Method for improving yield of dopamine synthesized by enzymic method

Abstract

The invention discloses a method for increasing the yield of dopamine synthesized by an enzymic method, and belongs to the field of dopamine synthesis. The method comprises the steps of thallus fermentation, preparation of immobilized dopa decarboxylase, preparation of a basic reaction solution, cation exchange resin activation, reaction, chromatographic desorption, reactivation of immobilized enzyme and resin and the like, so that the aim of improving the yield of the dopamine synthesized by the enzyme method is fulfilled. According to the method, the yield of dopamine can reach 90 g / L or above, the purification effect is good, the yield is high, the synthesis capacity of immobilized dopa decarboxylase can be repeatedly used for 50 times, the purification capacity of ion exchange resin can be repeatedly used for 100 times, and the method is easy to operate, low in toxicity, low in harm to the human body of an operator and more suitable for industrial production.

Description

Technical Field

[0001] The invention relates to dopamine synthesis, and in particular to a method for increasing the yield of dopamine synthesized by enzymatic method. Background Art

[0002] Dopamine, chemical name 4-(2-aminoethyl)-1,2-benzenediol, is mainly used to treat shock symptoms such as septic shock and hypotensive shock. Dopamine is an ideal anti-shock drug. Its main function is to increase vascular tension, enhance cardiac contractility, and increase cardiac output, thereby achieving an anti-shock effect. At present, dopamine is a very commonly used drug in clinical practice, with a wide range of effects. Small doses can dilate the renal artery and increase urine volume. Medium doses have a cardiotonic effect, and large doses have a pressor effect. In 2012, scientists found that dopamine can help further treat Parkinson's disease. The treatment method is to restore the level of dopamine in the brain and control the disease. At the same time, dopamine can further treat Alzheimer's disease and improve patients' memory. Therefore, the future application prospects and market prospects of dopamine are very huge. The existing dopamine synthesis methods are usually chemical synthesis and enzymatic synthesis methods. Chemical synthesis methods generally involve a variety of organic solvents, which are highly polluting to the environment and have low yields. There are also disadvantages such as long process routes and large amounts of wastewater. The enzymatic synthesis method uses L-DOPA as a substrate and utilizes dopa decarboxylase to catalyze the synthesis of dopamine. The dopamine product synthesized by the enzymatic method has an inhibitory effect on dopa decarboxylase, resulting in a low yield of the synthesized dopamine. Summary of the invention

[0003] Purpose of the invention: The purpose of the present invention is to provide a method for increasing the yield of dopamine synthesized by enzymatic method.

[0004] Technical solution: A method for increasing the yield of dopamine synthesized by enzymatic method, comprising the following steps:

[0005] S1. Genetic engineering was used to introduce the DOPA decarboxylase gene into Escherichia coli to establish a strain library. During fermentation production, the working strain was inoculated into the seed culture medium, cultured at 200 rpm and 37°C for 12-16 hours in a shaking incubator. The qualified shake flask seeds were obtained when the OD600 was greater than 3 and no foreign bacteria were found under microscopic examination. The seed liquid was inoculated into a 50L fermentation tank containing 20L fermentation medium at a 5% inoculation rate, cultured at 250 rpm and 37°C with aeration of 1.3-1.5 m / s. 3 / h, when the strain grows to OD600 of 40-50, add IPTG with a final concentration of 0.2mmol / L to induce expression, lower the culture temperature to 20℃, and start adding feed medium at the same time, with a feed rate of 200-220mL / h; when the strain grows to OD600 of more than 60, end the fermentation and collect the bacterial cells by centrifugation;

[0006] S2. Escherichia coli expressing DOPA decarboxylase efficiently is dissolved in water at a concentration of 10% to 20%, and the intracellular DOPA decarboxylase is released by crushing with a high-pressure homogenizer. The crude enzyme solution is centrifuged at 4000 to 5000 rpm to obtain a crude enzyme solution. The crude enzyme solution is adsorbed by agarose separation medium, impurities are eluted with 20mM imidazole-50mM PBS-200mM NaCl, and then eluted with 500mM imidazole-50mM PBS-200mM NaCl to extract and purify the refined enzyme solution. The refined enzyme solution is stirred with an activated epoxy resin carrier at a concentration of 30 to 60 mg protein / 1 g for 5 hours or more to obtain immobilized DOPA decarboxylase, and the enzyme protein immobilization rate reaches 90%;

[0007] S3, adding immobilized dopa decarboxylase to the basic reaction solution, and then adding levodopa to the reaction system, stirring the enzyme catalyzed reaction at a temperature of 35-40° C. to generate dopamine; while the reaction is being carried out, the basic reaction solution is pumped to the activated cation exchange resin through a filter, the filter retains the immobilized enzyme and unmelted levodopa, the basic reaction solution through the filter is subjected to chromatographic adsorption in a resin column, and the basic reaction solution without dopamine after the resin treatment is refluxed into the reaction system;

[0008] S4, the basic reaction solution subjected to chromatographic adsorption in the resin column in step S3 is subjected to chromatographic desorption, and the resin column is washed with 3BV of water at a water absorption flow rate of 1-3BV / h. After the resin column is washed with water, 0.1-0.3mol / L sodium chloride is used as an eluent for desorption, and the desorption flow rate is 1-3BV / h. When the OD280 of the desorption effluent reaches 10, the desorption effluent is collected, and the desorption is stopped when the OD280 of the desorption effluent is lower than 10. The collected desorption effluent is the dopamine desorption liquid. The purity of dopamine in the desorption liquid reaches more than 99%. According to the volume of the enzyme synthesis reaction system, the dopamine yield can reach more than 90g / L.

[0009] Preferably, in step S1, during the fermentation reaction, the dissolved oxygen content is maintained at 25%, the rotation speed is controlled below 300, and the ventilation is 1.3-1.5 m 3 / h.

[0010] Preferably, in step S3, the basic reaction solution comprises 0.20-0.40 mol / L phosphate buffer and 0.25-0.50 mmol / L 5'-pyridoxal phosphate; wherein the pH of the phosphate buffer is 6.5-7.5.

[0011] Preferably, in step S3, the mass volume ratio of the immobilized DOPA decarboxylase to the basic reaction solution is 1:10 to 1:50.

[0012] Preferably, in step S3, the amount of levodopa used is 100-120 g of levodopa per 1 L of the basic reaction solution.

[0013] Preferably, in step S3, the reaction time is 18 to 20 hours, and then the temperature is lowered to 10 to 20° C. to terminate the reaction.

[0014] Preferably, in step S3, the activation process of the cation exchange resin is: the resin is loaded into a column, washed with 2BV of 1N sodium hydroxide for 1 to 2 hours; then washed with purified water until neutral; then washed with 2BV of 1N hydrochloric acid for 1 to 2 hours; then washed with purified water until neutral; finally washed with 2BV of the basic reaction solution for 1 hour to obtain an activated cation exchange resin column.

[0015] Beneficial effects:

[0016] (1) The present invention utilizes immobilized enzyme technology to immobilize dopa decarboxylase and then perform an enzymatic synthesis of dopamine. While the reaction is being carried out, the reaction solution is filtered through a filter and then pumped to a cation exchange resin column that can specifically adsorb dopamine. The filter can intercept the immobilized enzyme and undissolved levodopa. The reaction solution filtered through the filter flows through the resin column for chromatographic adsorption, and dopamine in the reaction solution is adsorbed into the resin column. The reaction solution that does not contain dopamine after resin treatment flows back into the reaction system. This method can maintain the concentration of the reaction product dopamine in the reaction system at a low level, thereby effectively reducing the inhibitory effect of dopamine on dopa decarboxylase and effectively increasing the dopamine yield. In addition, the enzymatic synthesis reaction of the present invention is carried out simultaneously with the resin column chromatography purification, which can effectively save the time cost of dopamine production. After the macroporous cation exchange resin column adsorbs dopamine, sodium chloride solution is used as a desorbent for desorption, and the purity of dopamine in the desorbed solution reaches more than 99%.

[0017] (2) The method provided by the present invention can achieve a dopamine production of more than 90 g / L, with good purification effect and high yield. The immobilized dopamine decarboxylase can be reused 50 times without loss of synthesis capacity, and the ion exchange resin can be reused 100 times without loss of purification capacity. The method is simple to operate, has low toxicity, and has low harm to the operator's body, and is more suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the device for the enzymatic synthesis of dopamine;

[0019] Figure 2 It is a high performance liquid chromatography analysis chart. DETAILED DESCRIPTION

[0020] In order to make the technical solution of the present invention clearer, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] Example

[0022] A use of Figure 1 The method for improving the production of dopamine synthesized by enzymatic method by the device shown specifically comprises:

[0023] (1) Fermentation of bacteria: Take one working strain and inoculate it into 750ml LB liquid medium, and culture it in a shaking incubator at 200rpm and 37℃ for 15 hours. If the OD600 is 3.8 and there are no foreign bacteria under microscopic examination, it is a qualified shake flask seed. At an inoculation rate of 5%, inoculate the seed liquid into a 50L fermentation tank containing 20L fermentation medium, and culture it at 250rpm and 37℃ with aeration of 1.3~1.5m 3 / h, culture for 11 hours, the strain grows to OD600 of 44, add IPTG (isopropylthiogalactoside) with a final concentration of 0.2mmol / L to induce expression, lower the culture temperature to 20℃, and start adding feed medium at the same time, with a feed rate of 200~220mL / h. During the reaction, the dissolved oxygen is maintained at 25%, the rotation speed is controlled below 300, and the ventilation is 1.30~1.5m 3 / h. After 25 hours of fermentation, the strain grew to an OD600 of 62, and the fermentation was terminated. The bacterial cells were collected by centrifugation.

[0024] (2) Preparation of immobilized DOPA decarboxylase: 300 g of Escherichia coli was taken and dissolved in 2 L of water to obtain a bacterial solution. The bacterial solution was crushed by a 550 bar high-pressure homogenizer and centrifuged at 4500 rpm to obtain 2 L of crude enzyme solution. 120 g of agarose medium was weighed and added to the crude enzyme solution. The DOPA decarboxylase was adsorbed for 4 hours under stirring. The filtrate was filtered and the residual protein in the filtrate was measured. The calculated amount of enzyme protein adsorbed by the medium was 8.26 g. The solution was washed with 600 ml of 20 mM imidazole-50 mM PBS-200 mM NaCl solution and then washed with 300 ml of 500 mM imidazole-50 mM PBS-200 mM The NaCl solution was used to elute to obtain about 300 ml of refined enzyme solution, and the protein content in the enzyme solution was detected to be 7.62 g. 150 g of activated epoxy resin carrier was weighed and added to the refined enzyme buffer, stirred for 6 hours, filtered, and the residual protein content of the filtrate was determined and the protein immobilization rate was calculated to be 96%. The immobilized protein content per unit weight of the epoxy resin carrier was 48.77 mg / g. The immobilized dopa decarboxylase was washed 3 times with 300 ml of water for standby use.

[0025] (3) Prepare basic reaction solution: Prepare 15 L of basic reaction solution. The basic reaction solution formula contains 0.20~0.40 mol / L phosphate buffer (pH 6.5~7.5) and 0.25~0.5 mmol / L 5'-pyridoxal phosphate.

[0026] (4) Activation of cation exchange resin: Select LXT-101 resin produced by Xi'an Lanxiao Technology New Materials Co., Ltd., load 5 L of resin into the column, wash with 2 BV of 1N sodium hydroxide for 1 to 2 h; then wash with purified water until neutral, then wash with 2 BV of 1N hydrochloric acid for 1 to 2 h, then wash with purified water until neutral, wash with 2 BV of basic reaction solution for 1 h, and obtain an activated cation exchange resin column.

[0027] (5) Reaction: Measure 1L of the basic reaction solution into the reactor, weigh 80g of immobilized dopa decarboxylase and add it to the basic reaction solution, and add 120g of levodopa. Stir the enzyme catalyzed reaction at a temperature of 37°C to generate dopamine. While the reaction is in progress, the reaction solution is pumped through a filter at a flow rate of 5L / h to an activated cation exchange resin column for chromatographic adsorption, and the adsorption effluent of the resin column is refluxed into the reaction system. After the reaction time is 18 hours, the temperature is lowered to 10-20°C to terminate the reaction.

[0028] (6) Chromatographic desorption: 15 L of water was used to wash the resin column at a flow rate of 1-3 BV / h. After the resin column was washed, 0.1-0.3 mol / L sodium chloride was used as the eluent for desorption at a flow rate of 1-3 BV / h. When the OD280 of the desorption effluent reached 11.2, the desorption effluent was collected until the OD280 of the desorption effluent reached 9.6. A total of 8.6 L of dopamine desorption liquid was collected, and the dopamine content in the desorption liquid was 91.3 g. The purity of dopamine in the desorption liquid was 99.97% (e.g., 99.97%) detected by high performance liquid chromatography. Figure 2 Calculated by the volume of the reaction system, the dopamine production reached 91.3 g / L.

[0029] The HPLC analysis instruments and parameters are as follows:

[0030] Instrument: Thermo Instrument 3000

[0031] Chromatographic column: Inertsil ODS-3 5um, 4.6×150mm

[0032] Instrument parameters:

[0033] Mobile phase: deionized water: acetonitrile: trifluoroacetic acid = 98:2:0.1

[0034] Collection time: 15min

[0035] Flow rate: 1ml / min

[0036] Column temperature: 30°C

[0037] Detection wavelength: UV, 280nm

[0038] Injection volume: 10 μL

[0039] The retention time of dopamine hydrochloride is 4.2~4.3min.

[0040] (7) Reactivation of immobilized enzyme and resin: After the synthesis reaction is completed, the immobilized enzyme can be reused 50 times after washing with purified water without loss of synthesis capacity; after desorption is completed, the ion exchange resin can be reused 100 times after reactivation without loss of purification capacity.

[0041] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for increasing the yield of dopamine synthesized by enzymatic method, characterized in that: The following steps are involved: S1. Introduce the DOPA decarboxylase gene into Escherichia coli and establish a strain library. During fermentation production, take the working strain and inoculate it into the seed culture medium, culture it at a speed of 200 rpm and 37°C in a shaking incubator for 12-16 hours. If the OD600 is greater than 3 and there is no foreign bacteria under microscopic examination, it is a qualified shake flask seed. According to the inoculation amount of 5%, inoculate the seed liquid into a 50L fermentation tank containing 20L fermentation medium, culture it at a speed of 250 rpm and 37°C, and aerate it at 1.3-1.5m 3 / h, when the strain grows to OD600 of 40-50, add IPTG with a final concentration of 0.2mmol / L to induce expression, lower the culture temperature to 20℃, and start adding feed medium at the same time, with a feed rate of 200-220mL / h; when the strain grows to OD600 of more than 60, end the fermentation and collect the bacterial cells by centrifugation; S2, dissolving Escherichia coli expressing DOPA decarboxylase efficiently in water at a concentration of 10% to 20%, breaking with a high-pressure homogenizer to release intracellular DOPA decarboxylase, centrifuging at 4000 to 5000 rpm to obtain a crude enzyme solution, adsorbing the crude enzyme solution with agarose separation medium, eluting impurities with 20mM imidazole-50mM PBS-200mM NaCl, and then eluting with 500mM imidazole-50mM PBS-200mM NaCl to extract and purify the refined enzyme solution; stirring the refined enzyme solution with an activated epoxy resin carrier at a concentration of 30 to 60 mg protein / 1 g for 5 hours or more to obtain immobilized DOPA decarboxylase; S3, adding immobilized dopa decarboxylase to the basic reaction solution, and then adding levodopa to the reaction system, stirring the enzyme catalyzed reaction at a temperature of 35-40° C. to generate dopamine; while the reaction is being carried out, the basic reaction solution is pumped to the activated cation exchange resin through a filter, the filter retains the immobilized enzyme and unmelted levodopa, the basic reaction solution through the filter is subjected to chromatographic adsorption in a resin column, and the basic reaction solution without dopamine after the resin treatment is refluxed into the reaction system; S4, performing chromatographic desorption on the basic reaction solution subjected to chromatographic adsorption in the resin column in step S3, washing the resin column with 3BV of water at a water absorption flow rate of 1-3BV / h, and after the resin column is washed, using 0.1-0.3mol / L sodium chloride as an eluent for desorption, and the desorption flow rate is 1-3BV / h, when the OD280 of the desorption effluent reaches 10, collecting the desorption effluent, and stopping the desorption when the OD280 of the desorption effluent is lower than 10, and the collected desorption effluent is the dopamine desorption solution.

2. The method for increasing the yield of dopamine synthesized by enzymatic method according to claim 1, characterized in that: In step S1, during the fermentation reaction, the dissolved oxygen content is maintained at 25%, the rotation speed is controlled below 300, and the ventilation is 1.3-1.5m 3 / h.

3. The method for increasing the yield of dopamine synthesized by enzymatic method according to claim 1, characterized in that: In step S3, the basic reaction solution comprises 0.20-0.40 mol / L phosphate buffer and 0.25-0.50 mmol / L 5'-pyridoxal phosphate; wherein the pH of the phosphate buffer is 6.5-7.

5.

4. The method for increasing the yield of dopamine synthesized by enzymatic method according to claim 1, characterized in that: In step S3, the mass volume ratio of the immobilized DOPA decarboxylase to the basic reaction solution is 1:10-1:

50.

5. The method for increasing the yield of dopamine synthesized by enzymatic method according to claim 1, characterized in that: In step S3, the amount of levodopa used is 100-120 g of levodopa per 1 L of the basic reaction solution.

6. The method for increasing the yield of dopamine synthesized by enzymatic method according to claim 1, characterized in that: In step S3, the reaction time is 18 to 20 hours, and then the temperature is lowered to 10 to 20° C. to terminate the reaction.

7. The method for increasing the yield of dopamine synthesized by enzymatic method according to claim 1, characterized in that: In step S3, the activation process of the cation exchange resin is as follows: the resin is loaded into a column, washed with 2BV of 1N sodium hydroxide for 1 to 2 hours, and then washed with purified water until neutral; Then wash the column with 2BV of 1N hydrochloric acid for 1 to 2 hours; then wash with purified water until neutral; finally wash the column with 2BV of the basic reaction solution for 1 hour to obtain an activated cation exchange resin column.

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