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A kind of method for preparing p-coumaric acid by multi-enzyme cascade

A p-coumaric acid and cascading technology, applied in the field of enzyme catalysis, can solve the problems of high preparation cost, single enzyme source, and low yield

Active Publication Date: 2022-05-20
CHINA THREE GORGES UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Among them, the enzyme-catalyzed patents include: (1) "A method for synthesizing trans-4-coumaric acid by enzymatic method", the patent application number is 201911167675.4. This patent application uses cinnamic acid as raw material and uses trans-4-coumaric acid Cinnamic acid-4-monooxygenase (EC1.14.14.91) catalyzes the synthesis, adding a reduced coenzyme donor, the price is very expensive, the substrate concentration can reach 100g / L, and the yield has not been reported
(2) "A trans-cinnamic acid-4-hydroxylase and its coding gene and application", the patent number is: 201710136604, the invention utilizes the trans-cinnamic acid-4-hydroxylase of the genus Amaryllis huddixiao Carry out catalytic synthesis, all hydroxylases in this pathway also belong to plant cytochrome P450 enzyme EC1.14.14.91, but its yield is only 0.4g / L
[0008] The above methods all have problems such as low yield, single enzyme source, and high preparation cost.

Method used

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  • A kind of method for preparing p-coumaric acid by multi-enzyme cascade
  • A kind of method for preparing p-coumaric acid by multi-enzyme cascade
  • A kind of method for preparing p-coumaric acid by multi-enzyme cascade

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] At a temperature of 30°C and a pH of 7, add 50kg / h of p-hydroxyphenylpyruvate, 40kg / h of glucose, 2kg / h of liquid enzyme preparation of carbonyl reductase, 1.5kg / h of glucose dehydrogenase, and use polystyrene as the reaction medium. A two-phase aqueous system composed of ethanol and water, the reaction residence time is 2 hours, the organic membrane ultrafiltration equipment with a membrane pore size of 15kD and a water flux of 100kg / h is selected to recover 85% of carbonyl reductase and glucose dehydrogenase, return In the first-stage tubular reactor, the number of recycling times is 7 times; in the second-stage tubular reactor, at a temperature of 20°C and a pH of 8, the permeate containing p-hydroxyphenyl lactic acid has a flow rate of 90kg / h, add dehydratase liquid enzyme preparation 2kg / h, the reaction residence time is 2 hours, select 30kD membrane aperture for use, water flux is the organic membrane ultrafiltration equipment of 90kg / h, reclaim 75% of dehydratase...

Embodiment 2

[0058] At a temperature of 35°C and a pH of 8, add 40kg / h of p-hydroxyphenylpyruvate, 40kg / h of glucose, 2kg / h of liquid enzyme preparation of carbonyl reductase, and 2kg / h of glucose dehydrogenase. The reaction medium is ethanol and A single-phase system composed of 50% water, the reaction residence time is 1.5 hours, the organic membrane ultrafiltration equipment with a 20kD membrane pore size and a water flux of 90kg / h is selected to recover 82% of carbonyl reductase and glucose dehydrogenase, return In the first-stage tubular reactor, the recycling times are 6 times; in the second-stage tubular reactor, at a temperature of 25°C and a pH of 8, the permeate containing p-hydroxyphenyl lactic acid has a flow rate of 80kg / h, add dehydratase liquid enzyme preparation 3kg / h, the reaction residence time is 1.5 hours, select 20kD membrane aperture for use, water flux is the organic membrane ultrafiltration equipment of 90kg / h, reclaim 80% of dehydratase, return to the second In th...

Embodiment 3

[0059] Example 3: At a temperature of 25°C and a pH of 8, add 50kg / h of p-hydroxyphenylpyruvate, 40kg / h of glucose, 2.5kg / h of liquid enzyme preparation for carbonyl reductase, and 2.3kg / h of glucose dehydrogenase , the reaction medium is a two-phase system composed of cyclohexane and water, the reaction residence time is 2 hours, the organic membrane ultrafiltration equipment with a 25kD membrane pore size and a water flux of 90kg / h is used to recover 84% of carbonyl reductase and glucose Dehydrogenase, returned to the first-stage tubular reactor, recycled 7 times; entered the second-stage tubular reactor, at a temperature of 25°C and a pH of 8, permeated with p-hydroxyphenyl lactic acid liquid, the flow rate is 80kg / h, add dehydratase liquid enzyme preparation 3.3kg / h, the reaction residence time is 2.5 hours, select 30kD membrane pore size, the organic membrane ultrafiltration equipment with water flux of 80kg / h, recover 75% dehydrated The enzyme is returned to the second-s...

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Abstract

The invention provides a method for synthesizing p-coumaric acid by multi-enzyme cascade catalysis. Using p-hydroxyphenylpyruvate as raw material, it is reduced to p-hydroxyphenyl lactic acid by carbonyl reductase, and then p-coumaric acid is prepared by dehydratase. The method can be used for continuous production in a tubular reactor-membrane separation coupling device, or in a batch reactor method, where the coenzyme NAD(P)H required by carbonyl reductase is regenerated through the reaction of glucose dehydrogenase and glucose. Each batch of carbonyl reductase, glucose dehydrogenase, and dehydratase can be recycled more than 5 times, and the production efficiency can reach up to 50 g / L.h. The invention has low production cost, high efficiency and good product purity.

Description

technical field [0001] The invention relates to the field of enzyme catalysis; more specifically, it relates to a method for synthesizing p-coumaric acid by multi-enzyme cascade catalysis. Background technique [0002] P-coumaric acid (4-hydroxycinnamic acid or trans-4-coumaric acid, p-Coumaric acid, p-CA), widely exists in plants, is the active ingredient of Hedyotis diffusa, Eucommia leaves and other plants, has Prevention of cardiovascular disease, anti-oxidation and other biological activities. It is an intermediate in the synthesis of high-value health products such as flavonoids, isoflavones, and stilbenes, such as naringenin, anthocyanins, quercetin, catechins, and resveratrol. Alcohol, hydroxytyrosol and caffeic acid, etc.; in the agricultural industry for plant growth promoters; in the food industry for long-acting fungicides and fruit and vegetable preservation preservatives. In the cosmetics industry, p-coumaric acid can inhibit both tyrosine monophenolase and di...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/42
CPCC12P7/42Y02P20/10
Inventor 龚大春张书银肖开兴万里宋婷杨潇任立伟郭金玲
Owner CHINA THREE GORGES UNIV