阅读说明：本技术 一种酶催化法合成反式-4-香豆酸的方法 (Method for synthesizing trans-4-coumaric acid by enzyme catalysis method ) 是由 陈江 孙军亭 徐光彩 于 2019-11-25 设计创作，主要内容包括：本发明提供了一种酶催化法合成反式-4-香豆酸的方法。反式-肉桂酸-4-单加氧酶(EC 1.14.14.91)可催化反式-肉桂酸合成反式-4-香豆酸,同时消耗一分子O<Sub>2</Sub>和一个NAD(P)H,生成一个H<Sup>+</Sup>和一个NAD(P)<Sup>+</Sup>。在单水相或水/有机溶剂的双相反应体系中,加入反式-肉桂酸(原料),加入微量NAD(P)<Sup>+</Sup>或NAD(P)H,加入O<Sub>2</Sub>源,加入还原型氢供体,然后加入反式-肉桂酸4-单加氧酶和NAD(P)<Sup>+</Sup>还原酶,滴加碱液控制pH,控制反应温度；反应结束后反应液过滤除酶后浓缩,降温结晶可得反式-4-香豆酸。(The invention provides a method for synthesizing trans-4-coumaric acid by an enzyme catalysis method. Trans-cinnamic acid-4-monooxygenase (EC1.14.14.91) can catalyze trans-cinnamic acid to synthesize trans-4-coumaric acid while consuming one molecule of O 2 And one NAD (P) H to produce one H + And one NAD (P) + . Adding trans-cinnamic acid (raw material) and trace amount of NAD (P) into a single-water-phase or water/organic solvent two-phase reaction system + Or NAD (P) H, O 2 Source, addition of reduced hydrogen donor, followed by addition of trans-cinnamic acid 4-monooxygenase and NAD (P) + Reductase, dripping alkali liquor to control pH and control reaction temperature; after the reaction is finished, the reaction solution is filtered, enzyme is removed, concentrated, cooled and crystallized to obtain the trans-4-coumaric acid.)

1. A method for synthesizing trans-4-coumaric acid by an enzyme catalysis method is characterized by comprising the following steps: adding trans-cinnamic acid and trace amount of NAD (P) into a single-water-phase or water/organic solvent two-phase reaction system⁺Or NAD (P) H, O₂Source of reduced hydrogen donor, followed by addition of trans-cinnamic acid-4-monooxygenase and NAD (P)⁺Reductase, dripping alkali liquor to control pH and control reaction temperature; after the reaction is finished, filtering the reaction solution to remove enzyme, concentrating, cooling and crystallizing to obtain trans-4-coumaric acid; wherein the trans-cinnamic acid-4-monooxygenase is numbered as follows: EC 1.14.14.91.

2. The process for the enzymatic synthesis of trans-4-coumaric acid according to claim 1, characterized in that: when the coenzyme required by trans-cinnamic acid-4-monooxygenase is NADH, a trace amount of NAD is added into the reaction system⁺Or NADH; when the coenzyme required by trans-cinnamic acid-4-monooxygenase is NADPH, a trace amount of NADP is added into the reaction system⁺Or NADPH.

3. The process for the enzymatic synthesis of trans-4-coumaric acid according to claim 1, characterized in that: said O is₂The source being air, oxygen or H₂O₂。

4. The process for the enzymatic synthesis of trans-4-coumaric acid according to claim 1, characterized in that: the alkali is ammonia water, NaOH or KOH; the pH control range is 5.0-10.0, preferably 6.0-8.0, and preferably 6.8-7.5.

5. The process for the enzymatic synthesis of trans-4-coumaric acid according to claim 1, characterized in that: the reduced hydrogen donor is glucose, isopropanol, formic acid or formate.

6. The process for the enzymatic synthesis of trans-4-coumaric acid according to claim 5, characterized in that: when the reduced hydrogen donor is glucose, NAD (P) is added in the reaction⁺The reductase is glucose dehydrogenase; when the reduced hydrogen donor is isopropanol, NAD (P) is added in the reaction⁺The reductase is isopropanol dehydrogenase; when the reduced hydrogen donor is formic acid or formate, NAD (P) is added in the reaction⁺The reductase is formate dehydrogenase.

7. The process for the enzymatic synthesis of trans-4-coumaric acid according to any one of claims 1-6, characterized in that: the trans-cinnamic acid-4-monooxygenase comprises, but is not limited to, trans-cinnamic acid-4-monooxygenase having an amino acid sequence which can be SEQ01, SEQ02, SEQ03, SEQ04, SEQ05, SEQ06, SEQ07, SEQ08, SEQ09, SEQ10, SEQ11, SEQ12, SEQ13, SEQ14, SEQ15 or SEQ 16; or the trans-cinnamic acid-4-monooxygenase is a trans-cinnamic acid-4-monooxygenase which is a single and/or multiple point mutant having the amino acid sequence; or the trans-cinnamic acid-4-monooxygenase is a trans-cinnamic acid-4-monooxygenase of an amino acid sequence having a homology of 80% or more with the amino acid sequence.

8. The enzymatic synthesis of trans-4-coumaric acid according to claim 7, characterized in that: the host used for expressing the trans-cinnamic acid-4-monooxygenase is one of escherichia coli, bacillus, mould or yeast; preferably, the expression system is an escherichia coli BL21 expression system or a pichia pastoris GS115 expression system.

9. The enzymatic synthesis of trans-4-coumaric acid according to claim 8, characterized in that: the trans-cinnamic acid-4-monooxygenase can be fermentation liquor or bacterial sludge after the expression of the constructed host bacteria, crude enzyme subjected to simple treatment or pure enzyme subjected to purification treatment, liquid enzyme filtered by a flocculating agent and a ceramic membrane or immobilized enzyme subjected to immobilization treatment.

Technical Field

The present invention relates to the fields of biotechnology and enzyme catalysis; more specifically, the invention relates to a method for synthesizing trans-4-coumaric acid by an enzyme catalysis method.

Background

P-coumaric acid, i.e., coumaric acid (p-coumaric acid), 4-coumaric acid, p-hydroxycinnamic acid, and the like, is the most predominant of hydroxycinnamic acid compounds; the system was named 3- (4-hydroxyphenyl) -2-propenoic acid. The p-coumaric acid has cis-form and trans-form, and the trans-p-coumaric acid is trans-4-coumaric acid, is white fine needle crystal, has melting point of 212-214 ℃, is slightly soluble in cold water, hot water, methanol and ethanol, is slightly soluble in benzene and chloroform, and is insoluble in dichloromethane and petroleum ether.

Trans-4-coumaric acid can form resonance stable phenol free radical, so that the antioxidant activity is good; it has strong scavenging effect on hydrogen peroxide, superoxide radical, hydrogen radical and nitroso peroxide, and has quenching effect on singlet oxygen; the coumaric acid can inhibit enzymes generating free radicals and promote enzymes generating free radicals to be generated while quenching the free radicals. Research shows that the trans-4-coumaric acid has different degrees of inhibition effects on staphylococcus aureus, dysentery bacillus, escherichia coli and pseudomonas aeruginosa; bodini et al found that trans-4-coumaric acid was able to inhibit quorum sensing by Chromobacterium violaceum, Agrobacterium and Pseudomonas chlororaphis. The trans-4-coumaric acid also has good inhibition effect on salmonella mutation induced by bleomycin, hydrogen peroxide and IQ type chirp peptide mutation; trans-4-coumaric acid also inhibits mixed-function oxidase activity, as well as somatic and sexual cell mutations caused by Cyclophosphthalimide (CP).

The trans-4-coumaric acid can be applied to the industries of medicine, agriculture, food, cosmetics and the like. As a medicine, the trans-4-coumaric acid has different degrees of inhibition effects on staphylococcus aureus, dysentery bacillus, escherichia coli and pseudomonas aeruginosa; the trans-4-coumaric acid has effects of reducing blood lipid, preventing gastric cancer, and reducing atherosclerosis. Is used as plant growth promoter in agricultural industry. Is used as a long-acting bactericide and a fruit and vegetable fresh-keeping preservative in the food industry. In the cosmetic industry, trans-4-coumaric acid has inhibitory effect on tyrosine monophenolase and diphenolase, so that it can be added into cosmetics for inhibiting melanin generation.

In addition, trans-4-coumaric acid is a precursor or intermediate of many compounds with higher application value. For example, p-coumaric acid can be synthesized into coumarin through oxidation and cyclization reactions; trans-4-coumaric acid generates p-hydroxybenzoic acid through degradation reaction; the trans-4-coumaric acid can also be used as intermediate for synthesis of substances such as rhododendron as expectorant and esmolol as anti-epinephrine, and can also be used for synthesis of Gannuo as medicine for clinically treating light and middle-degree senile dementia.

The wide application of trans-4-coumaric acid causes the trans-4-coumaric acid to have strong market demand, but the existing industrial production method of trans-4-coumaric acid cannot meet the demand. The existing production main methods mainly comprise the following methods: the extraction method comprises the following steps: the trans-4-coumaric acid extracted by the method is pure natural, but has low yield and high cost. Chemical synthesis: prepared by condensation reaction of p-hydroxybenzaldehyde and malonic acid, the method has the main defects of non-natural products, large chemical pollution in the production process and high cost.

The method for producing trans-4-coumaric acid by the biotransformation method has two ways: one is the tyrosine/(phenylalanine) ammonialyase pathway, which belongs to the enzymes under EC 4.3.1.23 and EC 4.3.1.25; the patent includes: a phenylalanine ammonia lyase derived from star-of-Bethlehem, nucleotide sequence and application thereof, the patent number is: 201310466953.2, now in an unauthorized-deemed withdrawn state. The use of saccharomycete and the production process of natural p-coumaric acid includes the following steps: 201510411910. the method has an enzyme (whole cell) catalysis method and a fermentation method for producing trans-4-coumaric acid, wherein the 201510411910 patent is a fermentation method, and the yield can reach about 10 g/L. The other is the trans-cinnamate-4-hydroxylase pathway, which is an enzyme under EC1.14.14.91; the patent includes: trans-cinnamic acid-4-hydroxylase, its coding gene and application are disclosed in the patent numbers: 201710136604, currently in the mid-trial-to-trial phase. The method described in the patent is a fermentation method, and the circulation of coenzyme and substrate are provided by host bacteria; the method has low yield and high purification cost.

Disclosure of Invention

The invention provides a method for synthesizing trans-4-coumaric acid by an enzyme catalysis method; trans-cinnamic acid-4-monooxygenase(EC1.14.14.91) can catalyze trans-cinnamic acid to synthesize trans-4-coumaric acid while consuming one molecule of O₂And one NAD (P) H to produce one H⁺And one NAD (P)⁺. Adding trans-cinnamic acid (raw material) and trace amount of NAD (P) into a single-water-phase or water/organic solvent two-phase reaction system⁺Or NAD (P) H, O₂Source, addition of reduced hydrogen donor, followed by addition of trans-cinnamic acid 4-monooxygenase and NAD (P)⁺Reductase, dripping alkali liquor to control pH and control reaction temperature; after the reaction is finished, the reaction solution is filtered, enzyme is removed, concentrated and crystallized by cooling to obtain the trans-4-coumaric acid, and the reaction formula is as follows:

the invention provides the following technical scheme:

a method for synthesizing trans-4-coumaric acid by enzyme catalysis comprises adding trans-cinnamic acid and trace amount of NAD (P) into a single-water phase or water/organic solvent two-phase reaction system⁺Or NAD (P) H, O₂Source of reduced hydrogen donor, followed by addition of trans-cinnamic acid-4-monooxygenase and NAD (P)⁺Reductase, dripping alkali liquor to control pH and control reaction temperature; after the reaction is finished, filtering the reaction solution to remove enzyme, concentrating, cooling and crystallizing to obtain trans-4-coumaric acid; wherein the trans-cinnamic acid-4-monooxygenase is numbered as follows: EC 1.14.14.91.

When the coenzyme required by trans-cinnamic acid-4-monooxygenase is NADH, a trace amount of NAD is added into the reaction system⁺Or NADH; when the coenzyme required by trans-cinnamic acid-4-monooxygenase is NADPH, a trace amount of NADP is added into the reaction system⁺Or NADPH.

Said O is₂The source being air, oxygen or H₂O₂Introducing air or oxygen into the reaction system or adding the air or the oxygen into the reaction system to supplement the consumed oxygen; the concentration of oxygen in the reaction system can be monitored by an oxygen dissolving electrode; the solubility of oxygen in the reaction solution can be increased by means of pressurization.

The alkali is ammonia water, NaOH or KOH; the pH control range is 5.0-10.0, preferably 6.0-8.0, and preferably 6.8-7.5.

The reduced hydrogen donor is glucose, isopropanol, formic acid or formate.

When the reduced hydrogen donor is glucose, NAD (P) is added in the reaction⁺The reductase is glucose dehydrogenase, which is capable of producing NAD (P) H by glucose hydrogen supply; when the reduced hydrogen donor is isopropanol, NAD (P) is added in the reaction⁺The reductase is an isopropanol dehydrogenase enzyme capable of producing NAD (P) H by hydrogen donation from isopropanol; when the reduced hydrogen donor is formic acid or formate, NAD (P) is added in the reaction⁺The reductase is a formate dehydrogenase enzyme capable of producing NAD (P) H by hydrogen donation from formate; when the coenzyme required for trans-cinnamic acid-4-monooxygenase is NADH, the NAD (P)⁺The reductase is NAD⁺Reductase, NAD (P) when NADPH is the coenzyme required for trans-cinnamic acid-4-monooxygenase⁺The reductase is NADP⁺A reductase.

The trans-cinnamic acid-4-monooxygenase comprises, but is not limited to, trans-cinnamic acid-4-monooxygenase having an amino acid sequence which can be SEQ01, SEQ02, SEQ03, SEQ04, SEQ05, SEQ06, SEQ07, SEQ08, SEQ09, SEQ10, SEQ11, SEQ12, SEQ13, SEQ14, SEQ15 or SEQ 16; or the trans-cinnamic acid-4-monooxygenase is a trans-cinnamic acid-4-monooxygenase which is a single and/or multiple point mutant having the amino acid sequence; or the trans-cinnamic acid-4-monooxygenase is a trans-cinnamic acid-4-monooxygenase of an amino acid sequence having a homology of 80% or more with the amino acid sequence.

The host used for expressing the trans-cinnamic acid-4-monooxygenase is one of escherichia coli, bacillus, mould or yeast; preferably, the expression system is an escherichia coli BL21 expression system or a pichia pastoris GS115 expression system.

The trans-cinnamic acid-4-monooxygenase can be fermentation liquor or bacterial sludge after the expression of the constructed host bacteria, crude enzyme subjected to simple treatment or pure enzyme subjected to purification treatment, liquid enzyme filtered by a flocculating agent and a ceramic membrane or immobilized enzyme subjected to immobilization treatment.

Compared with the prior art, the invention has the beneficial effects that:

the method belongs to a trans-cinnamic acid-4-hydroxylase pathway, and the method of applying enzyme catalysis directly takes trans-cinnamic acid as a substrate through NAD (P)⁺The reductase completes the circulation of coenzyme and efficiently synthesizes trans-4-coumaric acid outside cells. Compared with patent 201710136604, a more efficient trans-cinnamic acid-4-hydroxylase expression system can be constructed without considering the balance between cell metabolism and energy metabolism, and the simple improvement of the expression level of the enzyme is much easier; the amount of cells used in the enzyme-catalyzed process is 1/10, even 1/100 or less for the fermentation process at the same yield; the enzyme catalysis method has the advantages of simple reaction system, high substrate concentration of more than 100g/L, high molar conversion rate of more than 98%, simple reaction system, less impurities and low product extraction and purification cost.

Detailed Description

The following examples are merely illustrative of the practice of the present invention and the present invention includes, but is not limited to, the following examples.