that of the parent strain, and has the following properties:

[0119] 1. Colony Morphological Characteristics:

[0120] The bacterial strain of the present invention is cultured and...

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Abstract

The invention provides mycobacterium neoaurum and an application thereof. The invention provides mycobacterium neoaurum MN HIL-4 used for converting phytosterol into HIL. The preservation number of the mycobacterium neoaurum MN HIL-4 is CGMCC No. 17948. The invention further provides an HIL preparation method. The method comprises converting phytosterol into HIL by using the mycobacterium neoaurumMN HIL-4 or the seed culture of the mycobacterium neoaurum MN HIL-4. According to the strain, phytosterol is used as a substrate, the weight yield of converted HIL is 53.83% (g / g%) phytosterol (pure), and the yield of HIL is 7.88 times that of a disclosed strain HIL. According to the invention, phytosterol is used as a substrate, the HIL product in the fermentation broth accounts for 99.29%, common byproducts HIP and HK do not exist in the product, and the yield of the byproducts is greatly lower than that of the disclosed strain.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a new mycobacterium aureus and its application in preparing steroid intermediate HIL. Background technique [0002] Steroid hormone drugs are an important category in the field of medicine in my country and are widely used clinically. However, due to the complex structure of steroidal compounds, many synthesis steps, complicated production process, heavy pollution and low yield, the cost is high and the economic benefits are weak. In recent years, it has been found that some microorganisms, such as Nocardia, Pseudomonas, Mycobacterium, Arthrobacter, etc., can grow with sterol as the only carbon source, and then some of the bacteria, actinomycetes, yeast, and mold The strain can transform specific parts of steroidal compounds, and its response specificity shows a great advantage. Therefore, the production of steroid drug intermediates by microbial degradation of sterols has gradually...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P17/06C12R1/32
CPCC12N1/20C12P17/06C12R2001/32C12N1/205
Inventor 常慧
Owner SHENYANG BOTAI PHARM CO LTD
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