Method of preparing o-phenol compound by enzyme method

A technology for enzymatic preparation and compound application in biochemical equipment and methods, enzymes, immobilized enzymes, etc., which can solve the problems of high environmental pollution, poor specificity, complex reaction process, etc., and achieve high reaction specificity and cycle time. The effect of short and simple process steps

Active Publication Date: 2017-05-31
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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  • Method of preparing o-phenol compound by enzyme method
  • Method of preparing o-phenol compound by enzyme method
  • Method of preparing o-phenol compound by enzyme method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 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, continue shaking, after ammonium sulfate is completely dissolved, add 25% glutaraldehyde (wt.%) to make the glutaraldehyde in the enzyme solution The amount reached 0.25% (wt.%), and the stirring was continued 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 completely Until there is no enzyme acti...

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 * 150mm, 5 μ m) to measure the content of piceatanol and 3'-hydroxy pterostilbene, and the liquid phase condition is: mobile 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: 10 μL; total flow rate is 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 ...

Embodiment 3

[0051] Example 3 Preparation of 3'-hydroxy pterostylbene by enzymatic method

[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 aid dissolution, and the reaction was carried out at 20° C., in a constant temperature shaking incubator at 220 rpm. 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, filtered, and the content of the 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 ...

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Abstract

The invention discloses a method of preparing an o-phenol compound by an enzyme method. The method comprises the step of performing a reaction for 1-7h at 15-40 DEG C to obtain the o-phenol compound by taking a carrier-free immobilized tyrosinase as a catalyst, a monophenol compound as a primer, dimethyl sulfoxide as a cosolvent and L-ascorbic acid as a reducing agent. According to the method disclosed by the invention, by taking the carrier-free immobilized tyrosinase as the biological catalyst and monophenol compound as the primer, the o-phenol compound is synthesized. The method has the advantages of being mild in reaction condition, short in period, high in specificity of reaction, simple in process step, safe to operate and the like. The invention provides a novel method of greenly producing piceatannol, 3'-hydroxyl pterostilbene 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, resveratrol, 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 Applications(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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