阅读说明：本技术 一种咖啡酸和香兰素的制备方法及其反应催化剂的制备方法 (Preparation method of caffeic acid and vanillin and preparation method of reaction catalyst thereof ) 是由 林涛 徐明文 蒋丽丽 曹学松 于 2019-12-31 设计创作，主要内容包括：本发明公开了一种咖啡酸和香兰素的生物制造方法,第一步以葡萄糖等为起始原料,通过大肠杆菌菌种,发酵生产中间体3-脱氢莽草酸,中间体的浓度大于50g/L,糖质量转化率(A/G)为30-40%,可达到最大的理论转化率；第二步采用酶催化过程,通过自主开发合适的酶,以发酵过程获得的3-脱氢莽草酸为原料,生物酶催化得到中间体原儿茶酸；第三步采用酶催化过程,通过自主开发合适的酶,以前一步获得的原儿茶酸为原料,生物酶催化得到中间体咖啡酸；第四步采用酶催化过程,通过自主开发合适的酶,以前一步获得的咖啡酸为原料,生物酶催化得到产品香兰素。整个过程采用一步发酵加三步生物酶催化,合成产品香兰素。本发明的有益效果：以葡萄糖为原料,其价格实惠,来源广泛,而且整个工艺得到了简化,产物浓度高,可达到20-80g/L,降低香兰素的生产成本且减少环境污染；本路线副反应少,后处理简便,总收率高,可达25%；反应时间短,全部过程在72-96h内完成。(The invention discloses a biological preparation method of caffeic acid and vanillin, comprising the following steps of taking glucose and the like as initial raw materials, fermenting and producing an intermediate 3-dehydroshikimic acid by using an escherichia coli strain, wherein the concentration of the intermediate is more than 50G/L, the sugar mass conversion rate (A/G) is 30-40%, and the maximum theoretical conversion rate can be achieved; secondly, an enzyme catalysis process is adopted, a proper enzyme is independently developed, 3-dehydroshikimic acid obtained in the fermentation process is used as a raw material, and the intermediate protocatechuic acid is obtained through biological enzyme catalysis; thirdly, an enzyme catalysis process is adopted, the protocatechuic acid obtained in the previous step is used as a raw material, and the intermediate caffeic acid is obtained through biological enzyme catalysis by independently developing a proper enzyme; and step four, adopting an enzyme catalysis process, wherein the caffeic acid obtained in the previous step is used as a raw material, and the vanillin product is obtained by biological enzyme catalysis through self development of a proper enzyme. The whole process adopts one-step fermentation and three-step biological enzyme catalysis to synthesize the product vanillin. The invention has the beneficial effects that: the glucose is used as a raw material, the price is substantial, the source is wide, the whole process is simplified, the product concentration is high and can reach 20-80g/L, the production cost of the vanillin is reduced, and the environmental pollution is reduced; the route has less side reaction, simple and convenient post-treatment and high total yield which can reach 25 percent; the reaction time is short, and the whole process is completed within 72-96 h.)

1. A method for preparing caffeic acid comprises the following steps:

the method comprises the following steps: a method for obtaining a 3-dehydroshikimic acid raw material by a fermentation method, namely a process for synthesizing the raw material by microorganisms by adopting natural source sugar;

step two: fermenting glucose to obtain 3-dehydroshikimic acid, and catalyzing by using a 3-dehydroshikimic acid dehydratase catalyst to obtain protocatechuic acid;

step three: dissolving the protocatechuic acid obtained in the step two, adding phosphate, adjusting the pH to 6-11, and adding an o-methyltransferase catalyst (OMT) and a cofactor S-adenosylmethionine (SAM);

step four: stirring the reaction system in the third step at a constant temperature of 25-40 ℃ for reacting for 16-24 hours to obtain the caffeic acid.

2. A preparation method of vanillin comprises the following specific steps:

the method comprises the following steps: a method for obtaining a 3-dehydroshikimic acid raw material by a fermentation method, namely a process for synthesizing the raw material by microorganisms by adopting natural source sugar;

step two: fermenting glucose to obtain 3-dehydroshikimic acid, and catalyzing by using a 3-dehydroshikimic acid dehydratase catalyst to obtain protocatechuic acid;

step three: adding an o-methyltransferase catalyst (OMT) and a cofactor SAMS-adenosylmethionine into the reaction system of the second step, and catalyzing to obtain a caffeic acid system liquid;

step four: adding an aromatic carboxylic acid reductase catalyst (ACAR) and an auxiliary factor NAD into the reaction system in the third step, stirring at a constant temperature of 25-40 ℃, reacting for 16-24 hours, and catalyzing to obtain vanillin.

3. The method for preparing the o-methyltransferase catalyst of claim 1, comprising the steps of:

the method comprises the following steps: recombining o-methyltransferase (OMT) fragments to a pET21a vector, and transforming an expression host bacterium BL21(DE3) by a positive recombinant plasmid pET21a (+) to obtain a prokaryotic expression strain pET21a (+)/BL 21(DE 3);

step two: after the above expression strain was cultured overnight with shaking at 37 ℃ and 200rpm in 5mL LB liquid medium containing 100ug/mL ampicillin at the final concentration, it was inoculated at 1% (V/V) ratio to 500mL LB liquid medium containing 100ug/mL ampicillin, cultured with shaking at 37 ℃ and 200rpm, and when OD600 was between 0.8 and 1.0, inducer IPTG (isopropyl-. beta. -D-thiogalactoside, IPTG) was added at the final concentration of 0.1mM, and it was induced overnight at 25 ℃;

step three: centrifuging the solution obtained in step two at 8000rpm, collecting, suspending in 50mM sodium phosphate buffer solution with pH7.0, ultrasonicating (200W, 3s/5s, 10min), centrifuging at 8000rpm for 20min at 4 deg.C to obtain o-methyltransferase catalyst (OMT).

4. The method for producing an ortho-methyltransferase catalyst according to claim 3, characterized in that: the culture medium in the second step comprises: 10g/L tryptone (OXOD), 5g/L yeast powder (OXOD), 10g/L sodium chloride (national reagent).

5. The method for preparing an aromatic carboxylic acid reductase catalyst (ACAR) according to claim 2, comprising the steps of:

the method comprises the following steps: respectively recombining Aromatic Carboxylic Acid Reductase (ACAR) to pET21a vectors, and transforming expression host bacteria BL21(DE3) by using a positive recombinant plasmid-pET 21a (+) to obtain a prokaryotic expression strain pET21a (+)/BL 21(DE 3);

step two: after the above expression strain was cultured overnight with shaking at 37 ℃ and 200rpm in 5mL LB liquid medium containing 100ug/mL ampicillin at the final concentration, it was inoculated at 1% (V/V) ratio to 500mL LB liquid medium containing 100ug/mL ampicillin, cultured with shaking at 37 ℃ and 200rpm, and when OD600 was between 0.8 and 1.0, inducer IPTG (isopropyl-. beta. -D-thiogalactoside, IPTG) was added at the final concentration of 0.1mM, and it was induced overnight at 25 ℃;

step three: centrifuging the solution obtained in step two at 8000rpm, collecting, suspending in 50mM sodium phosphate buffer solution with pH7.0, ultrasonicating (200W, 3s/5s, 10min), centrifuging at 8000rpm for 20min at 4 deg.C to obtain aromatic carboxylic acid reductase catalyst (ACAR).

Technical Field

The invention relates to a technology for preparing aromatic ring compound derivatives by an enzyme catalysis method, in particular to a process for producing vanillin by a biological method.

Background

Vanillin is commonly known as vanilla powder, vanillin, Yunnan powder, vanilla essence, vanillin, etc. Is extracted from Vanilla planifolia of Rutaceae. White to yellowish crystalline or crystalline powder, slightly sweet. It is soluble in hot water, glycerol and alcohol, and is not easily dissolved in cold water and vegetable oil. The fragrance is stable and is not easy to volatilize at higher temperature. It is easy to oxidize in air and is easy to change color when it is exposed to alkaline substances.

The 3-methoxy-4-hydroxybenzaldehyde has a chemical name of 3-methoxy-4-hydroxybenzaldehyde, has vanilla bean fragrance and strong milk fragrance, plays roles in enhancing and fixing fragrance, is widely applied to industries such as cosmetics, tobacco, cakes, candies, baked foods and the like, is one of synthetic spice varieties with the largest global yield, and has a history of more than 100 years for industrial production of vanillin. Vanillin can also be used as plant growth promoter, bactericide, lubricant defoamer, and is also an important intermediate for synthesizing medicaments and other spices. Besides, it can be used as glazing agent in electroplating industry, ripener in agriculture, deodorant in rubber products, anti-hardening agent in plastic products and medical intermediate, etc. and is widely used.

At present, the production process of vanillin mainly comprises two processes:

chemical synthesis method: the main route is a guaiacol-glyoxylic acid synthesis method which is shown as the following route and is represented by a reaction formula 1:

route for chemically synthesizing vanillin from guaiacol-glyoxylic acid

Guaiacol and glyoxylic acid are used as raw materials, condensation reaction is carried out by using strong base as a catalyst, and then a series of chemical processes such as oxidation, acidification, hydrolysis and the like are carried out to prepare the product. The process is simple to operate, but the strong acid and the strong base have great environmental pollution and limited raw material sources, so the costs of the raw materials of guaiacol (more than 4 ten thousand/ton) and glyoxylic acid (more than 2 ten thousand/ton) are higher.

Biosynthesis of natural vanillin: the method for synthesizing vanillin by adopting natural products as sources is numerous, and the raw material sources are used for distinguishing ferulic acid extracted from vanilla pods, rice bran and the like, eugenol extracted from clove, turmeric, corn starch sugar and the like. The vanillin prepared from the vanillin pod source is expensive, the vanillin prepared from ferulic acid extracted from rice bran and the like belongs to a medium price, the large-scale production of vanillin from the last three raw material sources is rarely reported, the vanillin is obtained by directly fermenting American international essence and flavor (IFF) from corn sugar in 2014, and the natual vanillin is marked to enter the market, but the product obtained by directly fermenting the corn sugar has low concentration, only 0.5-1g/L, and the cost of the raw material sugar for production and the cost of post-extraction concentration are high.

The literature reports that: european (European Directive 88/388/CEE, JO number L184, 22 June1988) and US regulations stipulate that vanillin produced by microbial conversion of ferulic acid is a natural product, but European regulations are more stringent, for example, corn cob obtained by alkaline hydrolysis of ferulic acid, which is not considered natural in European regulations, while US regulations are still considered natural. Therefore, to prepare vanillin from natural sources, chemical treatment processes should be avoided as much as possible, if the vanillin is to be introduced into the European market.

The two methods for preparing vanillin have defects, and the chemical synthesis method has great environmental pollution and is difficult to enter the European market. The biosynthesis method uses plant resources as raw materials, and has high cost.

Disclosure of Invention

In order to solve the problems, the invention provides a preparation method of caffeic acid and vanillin and a preparation method of a reaction catalyst thereof, and the specific technical scheme is as follows:

a method for preparing an o-methyltransferase catalyst and an aromatic carboxylic acid reductase comprises the following steps:

the method comprises the following steps: respectively recombining o-methyltransferase (OMT) fragments or Aromatic Carboxylic Acid Reductase (ACAR) fragments to a pET21a vector, and transforming an expression host bacterium BL21(DE3) by using a positive recombinant plasmid pET21a (+) to obtain a prokaryotic expression strain pET21a (+)/BL 21(DE 3);

step two: the above-mentioned expression strain was cultured overnight with shaking at 37 ℃ and 200rpm in 5mL of LB liquid medium to which 100ug/mL of ampicillin was added at the final concentration, and then inoculated at 1% (V/V) into 500mL of LB liquid medium containing 100ug/mL of ampicillin at 37 ℃ and 200 rpm. When OD600 is between 0.8-1.0, adding inducer IPTG (isopropyl-beta-D-thiogalactoside, IPTG) with final concentration of 0.1mM, and inducing at 25 deg.C overnight;

step three: centrifuging the solution obtained in step two at 8000rpm, collecting, suspending in 50mM sodium phosphate buffer solution with pH7.0, ultrasonicating (200W, 3s/5s, 10min), centrifuging at 8000rpm at 4 deg.C for 20min to obtain o-methyltransferase catalyst (OMT) or aromatic carboxylic acid reductase catalyst (ACAR).

Preferably, it is characterized in that: the culture medium in the second step comprises: 10g/L tryptone (OXOD), 5g/L yeast powder (OXOD), 10g/L sodium chloride (national reagent).

In the present invention, the o-methyltransferase is derived from human (Homo sapiens) and the aromatic carboxylate reductase is derived from escherichia coli (e.

The enzyme may be in the form of a catalyst: pure enzyme, corresponding recombinant bacteria resting cells, crude enzyme liquid or crude enzyme powder and the like.

A method for preparing caffeic acid comprises the following steps:

the method comprises the following steps: glucose is fermented to obtain 3-dehydroshikimic acid, and then protocatechuic acid is obtained by catalysis of a 3-dehydroshikimic acid dehydratase catalyst (Chinese patent application No. 2019104520898).

Step two: dissolving the protocatechuic acid obtained in the first step and adding phosphate to adjust the pH to 6-11, adding the o-methyltransferase catalyst and the cofactor S-adenosylmethionine (SAM) of claim 1;

step three: and (5) stirring the reaction system in the second step at a constant temperature of 25-40 ℃ for reacting for 16-24 hours to obtain the caffeic acid.

Preferably, it is characterized in that: phosphate was added to a final concentration of 50mM in step two.

The caffeic acid is a biocatalytic reaction which takes protocatechuic acid (PCA) as a raw material and uses an o-methyltransferase catalyst (OMT), and the reaction equation is as follows:

the reaction system is as follows: o-methyltransferase catalyst (OMT), phosphate buffer, protocatechuic acid, cofactor S-adenosylmethionine (SAM), sodium chloride. Specifically, the dosage of enzyme is 1-10g/L, the concentration of buffer is 50-200mM, the pH value of buffer is 6.0-11, the concentration of sodium chloride is 10-100mmol, and the concentration of substrate is 20-80 g/L. After the reaction, the product is quenched by acetonitrile and then is verified by High Performance Liquid Chromatography (HPLC), the reaction conversion rate can reach 80%, 90%, or 95%, or more than 99%, and the purity value of the product can reach 90%, or 95%, or more than 99%.

A preparation method of vanillin comprises the following specific steps:

the method comprises the following steps: dissolving caffeic acid obtained in claim 1 in an aqueous solution of phosphate, adjusting pH to 6-11;

step two: adding the aromatic carboxylic acid reductase catalyst (ACAR) of claim 1 and the cofactor NAD to the system of step one;

step three: and (5) stirring the reaction system in the second step at a constant temperature of 25-40 ℃ for reacting for 16-24 hours to obtain vanillin.

Preferably, it is characterized in that: the phosphate concentration in step one was 50 mM.

The vanillin is subjected to a biocatalytic reaction by using an aromatic carboxylic acid reductase catalyst (ACAR) on the basis of caffeic acid, and the reaction equation is as follows:

the reaction system is as follows: an aromatic carboxylic acid reductase catalyst (ACAR), phosphate buffer, caffeic acid, a cofactor such as NAD or NADP. Specifically, the dosage of enzyme is 1-10g/L, the concentration of buffer is 50-200mM, the pH value of buffer is 6.0-11, and the concentration of substrate is 20-80 g/L. After the reaction, the product is quenched by acetonitrile and then is verified by High Performance Liquid Chromatography (HPLC), the reaction conversion rate can reach 90 percent, or 95 percent, or more than 99 percent, and the purity value of the product can reach 90 percent, or 95 percent, or more than 99 percent.

According to the invention, 3-dehydroshikimic acid obtained by fermenting glucose is used as a substrate, protocatechuic acid is generated through enzyme catalysis, caffeic acid can be obtained through enzyme catalysis, and vanillin is obtained through further enzyme catalysis, so that the problem of raw material source in the existing method is solved, the process is obviously simplified, the environmental pollution is reduced, and the production cost of vanillin is reduced.

Drawings

FIG. 1 is a HPLC chromatogram of the catalytic production of protocatechuic acid from 3-dehydroshikimic acid in example 3/4;

FIG. 2 is a HPLC chromatogram of the catalytic production of caffeic acid from protocatechuic acid in example 5/6/7;

FIG. 3 is a HPLC survey of the caffeic acid catalyzed production of vanillin of example 8/9/10/11;

Detailed Description

In order to enhance the understanding of the present invention, the present invention will be further described in detail with reference to the following examples, which are provided for the purpose of illustration only and are not intended to limit the scope of the present invention.