A kind of method for enzymatically preparing o-phenolic compounds

A technology for enzymatic preparation and compounds, applied in biochemical equipment and methods, enzymes, immobilized enzymes, etc., can solve the problems of complex reaction process, large environmental pollution, poor specificity, etc., and achieve high specificity of reaction and easy operation Safe, short cycle effect

Active Publication Date: 2021-05-25
SHENZHEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the preparation of 3,4-dihydroxyphenylacetic acid is mainly based on chemical methods, the reaction process is complicated, the specificity is poor, a large amount of toxic and harmful reagents are used, and the environmental pollution is relatively large.

Method used

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  • A kind of method for enzymatically preparing o-phenolic compounds
  • A kind of method for enzymatically preparing o-phenolic compounds
  • A kind of method for enzymatically preparing o-phenolic compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Preparation of carrier-free immobilized tyrosinase

[0047] Wash and dry the fresh white Agaricus bisporus bought in the market, and mash it with a homogenizer according to the ratio of mushroom: phosphate buffer (50mM, pH6.0) = 1:2 (mass volume ratio, g / ml), The residue was removed by filtration, and the enzyme solution was divided and stored in a -20°C refrigerator. Thaw the frozen enzyme solution at room temperature, add ammonium sulfate to 50% saturation, and continue to shake. After the ammonium sulfate is completely dissolved, add 25% glutaraldehyde (wt.%) to make the concentration of glutaraldehyde in the enzyme solution The amount reached 0.25% (wt.%), and continued to stir at 4°C for 16h. Centrifuge the resulting turbid solution (10min, 8000rmp, 4°C), remove the supernatant, add phosphate buffer (pH6.0, 50mM) to wash the precipitate, then centrifuge (10min, 8000rmp, 4°C) and repeat the washing 4 times until the supernatant is complete Until there is...

Embodiment 2

[0048] Example 2 HPLC Determination of Picetanol and 3'-Hydroxy Pterostilbene Concentrations

[0049] High performance liquid chromatography adopts reverse C18 column (Japan GL Sciences, Inc company, Inertsil ODS-SP, 4.6 × 150 mm, 5 μm) to determine the content of piceatanol and 3'-hydroxy pterostilbene, the liquid phase conditions are: flow Phase A is: 0.5% acetic acid / acetonitrile = 95 / 5(v / v), mobile phase B is: acetonitrile / 0.5% acetic acid = 95 / 5(v / v), A / B=75 / 25(v / v ); injection volume: 10μL; total flow rate: 1ml / min; detection wavelength of piceatanol is 320nm, detection wavelength of 3'-hydroxy pterostilbene is 306nm; column temperature is 29°C.

[0050] Precisely prepare standard solutions of piceatanol and 3'-hydroxy pterostilbene with acetonitrile, each series is repeated 3 times, and the peak area is obtained by HPLC detection and the average value is obtained. The concentration (X, mM) is the abscissa, and the peak area (Y) is the ordinate to draw the standard curv...

Embodiment 3

[0051] Example 3 Enzymatic preparation of 3'-hydroxy pterostylbene

[0052] Accurately weigh 29% (wt.%) of carrier-free immobilized tyrosinase, the amount of pterostilbene added is 30% (wt.%), and the amount of L-ascorbic acid is 41% (wt.%). 25% (v / v%) DMSO was used to help dissolve, and reacted in a constant temperature shaking incubator at 20°C and 220rpm. After reacting for 1, 2, 3, 4, 5, 6, and 7 hours, centrifuge at 6000 rpm for 2 minutes, take 20 μl of supernatant, and put the rest of the reaction solution back into the constant temperature shaking incubator to continue the reaction. Figure 4 shown. The supernatant was diluted 20 times (diluted to 400 μl), vortexed and shaken to mix evenly, then filtered, and the content of substrate and product was detected by HPLC, such as Figure 5 As shown, the yield of 3'-hydroxy pterostylbene reached the maximum when the reaction was carried out to 5h, which was 61.8%. react as figure 1 and figure 2 shown.

[0053] Collect ...

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Abstract

The invention discloses a method for enzymatically preparing o-phenolic compounds, which uses carrier-free immobilized tyrosinase as a catalyst, a single phenolic compound as a substrate, dimethyl sulfoxide as a cosolvent, and L-ascorbic acid As a reducing agent, react at 15-40°C for 1-7 hours to obtain o-phenolic compounds. The present invention innovatively uses carrier-free immobilized tyrosinase as a biocatalyst and single phenolic compound as a substrate to synthesize o-phenolic compounds, which has the advantages of mild reaction conditions, short cycle, high reaction specificity, simple process steps, The advantages of safe operation provide a new method for the green production of piceatanol, 3'-hydroxy pterostylbene and 3,4-dihydroxyphenylacetic acid.

Description

technical field [0001] The invention relates to the field of biocatalysis, in particular to a method for enzymatically preparing o-phenolic compounds. Background technique [0002] Polyphenolic compounds widely exist in plants, have good antioxidant properties, and good anti-inflammatory and anti-tumor activities, and have received more and more attention. Ortho-phenolic compounds belong to polyphenolic compounds. Its unique ortho-dihydroxy structure makes its properties more active and has good anti-inflammatory, anti-tumor, anti-diabetic, anti-cardiovascular disease and anti-aging activities, such as 3'- Hydroxy pterostylbene, piceatanol, 3,4-dihydroxyphenylacetic acid, etc. have great application and research value. Therefore, it is of great significance to explore the methods and processes for the green preparation of ortho-phenolic compounds. [0003] 3'-Hydroxypterostilbene (3'-Hydroxypterostilbene, (E)-3,5-dimethoxy-3',4'-dihydroxystilbene), is an analogue of the na...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/22C12P7/42C12N9/02C12N11/00
CPCC12N9/0059C12N11/00C12P7/22C12P7/42C12Y110/03001
Inventor 杨缜程辉邹永
Owner SHENZHEN UNIV
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