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Method for preparing 1-deoxy-D-xylulose with chemical method-enzymatic method

A technology for enzymatic preparation of xylulose, applied in chemical instruments and methods, deoxygenated/unsaturated sugars, organic chemistry, etc., can solve the problems of expensive reaction raw materials, cumbersome reaction steps, and low total yield, and achieve purification steps Simple and easy to implement, low price, and the effect of increasing conversion rate

Inactive Publication Date: 2010-06-23
NORTHWEST UNIV(CN)
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0011] The object of the present invention is to provide a kind of efficient, simple, low-cost chemical method-enzymatic method to prepare the method for 1-deoxy-D-xylulose (DX), solve the problem that the reaction raw materials are expensive and expensive in the method for preparing DX in the background technology Unstable, cumbersome reaction steps, long time-consuming and low overall yield technical problems

Method used

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  • Method for preparing 1-deoxy-D-xylulose with chemical method-enzymatic method
  • Method for preparing 1-deoxy-D-xylulose with chemical method-enzymatic method
  • Method for preparing 1-deoxy-D-xylulose with chemical method-enzymatic method

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Embodiment 1

[0038] Take 148mg Na 2 HPO 4 In a 15mL centrifuge tube, add 800μl ultrapure water to dissolve it, then add 0.5mmol glycidol or 0.25mmol (R)-glycidol, and react in a water bath at 100°C for 3h; after the centrifuge tube is cooled to room temperature, adjust the pH to 7.5, Add catalase 180units, L-GPO 3units, sodium pyruvate 55mg, TIM 25units, ThPP 2mM, MgCl 2 10mM, β-mercaptoethanol 5mM, DXS 500μg, the total reaction volume is 1mL, and react at 37°C for 12 hours; during the reaction process, use paper chromatography to detect the degree of reaction, the medium is qualitative filter paper commonly used in laboratories, and the developer is 1M acetic acid Ammonium: 99% ethanol: EDTA-Na 2 =70:30:1 (v / v / v), the developer is 10% ethylenediamine sulfate aqueous solution. After the reaction is complete, add 20 units of alkaline phosphatase to the reaction mixture and incubate at 37°C for 4 hours; remove the moisture in the reaction mixture by vacuum rotary evaporation, add (500 μl×...

Embodiment 2

[0040] Take 100mg Na 2 Add 500μl ultrapure water to dissolve HPO4 in a 15mL centrifuge tube, then add 0.25mmol (rac)-glycidol or 0.125mmol (R)-glycidol, and react in a 50°C water bath for 2 hours; after the reaction, the centrifuge tube After cooling to room temperature, adjust the pH to 7.0, add Catalase 100units, L-GPO 1units, sodium pyruvate 30mg, TIM 10units, ThPP 1mM, MgCl 2 2mM, β-mercaptoethanol 1mM, DXS 200μg, total reaction volume 600μL, 30°C for 6 hours; during the reaction, use paper chromatography to detect the extent of the reaction, the medium is qualitative filter paper commonly used in laboratories, and the developer is 1M acetic acid Ammonium: 99% ethanol: EDTA-Na 2 =70:30:1 (v / v / v), the developer is 10% ethylenediamine sulfate aqueous solution. After the reaction is complete, add 5 units of alkaline phosphatase to the reaction mixture and incubate at 30°C for 2 hours; remove the moisture in the reaction mixture by vacuum rotary evaporation, add (500 μl×2) m...

Embodiment 3

[0042] Take 200mg Na 2 HPO 4 In a 15mL centrifuge tube, add 1000μl ultrapure water to dissolve it, then add 1mmol (rac)-glycidol or 0.5mmol (R)-glycidol, and react in a water bath at 100°C for 5h; after the reaction, cool the centrifuge tube to After room temperature, adjust the pH to 8.0, add Catalase 200units, L-GPO20units, sodium pyruvate 60mg, TIM 30units, ThPP 5mM, MgCl 2 10mM, β-mercaptoethanol 5mM, DXS 800μg, the total reaction volume is 1.5mL, and react at 40°C for 14 hours; during the reaction process, the degree of reaction is detected by paper chromatography, the medium is qualitative filter paper commonly used in the laboratory, and the developer is 1M Ammonium acetate: 99% ethanol: EDTA-Na 2=70:30:1 (v / v / v), the developer is 10% ethylenediamine sulfate aqueous solution. After the reaction is complete, add 30 units of alkaline phosphatase to the reaction mixture and incubate at 30°C for 4 hours; remove the moisture in the reaction mixture by vacuum rotary evapor...

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Abstract

The invention relates to a method for preparing 1-deoxy-D-xylulose with the combination of a chemical method and an enzymatic method, which comprises the steps as follows: in a water phase system under certain temperature, the glycidol can form glycerol 3-phosphate by inorganic phosphate through ring opening, then the generated L-glycerol 3-phosphate is oxidized by L-GPO to form DHAP under the existence of catalase, the DHAP is converted into DXP under the existence of pyruvic acid, TIM enzyme and DXS, and target molecule DX can be obtained by hydrolyzing the compound through phosphatase. The method organically couples the simple method for preparing the DHAP by taking the stable, cheap and easily available compound as raw material with the method for preparing the DX by using the enzymatic method, the target molecule DX can be obtained through one-step reaction, the yield is high, the purification step is simple and feasible, the DX with high purity (more than 95%) can be obtained, the price of the raw material is low, the nature is stable, and the technical problems of expensive and unstable reaction material, complicated reaction steps, longer time consuming and lower overall yield of the method for preparing DX in the background technology are solved.

Description

technical field [0001] The invention belongs to the field of biosynthesis of terpenoids, and in particular relates to an MEP pathway for preparing terpenoids biosynthesis by using cheap, easy-to-obtain and stable raw materials as substrates and combining chemical and enzymatic methods Approach to potential intermediate 1-deoxy-D-xylulose (DX). Background technique [0002] Terpenoids are widely distributed in nature and are present in almost all life forms. Scientists have isolated and identified nearly 40,000 terpenoids from animals, plants and microorganisms, which constitute the largest family of natural products, and many compounds in this family also show important physiological activities. For example, dolichol diphosphates regulate cell wall and glycoprotein biosynthesis; quinone compounds (such as plastoquinone and coenzyme Q) can be used as carriers for electron transfer and redox reactions; carotenoids and chlorophyll Side chains are involved in photosynthesis; c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/02C07H3/08C07H1/06
Inventor 高文运李恒田洁廖娟王雪娇王辉杨少青
Owner NORTHWEST UNIV(CN)
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