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Autotrophic pseudonocardia and application thereof

A technology of pseudonocardia and strains, applied in the field of microorganisms, can solve the problems of low vitamin D3 concentration, unfavorable, and increase the production of calcifediol, etc.

Pending Publication Date: 2021-12-03
DONGGUAN HEC GENERIC DRUG R&D CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the concentration of the substrate vitamin D3 added in the existing method is low. In the method disclosed in the patent US5474923, the maximum concentration of added vitamin D3 is only 1000ug / mL, which is unfavorable for increasing the dosage of vitamin D3 to improve calcifediol output

Method used

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  • Autotrophic pseudonocardia and application thereof
  • Autotrophic pseudonocardia and application thereof
  • Autotrophic pseudonocardia and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1 Combination and screening of ultraviolet mutagenesis and ribosome engineering mutagenesis of Pseudonocardia autotrophica (preservation number CGMCC 4.1211, name: Pseudonocardia autotrophica)

[0086] According to the method of experiment 1, obtain the spore suspension of autotrophic pseudonocardia (preservation number CGMCC 4.1211, name: Pseudonocardia autotrophica); get 5mL spore concentration to be 10 9 spore suspension per mL, added to a sterile In the petri dish, irradiate for 60s under ultraviolet light (UV254, 19W) at 30cm while stirring. Use the irradiated spore suspension, press 100 μL / block, respectively inoculate to the corresponding concentration (1 times the MIC, the MIC of Gen is 5 mg / L, the MIC of SS is 5 mg / L, and the MIC of Rif is 0.1 mg / L). On the Gaoshi No. 1 plate of antibiotics, 20 plates were inoculated in parallel for each antibiotic concentration, and 10 plates without antibiotics (as a reference group) were inoculated at the same time...

Embodiment 2

[0094] Embodiment 2: Optimization and improvement of fermentation medium

[0095] Based on the original fermentation medium of Pseudonocardia autotrophica (preservation number CGMCC4.1211, name: Pseudonocardia autotrophica), optimization and improvement are carried out.

[0096] In order to optimize and improve the carbon source, glucose, fructose, mannitol, glycerin, maltose, dextrin, corn flour, corn steep liquor, soluble starch, galactose, lactose, sorbitol, xylose and sucrose were used as the initial carbon source respectively. And evaluate the conversion rate of calcifediol under each condition. The results show that when sucrose and glycerol are used as a single carbon source, the conversion rate can be improved. Considering that the price of glycerol is about 2 times that of sucrose, sucrose is used as the first available carbon source of the fermentation medium. After multiple optimization, corn Syrup is determined as the second long-term carbon source; for the connec...

Embodiment 3

[0110] Embodiment 3: Determination of fermentation, reaction time and reaction system

[0111] Both the substrate vitamin D3 and the product calcifediol are fat-soluble and inhibit the growth of microorganisms. Therefore, separating the bacteria, substrate and product, and minimizing contact can reduce toxicity, which is beneficial to the production of the product.

[0112] Investigated different fermentation time and reaction time, found that 5 days of fermentation and 4 days of reaction, the yield of the target product was relatively highest, and the optimal combination was determined to be 5 days of fermentation and 4 days of reaction.

[0113] Investigate the reaction system, compare the reaction system of n-hexane, n-heptane, cyclohexane, ethanol, ethyl acetate, isopropanol, soybean oil, peanut oil, or corn oil with the fermentation medium, the results show that n-heptane and The reaction system of the fermentation medium is more conducive to the generation of the target ...

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Abstract

The invention relates to autotrophic pseudonocardia and application thereof, and belongs to the technical field of microorganisms. The pseudonocardia autotrophicus can be used for preparing calcifediol and / or calcitriol, compared with an original strain, the yield is remarkably improved.

Description

technical field [0001] The invention relates to the technical field of microorganisms, in particular to an autotrophic pseudonocardia and its application. Background technique [0002] Pseudonocardiaceae are a group of evolutionarily related, morphologically diverse, peptidoglycan containing Meso-DAP (meso-diaminopimelic acid), galactose and arabinose in the whole cell hydrolyzate, no branch Bacteric acid actinomycetes. Many members of this family can ferment to produce important biologically active substances, including antibiotics, enzymes, vitamins, etc. At the same time, there are very few pathogenic bacteria in this family, which is a kind of safe microorganism. [0003] Calcifediol, also known as 25-hydroxyvitamin D3, is one of the representatives of active vitamin D3 drugs. It is especially suitable for patients with normal renal function but insufficient liver function. Calcitriol, also known as 1,25-dihydroxyvitamin D3, can promote intestinal absorption of calci...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N13/00C12N15/01C12P7/02C12R1/01
CPCC12N1/20C12N13/00C12N15/01C12P7/02
Inventor 杨虎丰亚辉王仲清张克闯林伟罗忠华黄芳芳
Owner DONGGUAN HEC GENERIC DRUG R&D CO LTD
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