CN114350717A - Method for preparing 3, 4-dihydroxy phenethyl alcohol by using biological enzyme catalysis - Google Patents
Method for preparing 3, 4-dihydroxy phenethyl alcohol by using biological enzyme catalysis Download PDFInfo
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- JUUBCHWRXWPFFH-UHFFFAOYSA-N Hydroxytyrosol Chemical compound OCCC1=CC=C(O)C(O)=C1 JUUBCHWRXWPFFH-UHFFFAOYSA-N 0.000 title claims abstract description 141
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- 238000000034 method Methods 0.000 title claims abstract description 17
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- YCCILVSKPBXVIP-UHFFFAOYSA-N 2-(4-hydroxyphenyl)ethanol Chemical compound OCCC1=CC=C(O)C=C1 YCCILVSKPBXVIP-UHFFFAOYSA-N 0.000 claims abstract description 79
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- 238000000855 fermentation Methods 0.000 claims abstract description 28
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Abstract
The invention discloses a method for preparing 3, 4-dihydroxy phenylethanol by bio-enzyme catalysis, which comprises the steps of taking escherichia coli fermentation liquor for inducing enzyme production as a raw material, centrifuging to obtain thalli, adding a buffer solution to suspend the thalli, introducing oxygen and the like, adding a substrate of p-hydroxyphenylethanol, and carrying out reaction by bio-enzyme catalysis to obtain the 3, 4-dihydroxy phenylethanol. According to the invention, a p-hydroxyphenylethanol substrate is used, the 3, 4-dihydroxyphenylethanol is obtained by biological synthesis through biological enzyme catalysis, the reaction condition is mild, no by-product is generated, and the residual quantity of the substrate is small; the biological enzyme catalysis efficiency is high and reaches 99 percent, and the yield of the product 3, 4-dihydroxy phenethyl alcohol reaches more than 13 g/L; the preparation method disclosed by the invention is green and environment-friendly, has a short reaction period, can be applied to large-scale industrial production, and meets the market demand.
Description
Technical Field
The invention relates to a method for preparing 3, 4-dihydroxy phenethyl alcohol by biological enzyme catalysis, belonging to the technical field of biological engineering.
Background
3, 4-Dihydroxyphenylethanol, also known as hydroxytyrosol, of the formula C8H10O3The compound is a natural polyphenol compound, is found in olive oil and wastewater generated by processing the olive oil, is mainly free 3, 4-dihydroxyphenyl ethanol obtained by hydrolyzing esterified oleuropein, and researches show that the 3, 4-dihydroxyphenyl ethanol has stronger antioxidant activity and multiple biological and pharmacological activities, can prevent osteoporosis, is beneficial to treating diseases such as diabetes, obesity and the like related to mitochondrial dysfunction, can reduce the morbidity of the diseases, and is widely appliedCan be used in food industry, nutrition, health promotion and cosmetics. Therefore, the extraction and preparation of the 3, 4-dihydroxy phenylethanol have certain practical significance and better economic benefit.
3, 4-dihydroxy benzene ethanol, the traditional preparation method is to extract from olive oil or olive leaves, a large amount of flammable and combustible organic reagents such as ethyl acetate, absolute ethyl alcohol, dichloromethane and the like are needed in the extraction process, the extraction yield is only 8.4%, the yield is low, and meanwhile, the extraction process contains more byproducts, so that the purity of the p-hydroxyphenylethanol is low; the currently commonly used preparation method is generally a synthetic preparation, for example, the synthetic method of hydroxytyrosol disclosed by xiaoyang, zhangyang and the like, and reports that catechol and glyoxylic acid are used as raw materials, 3, 4-dihydroxyphenyl ethanol is synthesized by 3, 4-dihydroxymandelic acid, 3, 4-dihydroxyphenyl ethanol is synthesized by four-step reaction, the total yield is 52.7%, and the yield is low; the other preparation method adopts a biological preparation method, for example, the engineering bacteria and the application disclosed in Chinese patent CN201710659225.1, reports that the four enzymes of L-amino acid oxidase, alpha-keto acid decarboxylase, alcohol dehydrogenase, NAD (P) reductase are co-expressed in escherichia coli to biosynthesize 2-phenethyl alcohol, tyrosol and hydroxytyrosol, wherein, the yield of the 3, 4-dihydroxyphenethyl alcohol is 435mg/L and is lower; chinese patent CN201710659225.1 discloses an Escherichia coli for expressing hydroxytyrosol and hydroxytyrosol glucoside, a construction method and application thereof, and reports that the constructed Escherichia coli contains and can express ARO10, HpaBC and UGT genes, recombinant Escherichia coli tyrosine is expressed through related genes to obtain 3, 4-dihydroxyphenyl ethanol, the yield can reach 401mg/L, and the yield is low. Therefore, in order to improve the yield and the conversion rate of the 3, 4-dihydroxy phenethyl alcohol, the invention provides a method for preparing the 3, 4-dihydroxy phenethyl alcohol by using biological enzyme catalysis.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for preparing 3, 4-dihydroxyphenyl ethanol by bio-enzyme catalysis, which takes a strain for inducing the production of 3, 4-dihydroxyphenyl ethanol as bio-enzyme and takes p-hydroxyphenylethanol as a substrate, and synthesizes the 3, 4-dihydroxyphenyl ethanol by bio-enzyme catalysis, has high catalytic efficiency and no by-product, and can be used for large-scale industrial production.
In order to achieve the purpose, the invention adopts the following technical scheme: a method for preparing 3, 4-dihydroxy phenethyl alcohol by bio-enzyme catalysis specifically comprises the following steps:
s1, carrying out centrifugal treatment on the escherichia coli fermentation liquor for inducing enzyme production, wherein the centrifugal rotation speed is 2000-8000 rpm/min, the centrifugal time is 1-10 min, and removing the centrifugal liquor to obtain thalli;
s2, adding a buffer solution into the thalli in the step S1 to enable the thalli to be suspended, moving the thalli into a four-neck flask, then carrying out magnetic stirring at the stirring speed of 200-1200 rpm/min, carrying out water bath heating at the heating temperature of 25-45 ℃, then introducing oxygen into the thalli suspension, and controlling the pH in real time;
s3, weighing a p-hydroxyphenylethanol substrate, dissolving and mixing uniformly by using a small amount of buffer solution, adding the p-hydroxyphenylethanol substrate into a four-neck flask for many times, continuously stirring for reaction, detecting the p-hydroxyphenylethanol substrate in a reaction solution by using a high performance liquid phase, stopping stirring and closing ventilation when the p-hydroxyphenylethanol substrate does not exist in the reaction solution, stopping the reaction, obtaining a reaction solution of a 3, 4-dihydroxyphenylethanol product, and storing the reaction solution in a cold storage way;
s4, detecting and analyzing the 3, 4-dihydroxy phenylethanol product in the reaction solution in the step S3 by adopting high performance liquid chromatography, so that the yield of the 3, 4-dihydroxy phenylethanol is over 13g/L, and the conversion rate of the 3, 4-dihydroxy phenylethanol is over 99%.
Preferably, the total adding mass of the p-hydroxyphenylethanol is less than or equal to 13.9g, the total adding mass of the p-hydroxyphenylethanol is controlled to be 12.0-12.5 g/L, and the total adding mass of the p-hydroxyphenylethanol is equal to the total adding mass of the p-hydroxyphenylethanol/the total volume of the buffer solution.
Preferably, the biomass of the thallus is 15-45 g/m3。
Preferably, the addition amount of the p-hydroxyphenylethanol is 0.5-5 g/30min each time.
Preferably, the escherichia coli fermentation broth is one of escherichia coli MG1655 fermentation broth, escherichia coli BL21(DE3) fermentation broth, or escherichia coli BMGA fermentation broth.
Preferably, the buffer is one of a PB buffer, a PBs buffer or a TRIS buffer
Preferably, the oxygen introduction amount is 0.5-5L/min.
Preferably, the pH value is 5.5-8.5.
Preferably, the temperature for refrigerated storage is 4 ℃.
The invention has the beneficial effects that: according to the invention, a p-hydroxyphenylethanol substrate is used, the 3, 4-dihydroxyphenylethanol is obtained by biological synthesis through biological enzyme catalysis, the reaction condition is mild, no by-product is generated, and the residual quantity of the substrate is small; the total adding quality of the p-hydroxyphenylethanol is controlled to be not more than 13.9g, so that the biological enzyme has higher catalytic efficiency, the enzyme catalytic efficiency is higher than 99%, the total adding quantity of the p-hydroxyphenylethanol is controlled to be 12.0-12.5 g/L, the yield of the product 3, 4-dihydroxyphenylethanol is increased to 13.78g/L, and the conversion rate of the product 3, 4-dihydroxyphenylethanol is increased to 99.4%; the preparation method can maximize the utilization of the hydroxyphenylethanol, the biological enzyme and the buffer solution, and can not cause resource waste; meanwhile, the preparation method is green and environment-friendly, has short reaction period, can be applied to large-scale industrial production, and meets the market demand.
Drawings
FIG. 1 is a schematic representation of the 3, 4-dihydroxybenzene ethanol biosynthesis scheme according to the present invention;
FIG. 2 is a standard graph of p-hydroxyphenylethanol according to the present invention;
FIG. 3 is a graph of a 3, 4-dihydroxybenzene ethanol standard curve according to the present invention;
FIG. 4 is a high performance liquid chromatogram of a reaction solution of a 3, 4-dihydroxybenzene ethanol product obtained in example 10 of the present invention.
Detailed Description
In order to more clearly and completely illustrate the present invention, the following examples are given by way of illustration of the present invention, and are not intended to limit the present invention.
In the following examples, the construction methods of Escherichia coli MG1655, Escherichia coli BL21(DE3) or Escherichia coli BMGA, which are used as a gene expression protease in the present invention, and Escherichia coli MG1655, Escherichia coli BL21(DE3) or Escherichia coli BMGA were used in accordance with the reports of patents (Escherichia coli expressing hydroxytyrosol and hydroxytyrosol glucoside, construction methods and applications, CN 201610158902.7).
The constructed recombinant escherichia coli is placed in a culture solution for overnight culture to obtain an escherichia coli fermentation culture solution for expressing protease, and the culture process is as follows:
firstly, placing the constructed recombinant Escherichia coli MG1655 into a culture solution, wherein the culture solution comprises tryptone 10g, yeast extract 5g, NaCl 10g and pure water to a constant volume of 1.0L, and carrying out overnight culture at 37 ℃ and pH7.0 to obtain Escherichia coli MG1655 fermentation liquor.
Secondly, the constructed escherichia coli BL21(DE3) is placed in a culture solution, the culture solution comprises 10g of tryptone, 5g of yeast extract, 10g of NaCl and pure water with constant volume of 1.0L, and the fermentation solution is cultured overnight at 37 ℃ and pH of 7.0 to obtain escherichia coli BL21(DE3) fermentation solution.
And thirdly, placing the constructed escherichia coli BMGA into a culture solution, wherein the culture solution comprises 10g of tryptone, 5g of yeast extract, 10g of NaCl and pure water with constant volume of 1.0L, and performing overnight culture at 37 ℃ and pH of 7.0 to obtain the escherichia coli BMGA fermentation liquor.
The gene-expressed proteases of Escherichia coli obtained by obtaining the fermentation broth of Escherichia coli MG1655, Escherichia coli BL21(DE3) or Escherichia coli BMGA as described above were used in the following examples.
Example 1
(1) Putting the fermentation liquor of Escherichia coli MG1655 for inducing enzyme production into a centrifuge tube, centrifuging for 3min in a centrifuge with the rotation speed of 2000rpm/min, removing the centrifuged clear liquid, and collecting thallus;
(2) the cells were suspended by adding 90ml of PB buffer solution to the cells, and the biomass of the cells was 15g/m3Then pouring the mixture into a four-neck flask, putting the four-neck flask into a magnetic stirring water bath kettle with the rotating speed of 200rpm/min and the temperature of 25 ℃, introducing oxygen into the thallus suspension at the oxygen introduction rate of 0.5L/min, and regulating the pH value to 5.5 in real time by using ammonia water;
(3) weighing 0.5g of p-hydroxyphenylethanol substrate, dissolving in 10ml of PB buffer solution, after complete dissolution, dropwise adding into a four-neck flask at one time at a rate of 0.5g/30min, continuously stirring for reaction, detecting the p-hydroxyphenylethanol substrate in the reaction solution through a liquid phase, stopping stirring and closing ventilation when no p-hydroxyphenylethanol substrate exists in the reaction solution, stopping the reaction, obtaining a reaction solution of a 3, 4-dihydroxyphenylethanol product, and refrigerating and storing the reaction solution at a temperature of 4 ℃.
Example 2
(1) Putting the fermentation liquor of Escherichia coli MG1655 for inducing enzyme production into a centrifuge tube, centrifuging for 3min in a centrifuge with the rotation speed of 2000rpm/min, removing the centrifuged clear liquid, and collecting thallus;
(2) 40ml of PB buffer was added to the cells, and the cells were suspended to give biomass of 42g/m3Then pouring the mixture into a four-neck flask, putting the four-neck flask into a magnetic stirring water bath kettle with the rotating speed of 200rpm/min and the temperature of 25 ℃, introducing oxygen into the thallus suspension at the oxygen introduction rate of 0.5L/min, and regulating the pH value to 5.5 in real time by using ammonia water;
(3) weighing 0.5g of p-hydroxyphenylethanol substrate, dissolving in 1.5ml of PB buffer solution, completely dissolving and uniformly mixing, dripping into a four-neck flask at 0.5g/30min for one time, continuously stirring for reaction, detecting the p-hydroxyphenylethanol substrate in the reaction solution through a liquid phase, stopping stirring and closing ventilation when no p-hydroxyphenylethanol substrate exists in the reaction solution, stopping the reaction, obtaining the reaction solution of the 3, 4-dihydroxyphenylethanol product, and refrigerating and storing the reaction solution at the temperature of 4 ℃.
Example 3
(1) Putting the fermentation liquor of Escherichia coli MG1655 for inducing enzyme production into a centrifuge tube, centrifuging for 3min in a centrifuge with the rotation speed of 2000rpm/min, removing the centrifuged clear liquid, and collecting thallus;
(2) 250ml of PB buffer was added to the cells, and the cells were suspended to give a biomass of 15g/m3Then pouring the mixture into a four-neck flask, putting the four-neck flask into a magnetic stirring water bath kettle with the rotating speed of 200rpm/min and the temperature of 25 ℃, introducing oxygen into the thallus suspension at the oxygen introduction rate of 0.5L/min, and regulating the pH value to 5.5 in real time by using ammonia water;
(3) weighing 3.25g of p-hydroxyphenylethanol substrate, dissolving in 20ml of PB buffer solution, completely dissolving and uniformly mixing, dripping into a four-neck flask for five times at 0.65g/30min, continuously stirring for reaction, detecting the p-hydroxyphenylethanol substrate in the reaction solution through a liquid phase, stopping stirring and closing ventilation when no p-hydroxyphenylethanol substrate exists in the reaction solution, stopping the reaction, obtaining the reaction solution of the 3, 4-dihydroxyphenylethanol product, and refrigerating and storing the reaction solution at the temperature of 4 ℃.
Example 4
(1) Placing the fermentation broth of Escherichia coli BL21(DE3) for inducing enzyme production in a centrifuge tube, centrifuging for 3min in a centrifuge with the rotation speed of 2000rpm/min, removing the supernatant, and collecting the thallus;
(2) the cells were suspended by adding 350ml of PB buffer solution to the cells, and the biomass of the cells was 40g/m3Then pouring the mixture into a four-neck flask, putting the four-neck flask into a magnetic stirring water bath kettle with the rotating speed of 500rpm/min and the temperature of 25 ℃, introducing oxygen into the thallus suspension at the oxygen introduction rate of 1L/min, and regulating the pH value to 8.5 by ammonia water in real time;
(3) weighing 4.45g of p-hydroxyphenylethanol substrate, dissolving in 10ml of PB buffer solution, completely dissolving and uniformly mixing, dripping into a four-neck flask for five times at 0.89g/30min, continuously stirring for reaction, detecting the p-hydroxyphenylethanol substrate in the reaction solution through a liquid phase, stopping stirring and closing ventilation when no p-hydroxyphenylethanol substrate exists in the reaction solution, stopping the reaction, obtaining the reaction solution of the 3, 4-dihydroxyphenylethanol product, and refrigerating and storing the reaction solution at the temperature of 4 ℃.
Example 5
(1) Placing the fermentation liquor of Escherichia coli BMGA inducing enzyme production in a centrifuge tube, centrifuging for 3min in a centrifuge with the rotation speed of 3000rpm/min, removing the centrifuged clear liquid, and collecting thallus;
(2) the cells were suspended by adding 500ml of PB buffer solution to the cells to give a biomass of 20g/m3Then pouring the mixture into a four-neck flask, putting the four-neck flask into a magnetic stirring water bath kettle with the rotating speed of 500rpm/min and the temperature of 25 ℃, introducing oxygen into the thallus suspension at the oxygen introduction rate of 2L/min, and regulating the pH value to be 6.6 by ammonia water in real time;
(3) weighing 6.2g of p-hydroxyphenylethanol substrate, dissolving in 10ml of PB buffer solution, completely dissolving and uniformly mixing, dripping into a four-neck flask for two times at a speed of 3.1g/30min, continuously stirring for reaction, detecting the p-hydroxyphenylethanol substrate in the reaction solution through a liquid phase, stopping stirring and closing ventilation when no p-hydroxyphenylethanol substrate exists in the reaction solution, stopping the reaction, obtaining the reaction solution of the 3, 4-dihydroxyphenylethanol product, and refrigerating and storing the reaction solution at a temperature of 4 ℃.
Example 6
(1) Placing the fermentation liquor of Escherichia coli BMGA inducing enzyme production in a centrifuge tube, centrifuging for 5min in a centrifuge with the rotation speed of 4000rpm/min, removing the centrifuged clear liquid, and collecting thalli;
(2) 700ml of TRIS buffer was added to the cells, and the cells were suspended to give 20g/m biomass3Then pouring the mixture into a four-neck flask, putting the four-neck flask into a magnetic stirring water bath kettle with the rotating speed of 500rpm/min and the temperature of 25 ℃, introducing oxygen into the thallus suspension at the oxygen introduction rate of 4L/min, and regulating the pH value to be 6.5 by ammonia water in real time;
(3) weighing 8.5g of p-hydroxyphenylethanol substrate, dissolving in 8ml of TRIS buffer solution, completely dissolving and uniformly mixing, dripping into a four-neck flask for two times at 4.25g/30min, continuously stirring for reaction, detecting the p-hydroxyphenylethanol substrate in the reaction solution through a liquid phase, stopping stirring and closing ventilation when no p-hydroxyphenylethanol substrate exists in the reaction solution, stopping the reaction, obtaining the reaction solution of the 3, 4-dihydroxyphenylethanol product, and refrigerating and storing the reaction solution at the temperature of 4 ℃.
Example 7
(1) Placing the fermentation broth of Escherichia coli BL21(DE3) for inducing enzyme production in a centrifuge tube, centrifuging for 7min in a centrifuge with the rotation speed of 6000rpm/min, removing the supernatant, and collecting the thallus;
(2) 880ml of PBS buffer was added to the cells, and the cells were suspended to give biomass of 35g/m3Then pouring the mixture into a four-neck flask, putting the four-neck flask into a magnetic stirring water bath kettle with the rotating speed of 800rpm/min and the temperature of 45 ℃, introducing oxygen into the thallus suspension at the oxygen introduction rate of 5L/min, and regulating the pH value to 7.2 by ammonia water in real time;
(3) weighing 10.8g of p-hydroxyphenylethanol substrate, dissolving in 20ml of PBS buffer solution, completely dissolving and uniformly mixing, dropwise adding into a four-neck flask for ten times at 1.08g/30min, continuously stirring for reaction, detecting the p-hydroxyphenylethanol substrate in the reaction solution through a liquid phase, stopping stirring and closing ventilation when no p-hydroxyphenylethanol substrate exists in the reaction solution, stopping the reaction, obtaining the reaction solution of the 3, 4-dihydroxyphenylethanol product, and refrigerating and storing the reaction solution at the temperature of 4 ℃.
Example 8
(1) Putting the Escherichia coli MG1655 fermentation liquid for inducing enzyme production into a centrifuge tube, centrifuging for 9min in a centrifuge with the rotation speed of 8000rpm/min, removing the centrifuged clear liquid, and collecting thallus;
(2) the cells were suspended by adding 1000ml of PBS buffer solution to the cells, and the biomass of the cells was 40g/m3Then pouring the mixture into a four-neck flask, putting the four-neck flask into a magnetic stirring water bath kettle with the rotating speed of 1000rpm/min and the temperature of 35 ℃, introducing oxygen into the thallus suspension at the oxygen introduction rate of 3.1L/min, and regulating the pH value to be 8.5 by ammonia water in real time;
(3) weighing 12.24g of p-hydroxyphenylethanol substrate, dissolving in 10ml of PBS buffer solution, completely dissolving and uniformly mixing, dripping into a four-neck flask for six times at a speed of 2.04g/30min, continuously stirring for reaction, detecting the p-hydroxyphenylethanol substrate in the reaction solution through a liquid phase, stopping stirring and closing ventilation when no p-hydroxyphenylethanol substrate exists in the reaction solution, stopping the reaction, obtaining the reaction solution of the 3, 4-dihydroxyphenylethanol product, and refrigerating and storing the reaction solution at a temperature of 4 ℃.
Example 9
(1) Placing the fermentation broth of Escherichia coli BL21(DE3) for inducing enzyme production in a centrifuge tube, centrifuging for 10min in a centrifuge with the rotation speed of 8000rpm/min, removing the supernatant, and collecting thallus;
(2) 1100ml of TRIS buffer was added to the cells, and the cells were suspended to give a biomass of 45g/m3Then pouring the mixture into a four-neck flask, putting the four-neck flask into a magnetic stirring water bath kettle with the rotation speed of 1200rpm/min and the temperature of 27 ℃, then introducing oxygen into the thallus suspension at the oxygen introduction rate of 5L/min, and usingAmmonia water regulates and controls the pH value to be 6.3 in real time;
(3) weighing 13.9g of p-hydroxyphenylethanol substrate, dissolving in 16ml of TRIS buffer solution, completely dissolving and uniformly mixing, dripping into a four-neck flask for eight times at 1.7375g/30min, continuously stirring for reaction, detecting the p-hydroxyphenylethanol substrate in the reaction solution through a liquid phase, stopping stirring and closing ventilation when no p-hydroxyphenylethanol substrate exists in the reaction solution, stopping the reaction, obtaining the reaction solution of the 3, 4-dihydroxyphenylethanol product, and refrigerating and storing the reaction solution at the temperature of 4 ℃.
Example 10
(1) Placing the fermentation broth of Escherichia coli BL21(DE3) for inducing enzyme production in a centrifuge tube, centrifuging for 10min in a centrifuge with the rotation speed of 8000rpm/min, removing the supernatant, and collecting thallus;
(2) 1100ml of TRIS buffer was added to the cells, and the cells were suspended to give a biomass of 45g/m3Then pouring the mixture into a four-neck flask, putting the four-neck flask into a magnetic stirring water bath kettle with the rotation speed of 1200rpm/min and the temperature of 27 ℃, introducing oxygen into the thallus suspension at the oxygen introduction rate of 5L/min, and regulating the pH value to be 6.4 by ammonia water in real time;
(3) weighing 14g of p-hydroxyphenylethanol substrate, dissolving the substrate in 16ml of TRIS buffer solution, completely dissolving and uniformly mixing, dripping the substrate into a four-neck flask for eight times at a speed of 1.75g/30min, continuously stirring for reaction, detecting the p-hydroxyphenylethanol substrate in the reaction solution through a liquid phase, stopping stirring and closing ventilation when no p-hydroxyphenylethanol substrate exists in the reaction solution, stopping the reaction, obtaining the reaction solution of the 3, 4-dihydroxyphenylethanol product, and refrigerating the reaction solution at a temperature of 4 ℃.
Example 11
(1) Placing the fermentation broth of Escherichia coli BL21(DE3) for inducing enzyme production in a centrifuge tube, centrifuging for 10min in a centrifuge with the rotation speed of 8000rpm/min, removing the supernatant, and collecting thallus;
(2) 1250ml of TRIS buffer was added to the cells to suspend the cells, and the biomass of the cells was 60g/m3Then pouring the mixture into a four-neck flask, putting the four-neck flask into a magnetic stirring water bath kettle with the rotation speed of 1200rpm/min and the temperature of 27 ℃, and then introducing the mixture into the thallus suspensionIntroducing oxygen at an oxygen content of 6L/min, and regulating the pH value to 6.4 in real time by using ammonia water;
(3) weighing 15.2g of p-hydroxyphenylethanol substrate, dissolving in 16ml of TRIS buffer solution, completely dissolving and uniformly mixing, dripping into a four-neck flask for eight times at 1.9g/30min, continuously stirring for reaction, detecting the p-hydroxyphenylethanol substrate in the reaction solution through a liquid phase, stopping stirring and closing ventilation when no p-hydroxyphenylethanol substrate exists in the reaction solution, stopping the reaction, obtaining the reaction solution of the 3, 4-dihydroxyphenylethanol product, and refrigerating and storing the reaction solution at the temperature of 4 ℃.
Example of measurement
(1) Drawing standard curve of p-hydroxyphenylethanol
Weighing 0.1g of a p-hydroxyphenylethanol substrate standard sample, transferring the p-hydroxyphenylethanol substrate standard sample into a 50ml volumetric flask, diluting with methanol to a constant volume, then transferring 0.5ml, 1ml, 2ml, 4ml, 6ml and 8ml into 610 ml volumetric flasks respectively, diluting with methanol to a constant volume to obtain concentrations of 0.1g/L, 0.2g/L, 0.4g/L, 0.8g/L, 1.2g/L and 1.6g/L respectively, and detecting by a high performance liquid chromatograph, wherein chromatographic conditions are as follows: the column chromatography was C18, mobile phase a was 0.1% acetic acid water, mobile phase D was methanol, mobile phase a: mobile phase D (v: v) 80: 20, flow rate: 1ml/L, column temperature: 35 ℃, wavelength: 276nm, injection volume: 10 μ L, retention time: 18min, the p-hydroxyphenylethanol peak-off time is about 13min, the concentration (g/L) is used as an abscissa, the peak area is used as an ordinate, a p-hydroxyphenylethanol standard curve is drawn, and the result is shown in figure 2, wherein y is 7 × 106x-17381，R2＝1(R2Is a linear fit constant).
(2) Marking curve drawing of 3, 4-dihydroxy phenethyl alcohol
Weighing 0.1g of 3, 4-dihydroxyphenylethanol standard sample, transferring to a 50ml volumetric flask, diluting with methanol to a constant volume, transferring 0.5ml, 1ml, 2ml, 4ml, 6ml and 8ml to 610 ml volumetric flasks respectively, diluting with methanol to a constant volume to obtain standard samples with concentrations of 0.1g/L, 0.2g/L, 0.4g/L, 0.8g/L, 1.2g/L and 1.6g/L, respectively, detecting by a high performance liquid chromatograph, wherein the chromatographic conditions are consistent with those in the above (1) in the determination example, the 3, 4-dihydroxyphenylethanol peak time is about 7.5min, and the concentration (g/L) is taken as an abscissaThe peak area was plotted on the ordinate, and the result of plotting a 3, 4-dihydroxybenzene ethanol standard curve is shown in fig. 3, where y is 1 × 107x+14745，R2＝0.9999(R2Is a linear fit constant).
(3) Measurement of reaction solutions of 3, 4-Dihydroxybenzeneethanol products of examples 1 to 11
2ml of the reaction solutions of the 3, 4-dihydroxybenzene ethanol products obtained in examples 1 to 10 were respectively collected, centrifuged, and 1ml of the supernatant was respectively collected, transferred to 10ml volumetric flasks, diluted with methanol to a constant volume, and analyzed by HPLC, wherein the chromatographic conditions were identical to those in (1) above in this measurement example, the peak time was about 7.5min, and the chromatogram was as shown in FIG. 4, which is the chromatogram of the reaction solution of the product obtained in example 10. The yields and conversion rates of 3, 4-dihydroxybenzylethanol were obtained according to the 3, 4-dihydroxybenzylethanol standard curve, and the conversion rate of the present invention was the actual yield of the product/the theoretical yield of the product, the theoretical yield of the product was the total amount of the substrate added/the relative molecular mass of the substrate × the relative molecular mass of the product, and the total amount of the substrate added was the total mass of the substrate added/(volume of buffer in which the cells were suspended + volume of buffer in which the substrate was dissolved).
TABLE 3 yield, conversion of 4, 4-dihydroxyphenylethanol
As can be seen from the above table, the total adding mass of the p-hydroxyphenylethanol in the embodiments 2 to 9 is not more than 13.9g, and the total adding mass of the p-hydroxyphenylethanol is controlled to be 12.0 to 12.5g/L, so that the catalytic efficiency of the biological enzyme is high and reaches 99%, the conversion rate of the 3, 4-dihydroxyphenylethanol is high and is more than 99%, the yield of the 3, 4-dihydroxyphenylethanol is high and is more than 13g/L, and the p-hydroxyphenylethanol and the biological enzyme have good utilization rates; although the biological enzyme catalysis efficiency of the embodiment 1 reaches 99%, the total addition amount of the p-hydroxyphenylethanol in the embodiment 1 is less than 12.0g/L, and the actual yield of the obtained 3, 4-dihydroxyphenylethanol is also less than 13g/L, namely the volume consumption of the buffer solution is large, so that the buffer solution is wasted; the total mass of the added p-hydroxyphenylethanol in the example 10 is more than 13.9g, and the total amount of the added p-hydroxyphenylethanol exceeds 12.5g/L, but the biological enzyme catalysis efficiency is still reduced, and the conversion rate of the 3, 4-dihydroxyphenylethanol is also reduced to be less than 99 percent, which indicates that the biological enzyme catalysis efficiency is reduced and the conversion rate and the yield of the 3, 4-dihydroxyphenylethanol are influenced by excessive addition of the p-hydroxyphenylethanol; although the total amount of the added p-hydroxyphenylethanol in the embodiment 11 is controlled to be 12.0-12.5 g/L, so that the actual yield of the 3, 4-dihydroxyphenylethanol can reach 13g/L, the total mass of the added p-hydroxyphenylethanol is greater than 13.9g, and the conversion rate of the 3, 4-dihydroxyphenylethanol is less than 99%, although the dosage of the biological enzyme is increased, the biological enzyme catalysis efficiency is still reduced, which also well shows that the biological enzyme catalysis efficiency is affected by the excessive addition of the p-hydroxyphenylethanol, and further the conversion rate and the yield of the 3, 4-dihydroxyphenylethanol are reduced.
In conclusion, the total adding quality of the p-hydroxyphenylethanol is controlled to be less than or equal to 13.9g, the total adding quantity of the p-hydroxyphenylethanol is controlled to be 12.0-12.5 g/L, the biological enzyme can obtain higher enzyme catalysis efficiency which reaches 99%, the conversion rate of the 3, 4-dihydroxyphenylethanol is higher than 99%, and the yield of the 3, 4-dihydroxyphenylethanol is 13g/L, so that the p-hydroxyphenylethanol, the biological enzyme and a buffer solution are utilized to the maximum extent, and resource waste is avoided.
Finally, it should be noted that the above embodiments are only used for illustrating and not limiting the technical solutions of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the present invention without departing from the spirit and scope of the present invention, and all modifications or partial substitutions should be covered by the scope of the claims of the present invention.
Claims (9)
1. A method for preparing 3, 4-dihydroxy phenethyl alcohol by bio-enzyme catalysis is characterized by comprising the following steps:
s1, carrying out centrifugal treatment on the escherichia coli fermentation liquor for inducing enzyme production, wherein the centrifugal rotation speed is 2000-8000 rpm/min, the centrifugal time is 1-10 min, and removing the centrifugal liquor to obtain thalli;
s2, adding a buffer solution into the thalli in the step S1 to enable the thalli to be suspended, moving the thalli into a four-neck flask, then carrying out magnetic stirring at the stirring speed of 200-1200 rpm/min, carrying out water bath heating at the heating temperature of 25-45 ℃, then introducing oxygen into the thalli suspension, and controlling the pH in real time;
s3, weighing a p-hydroxyphenylethanol substrate, dissolving and mixing uniformly by using a small amount of buffer solution, adding the p-hydroxyphenylethanol substrate into a four-neck flask for many times, continuously stirring for reaction, detecting the p-hydroxyphenylethanol substrate in a reaction solution by using a high performance liquid phase, stopping stirring and closing ventilation when the p-hydroxyphenylethanol substrate does not exist in the reaction solution, stopping the reaction, obtaining a reaction solution of a 3, 4-dihydroxyphenylethanol product, and storing the reaction solution in a cold storage way;
s4, detecting and analyzing the 3, 4-dihydroxy phenylethanol product in the reaction solution in the step S3 by adopting high performance liquid chromatography, so that the yield of the 3, 4-dihydroxy phenylethanol is over 13g/L, and the conversion rate of the 3, 4-dihydroxy phenylethanol is over 99%.
2. The method for preparing 3, 4-dihydroxy phenethyl alcohol by using bio-enzyme catalysis as claimed in claim 1, characterized in that the total mass of the added p-hydroxyphenylethanol is less than or equal to 13.9g, the total mass of the added p-hydroxyphenylethanol is controlled to be 12.0-12.5 g/L, and the total mass of the added p-hydroxyphenylethanol is equal to the total mass of the added p-hydroxyphenylethanol per the total volume of the buffer solution.
3. The method for preparing 3, 4-dihydroxy phenethyl alcohol by using bio-enzyme catalysis as claimed in claim 2, wherein the addition amount of p-hydroxyphenylethanol is 0.5-5 g/30min each time.
4. The method for preparing 3, 4-dihydroxy phenylethanol by bio-enzyme catalysis according to claim 1, wherein the biomass of the thallus is 15-45 g/m3。
5. The method for preparing 3, 4-dihydroxyphenylethanol catalyzed by a bio-enzyme according to claim 1, wherein the buffer is one of PB buffer, PBS buffer or TRIS buffer.
6. The method for preparing 3, 4-dihydroxy phenylethanol by bio-enzyme catalysis according to claim 1, wherein the oxygen introduction amount is 0.5-5L/min.
7. The method for preparing 3, 4-dihydroxy phenylethanol by bio-enzyme catalysis according to claim 1, wherein the pH value is 5.5-8.5.
8. The method for preparing 3, 4-dihydroxy phenylethanol by bio-enzyme catalysis according to claim 1, wherein the escherichia coli fermentation broth is one of escherichia coli MG1655 fermentation broth, escherichia coli BL21(DE3) fermentation broth, or escherichia coli BMGA fermentation broth.
9. The method for preparing 3, 4-dihydroxy phenylethanol by bio-enzyme catalysis according to claim 1, wherein the temperature for cold storage is 4 ℃.
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