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A kind of bacterial strain and application thereof for efficiently converting vitamin D3 into its active form

A technology of vitamins and bacterial strains, which is applied in the direction of microorganism-based methods, measurement/testing of microorganisms, bacteria, etc., can solve the problems of low solubility, low substrate feeding concentration and low conversion efficiency, and achieve high substrate conversion rate and high area selective effect

Active Publication Date: 2022-04-08
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since vitamin D3 is a fat-soluble compound, its solubility in the water phase is extremely low, resulting in very low substrate feeding concentration and conversion efficiency during the conversion process. bottleneck in production
Therefore, it is of great significance to screen a microorganism that efficiently transforms vitamin D3. At present, Bacillus megaterium (Bacillus megaterium) H-1 is used to transform vitamin D3 to simultaneously generate 25-OH-VD3 and 1α,25-diOH-VD3. Research not reported

Method used

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  • A kind of bacterial strain and application thereof for efficiently converting vitamin D3 into its active form
  • A kind of bacterial strain and application thereof for efficiently converting vitamin D3 into its active form
  • A kind of bacterial strain and application thereof for efficiently converting vitamin D3 into its active form

Examples

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Embodiment 1

[0024] Example 1 Screening, Identification and Preservation of Vitamin D3 Hydroxylating Bacteria

[0025] (1) Screening of vitamin D3 hydroxylation strains

[0026] The present invention obtains samples from soil, weighs 5-10g of soil samples into a 250mL Erlenmeyer flask, suspends them with sterile water, and incubates them at 30°C and 220r / min for 2h.

[0027] Inoculate 1 mL of the suspension into 50 mL of enriched medium, and incubate at 30 °C, 220 r / min for 3 days. Transfer 1 mL of the culture to fresh enriched medium every 3 days for three consecutive subcultures.

[0028] The bacterial solution was diluted in gradient and spread on the separation medium, and cultured at 30°C until colonies grew.

[0029] As a preferred embodiment, in this example, colonies were inoculated into 50 mL of transformation medium, cultured at 30°C and 220 r / min, and after growing for 12-24 hours, 25 mg of vitamin D3 dissolved in 250 μL of ethanol was added to continue fermentation for 24 hou...

Embodiment 2

[0040] Example 2 Bacillus megaterium (Bacillus megaterium) H-1 bacterial strain transformation vitamin D3 (scheme 1)

[0041] (1) Prepare Bacillus megaterium (Bacillus megaterium) H-1 bacterial strain transforms the seed liquid of vitamin D3:

[0042] Pick the Bacillus megaterium (Bacillus megaterium) H-1 strain on the solid LB medium to transform vitamin D3, inoculate in a 100mL Erlenmeyer flask containing 10mL LB seed medium, and place it on a shaker at 30°C Cultivate at a rotational speed of 220r / min for 12h to the logarithmic phase, and then obtain a seed liquid for converting vitamin D3 by Bacillus megaterium H-1 strain.

[0043] (2) Bacterial culture: Inoculate the seed liquid of the above-mentioned Bacillus megaterium (Bacillus megaterium) H-1 strain into a 250mL cone containing 50mL of sterilized LB fermentation medium with an inoculum of 10% (w / w). Shaped flasks were cultured on a shaker at a speed of 220 r / min at 30°C, and fermented for 12 hours, and the cells enter...

Embodiment 3

[0048] Example 3 Bacillus megaterium (Bacillus megaterium) H-1 strain transforms vitamin D3 (scheme 2)

[0049] (1) Prepare Bacillus megaterium (Bacillus megaterium) H-1 bacterial strain transforms the seed liquid of vitamin D3:

[0050] Pick the Bacillus megaterium (Bacillus megaterium) H-1 strain on the solid LB medium to transform vitamin D3, inoculate it in a 100mL Erlenmeyer flask containing 10mL GPY seed medium, and place it on a shaker at 30°C Cultivate at a rotational speed of 220r / min for 12h to the logarithmic phase, and then obtain a seed liquid for converting vitamin D3 by Bacillus megaterium H-1 strain.

[0051] (2) Bacterial culture: inoculate the seed solution of the above-mentioned Bacillus megaterium (Bacillus megaterium) H-1 strain into a 250mL conical tube containing 50mL of sterilized fermentation medium with an inoculum of 10% (w / w). In the bottle, under the condition of 30°C, culture on a shaker at a rotation speed of 220r / min, ferment for 12h, and the b...

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Abstract

The invention discloses a bacterial strain for efficiently transforming vitamin D3 into its active form and its application. The invention belongs to the field of biotechnology, and relates to a strain of Bacillus megaterium H‑1 for efficiently transforming vitamin D3 and its application. The strains are preserved in the General Microbiology Center of the China Committee for the Collection of Microorganisms, and the preservation number is CGMCC No.20362. Bacillus megaterium (Bacillus megaterium) H‑1 can convert vitamin D3 into 25‑OH‑VD3 and 1α,25‑diOH‑VD3, and the substrate conversion rate reaches 95% under the condition that the substrate dosage is 1.0g / L , the concentration of 25‑OH‑VD3 reached 26 mg / L, and the molar yield was 4.90%; the concentration of 1α,25‑diOH‑VD3 reached 23 mg / L, and the molar yield was 4.25%.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a bacterial strain for efficiently converting vitamin D3 into its active form and its application, in particular to a strain of Bacillus megaterium H-1 and its efficient conversion of vitamin D3 into 25-OH- Method for VD3 and 1α,25-diOH-VD3. Background technique [0002] Vitamin D3 is a sterol derivative. In the World Health Organization's Essential Drugs List, vitamin D3 is the safest and most effective drug in the health system. Its main active forms in the human body are 25-OH-VD3 and 1α, 25-diOH-VD3. 25-OH-VD3 can inhibit cell proliferation, stimulate cell differentiation, inhibit renin synthesis, stimulate antimicrobial peptide synthesis in macrophages and promote calcium ion influx in skeletal muscle cells. It is used to treat bone Treatment of metabolic bone diseases such as porosis, rickets, and osteomalacia, as well as hypocalcemia caused by hemodialysis, can in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P7/02C12N1/02C12Q1/04C12R1/11
CPCC12P7/02C12N1/02C12Q1/04G01N2333/32C12R2001/11C12N1/205
Inventor 许正宏李会史劲松何鹏张晓梅龚劲松
Owner JIANGNAN UNIV