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Method for producing 25-hydroxy vitamin D3 by converting vitamin D3 hydroxylase

A technology for hydroxyvitamin and vitamin, applied in the field of bioengineering, can solve the problems of chemical synthesis method with many reaction steps, complicated separation and purification process, unfavorable industrial production, etc., and achieves the effects of increasing enzyme conversion efficiency, reducing cost and shortening conversion cycle.

Pending Publication Date: 2020-07-24
沈阳美得欣医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] 25-Hydroxyvitamin D3[25(OH)VD3] can be synthesized by chemical methods, but most of the methods used in chemical synthesis are multi-step reactions such as ring opening, bond breaking, ring fusion, reduction, coupling, oxidation, purification, etc. The chemical synthesis method has the disadvantages of many reaction steps, low conversion rate, and complicated separation and purification process, which is not conducive to large-scale industrial production.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Escherichia coli fermentation culture

[0046] Incline medium: peptone 10g / L, yeast extract 5g / L, maltose 1.5%, NaCl 10g / L, pH to 7.0;

[0047] Fermentation medium: peptone 1%, maltose 1.5%, yeast extract 0.6%-NaCl 0.2%, 0.005% NaF, pH to 7.0;

[0048]Use the culture medium at 37°C and 200rpm to cultivate to OD600=4, add IPTG (sterilized by filtration) with a final concentration of 1.2mM as an inducer, induce enzyme production at 28°C, and obtain E. coli bacterial liquid after culturing for 24 hours .

Embodiment 2

[0050] Enzymatic conversion to produce 25-hydroxyvitamin D3

[0051] Take 400mL of fermentation broth, centrifuge at 11000rpm for 5min, collect the bacteria, add 3 times the volume of the fermentation broth to wash the bacteria twice, add 80mL of 0.4mol / L phosphoric acid and nitric acid buffer (pH7.0) to make a bacterial suspension . Under the pressure of 900bar, the high-pressure homogenizer was used to break the wall for 25 minutes, and the breakage rate of Escherichia coli reached 96%. Centrifuge the broken wall solution at a speed of 10000 rpm for 6 minutes, and collect the supernatant, that is, the crude enzyme solution of vitamin D3 hydroxylase.

[0052] Dissolve 20mg of vitamin D3 in 1mL of ethyl acetate solution, slowly add to the crude enzyme solution, and add 5mg of NADP+ respectively, after mixing evenly, carry out enzyme conversion under 400rpm shaking condition, the conversion temperature is 35°C, and the conversion time is 48h ; after the transformation, carry ...

Embodiment 3

[0055] Enzymatic conversion to produce 25-hydroxyvitamin D3.

[0056] Take 0.8L of fermentation broth, centrifuge at 12000rpm for 8min, collect the bacteria, add deionized water 6 times the volume of the fermentation broth to wash the bacteria for 3 times, add 0.4mol / L phosphoric acid and nitric acid buffer (pH7.0) 100mL, and make a bacterial suspension liquid. Under the pressure of 1200bar, the high-pressure homogenizer was used to break the wall for 40 minutes, and at this time, the wall breaking rate of Escherichia coli reached 99.5%. Centrifuge the broken wall solution at a speed of 12000 rpm for 5 minutes, and collect the supernatant, that is, the crude enzyme solution of vitamin D3 hydroxylase.

[0057] Dissolve 40mg of vitamin D3 in 1mL of ethanol or ether solution, slowly add it to the crude enzyme solution, and add 10mg of NADP+, after mixing evenly, carry out enzyme conversion under the shaking condition of 600rpm, the conversion temperature is 35°C, and the convers...

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Abstract

The invention provides a method for producing 25-hydroxy vitamin D3 by converting vitamin D3 hydroxylase. The method comprises the following steps: a, fermentation: inoculating genetically engineeredbacteria for producing vitamin D3 hydroxylase into a fermentation medium, culturing until OD600 is equal to 1-5, and adding IPTG for inducing enzyme production, wherein the final concentration of IPTGis 1.2-2mM, the induction temperature is 28 DEG C, and the fermentation culture time is 16-24h to obtain a fermentation broth; and b, wall breaking: centrifuging the fermentation broth at 11000-13000rpm for 5-7min, collecting the bacteria, adding deionized water with the volume 4-6 times that of the fermentation broth to wash the bacteria for 1-3 times, resuspending the bacteria with a mixed buffer solution of phosphoric acid and nitric acid in a ratio of 1.5: 1, and carrying out mechanical wall breaking on the bacteria liquid by a high pressure homogenizer to obtain a cell wall breaking liquid. The conversion period is shortened from the flow, and hydroxy is directly introduced in a short time to generate an active product. The conversion conditions are mild.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for converting vitamin D3 hydroxylase to produce 25-hydroxyvitamin D3. Background technique [0002] Calcifediol, also known as 25-hydroxyvitamin D3[25(OH)VD3], is a biologically active derivative of vitamin D3. Vitamin D3 has no activity in the human body and must be converted into active biological substances by biological enzymes. ingredients to make a difference. Animal experiments have shown that calcifediol has obvious effects on metabolic bone diseases such as osteoporosis, rickets, and osteomalacia, and can also be used to treat hypocalcemia caused by hemodialysis. It can also be used as a health food raw material and a feed additive, and has a wide range of applications in health food, medicine and feed additives. [0003] 25-Hydroxyvitamin D3[25(OH)VD3] can be synthesized by chemical methods, but most of the methods used in chemical synthesis are multi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12P33/06C12R1/19C12R1/04
CPCC12N9/0073C12P33/06C12Y114/13013
Inventor 刘婷婷
Owner 沈阳美得欣医药科技有限公司
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