CN109943582A - Method for producing dopamine based on catalysis of dopamine decarboxylase - Google Patents

Abstract

The invention discloses a method for producing dopamine catalyzed by dopamine decarboxylase. The method specifically comprises the following steps: constructing a genetically engineered bacterium BL21 (DE3)/pET28a-DDC expressing dopa decarboxylase (DDC); culturing the genetically engineered bacterium BL21 (DE3)/pET28a-DDC and induced expression of DDC; collect the above-mentioned cells, resuspend the cells with buffer, sonicate, and centrifuge to remove impurities to obtain the crude enzyme liquid of DDC; using dopa as the substrate, add 0.1- After adjusting the pH of the reaction system with 1 mM pyridoxal phosphate, the DDC crude enzyme solution was added to react to obtain the product dopamine. The method of the invention is the first systematically made an enzyme catalysis production process from dopa to dopamine, the catalytic efficiency is as high as 7.4g/l, the conversion rate is as high as 95% or more, the environment is friendly, the raw materials are readily available, the production cost is low, the process is simple, and the reaction The conditions are mild and have good prospects for industrial production.

Description

A method for producing dopamine catalyzed by dopamine decarboxylase

technical field

The invention belongs to the technical field of biological enzyme catalysis, in particular to a method for catalyzing dopamine production based on dopamine decarboxylase.

Background technique

Dopa, also known as dihydroxyphenylalanine, levodopa, is an amino acid produced by the oxidation of tyrosine, which can pass through the blood-brain barrier and be converted into dopamine in the body. Medically used for the treatment of tremor paralysis.

Dopamine is a dopamine receptor agonist. Dopamine is an important neurotransmitter in the brain. The dopaminergic system is closely related to neuropsychiatric diseases. Dopamine is a substance that the human body can synthesize by itself. It can enhance myocardial contraction, increase cardiac output, increase arterial blood pressure, and resist shock. effect. Due to its prominent physiological function, the success of dopamine artificial synthesis makes it an anti-shock synthetic drug. Clinical application shows that dopamine has a significant effect on different types of shock. It has important theoretical research significance and application prospects for the development of drugs for the treatment of Parkinson's disease, drug dependence and schizophrenia. The synthesis of dopamine is difficult, expensive, requires high purity of the product, and yields less. Therefore, it is very necessary to provide a method that can efficiently and economically produce dopamine.

The method for producing dopamine in the prior art, wherein the chemical production conversion rate is low, the cost is high, the pollution is serious, the period of the biosynthesis method is long, and the extraction is difficult. It has good prospects and is a new type of enzyme catalysis method.

SUMMARY OF THE INVENTION

In view of the deficiencies of the prior art, the purpose of the present invention is to provide a method for producing dopamine catalyzed by dopamine decarboxylase, which is green and environmentally friendly, and has the advantages of simple chemical production method, low cost and suitable for industrialization.

In order to solve the prior art problem, the technical scheme adopted in the present invention is:

A method for producing dopamine based on dopamine decarboxylase catalysis, comprising the following steps:

Step 1, construct genetically engineered bacteria BL21(DE3)/pET28a-DDC expressing dopa decarboxylase;

Step 2, culturing dopa decarboxylase genetically engineered bacteria BL21(DE3)/pET28a-DDC;

Step 3, collecting the genetically engineered bacteria in step 2, resuspending with a pH 5-9 buffer, and crushing with an ultrasonic crusher; centrifuging to remove impurities to obtain a crude enzyme solution of dopa decarboxylase;

Step 4, select dopa as a substrate, add 0.1-1mM pyridoxal phosphate, then add a buffer solution to adjust the pH of the reaction system to 5-9, and finally add 0.5-2g/l of dopa decarboxylase crude enzyme solution, 40- Reaction at 45°C for 0.5h-3h to obtain the product dopamine, and the final concentration of the substrate dopa in the reaction system does not exceed 6g/l.

After the reaction, dopamine can be detected by high performance liquid chromatography. The detection method was Agilent1260 high performance liquid chromatography; Agilent TC-C18 column (150 mm×4.6 mm, 5 μm) was used; 0.1% trifluoroacetic acid solution-acetonitrile (96:4) was used as mobile phase; flow rate was 1.0 ml·min ^{- 1} ; detection wavelength is 280nm; column temperature: room temperature.

As an improvement, the method for constructing a genetically engineered bacterium expressing dopa decarboxylase described in step 1 is as follows: coding is performed according to the reported amino acid sequence of dopa decarboxylase DDC (GenBank: AMQ13055.1). After suboptimization, the whole gene was synthesized and subcloned into the vector pET28a to obtain the recombinant plasmid pET28a-DDC. The constructed recombinant plasmid pET28a-DDC was transformed into the E. coli expression host BL21 (DE3) by the calcium chloride method to obtain Dopa decarboxylase expressing strain BL21(DE3)/pET28a-DDC.

As an improvement, the method for culturing genetically engineered bacteria BL21(DE3)/pET28a-DDC in step 2 is as follows: picking a single colony of genetically engineered bacteria BL21(DE3)/pET28a-DDC expressing dopa decarboxylase, inoculating it on LB culture medium at 37°C until the OD value reaches above 0.6, then add 0.5mM IPTG, culture at 18°C for 10-12 hours, and collect the genetically engineered bacteria BL21(DE3)/pET28a-DDC expressing dopa decarboxylase by centrifugation.

As an improvement, in step 4, the final concentration of dopa is 5g/l, the amount of pyridoxal phosphate added is 0.4mM, the pH of the reaction system is 6.8-7.0, the reaction temperature is 42°C, and dopa decarboxylase is added. The concentration of crude enzyme solution was 1g/l, and the reaction time was 1h.

It is further improved that the buffer in step 4 is one of phosphate buffer, PBS buffer or Tris-hydrochloric acid buffer.

As an improvement, dopa is used as the substrate in step 4, and the initial concentration is 2 g/l, which is added to the reaction system in stages, and the final concentration does not exceed 6 g/l.

As an improvement, in the step 3, the power of the ultrasonic crusher is 300W, and the crushing process is intermittent crushing.

Beneficial effects:

Compared with the prior art, in the chemical process, vanillin is used as a raw material, which is first reacted with nitromethane to generate 4-hydroxy-3-methoxy-β-nitroethylbenzene (referred to as nitro) . The nitro compound is reduced with zinc and hydrochloric acid as a reducing agent to obtain 4-hydroxy-3-methoxyethylamine salt (reduced for short). The salt is dopamine. In chemical synthesis, the synthesis of dopamine is difficult, the purity required for the product is relatively high, the yield is small, and there are disadvantages such as high cost and serious pollution. The present invention is a method for producing dopamine catalyzed by dopamine decarboxylase. The biocatalytic process has never been specifically studied before. The method of the present invention is green and safe, the output of dopamine reaches 7.4 g/l, and the conversion rate is as high as more than 95%. The raw materials are readily available, the production cost is low, the process is simple, the reaction conditions are mild, the catalytic efficiency and the conversion rate are high, the environment is friendly, the industrialization prospect is good, and the production is safe.

Description of drawings

Fig. 1 is the high performance liquid chromatogram of the product in the end of reaction in embodiment 3, wherein, 1-dopamine standard substance, 2-dopa.

Detailed ways

Example 1

Construction of genetically engineered bacteria BL21(DE3)/pET28a-DDC expressing dopa decarboxylase

According to the reported amino acid sequence of dopa decarboxylase DDC derived from Coccinella axyridis (GenBank: AMQ13055.1), after codon optimization (the optimization steps were entrusted to GenScript), the whole gene was synthesized and the sequence was subcloned into On the vector pET28a, the recombinant plasmid pET28a-DDC was obtained, and the constructed recombinant plasmid pET28a-DDC was transformed into the E. coli expression host BL21 (DE3) by the calcium chloride method to obtain the dopa decarboxylase expression strain BL21 (DE3)/pET28a -DDC.

Example 2

Culturing the genetically engineered bacteria BL21(DE3)/pET28a-DDC expressing dopa decarboxylase, and inducing the expression

Pick a single colony of genetically engineered bacteria expressing dopa decarboxylase, inoculate it in LB medium, and cultivate at 37 °C; when the OD value reaches 0.6, the amount of IPTG added is 0.5 mM, and culture at 18 °C-30 °C for 10-12 hours; after the fermentation in the bottle was completed, the genetically engineered bacteria expressing dopa decarboxylase were collected by centrifugation and used for later use. Resuspend the collected cells with the corresponding volume of buffer, and use an ultrasonic cell disrupter (10min for sonication, 2s for sonication, 3s for stop, 300W power), and after the fragmentation is completed, centrifuge at 4000-8000rpm, 4°C for 8-10min The dopa decarboxylase crude enzyme solution was obtained, and the protein concentration was determined with a microplate reader.

The formula of LB medium: 10g/l peptone, 5g/l yeast powder, 5g/l sodium chloride.

Example 3

To catalyze the production of dopamine, the specific operation steps are as follows: the reaction system is 10ml, the initial concentration of dopa is 2g/l as the substrate, 0.4mM of PLP is added, the pH of the system is adjusted to 6.8 with PBS buffer, and then 1g/l of crude oil is added. Enzyme concentration, shake reaction at 40°C, and add substrate dopa every 10-15min, 1-3g/l each time, to ensure that the final concentration during the reaction does not exceed 6g/l. After the reaction, high performance liquid chromatography (using Agilent TC-C18 column (150 mm×4.6 mm, 5 μm); 0.1% trifluoroacetic acid solution-acetonitrile (96:4) was used as the mobile phase; the flow rate was 1.0 ml· min-1; detection wavelength is 280 nm; column temperature: room temperature) to detect the amount of dopamine generated. The yield of dopamine reached 7.4g/l, and the conversion rate was as high as 95%.

Example 4

Except that the addition amount of the crude enzyme solution was changed to 0.5 g/l, the rest were the same as in Example 3. After the reaction, as measured by high performance liquid chromatography, the output of dopamine reaches 7.0g/l, and the conversion rate reaches more than 94%.

Example 5

Except that the pH of the reaction system is 8, the rest are the same as in Example 1. After the completion of the reaction, as detected by high performance liquid chromatography, the output of dopamine reaches 4.0 g/l, and the conversion rate reaches more than 89%.

Example 6

Using dopa as a substrate, the genetically engineered bacteria BL21(DE3)/pET28a-DDC cultivated in step 2 were used to directly catalyze the production of dopamine. Except that the crude enzyme solution was replaced with 1 g/l genetically engineered bacteria BL21(DE3)/pET28a-DDC, the rest were the same as in Example 1. After the reaction was completed, the yield of dopamine was 0.5 g/l as detected by high performance liquid chromatography.

Example 7

Except that the substrate dopa was added into the reaction system at one time, and the initial concentration was 5 g/l, the rest were the same as in Example 1. After the reaction was completed, by high performance liquid chromatography detection, the output of dopamine reached 5.0 g/l, and the conversion rate reached 5.0 g/l. More than 91%.

Example 8

Except not containing PLP, the rest are the same as in Example 1. After the reaction, the output of dopamine is 0 by high performance liquid chromatography detection.

It can be seen from the results of Examples 3-8 that when the pH of the reaction system of the present invention is 6.8, the catalytic reaction efficiency is good, and PLP is an indispensable additive in the reaction system. The prior art includes a chemical method and a biosynthesis method. The chemical method has low conversion rate, high cost, serious pollution, and the biosynthesis method has a long cycle and is difficult to extract. Compared with the prior art, the method for producing dopamine catalyzed by a biological enzyme of the present invention is environmentally friendly, the yield of dopamine reaches 7.4 g/l, the conversion rate is as high as 95% or more, the raw materials of the present invention are easily available, the production cost is low, and the process is simple, The reaction conditions are mild, the catalytic efficiency and conversion rate are high, the environment is friendly, and the industrialization prospect is good.

The above are only preferred specific embodiments of the present invention, and the protection scope of the present invention is not limited thereto. Any person skilled in the art can obviously obtain the simplicity of the technical solution within the technical scope disclosed in the present invention. Variations or equivalent substitutions fall within the protection scope of the present invention.

Claims (7)

1. a kind of method for producing dopamine based on dopamine decarboxylase enzymatic, which comprises the following steps:

Step 1, the genetic engineering bacterium BL21(DE3 of building expression DOP Adecarboxylase)/pET28a-DDC；

Step 2, the genetic engineering bacterium BL21(DE3 of DOP Adecarboxylase is cultivated)/pET28a-DDC；

Step 3, the genetic engineering bacterium in collection step 2, after being resuspended with the buffer of pH5-9, Ultrasonic Cell Disruptor is broken；Centrifugation is gone Except impurity, the crude enzyme liquid of DOP Adecarboxylase is obtained；

Step 4, DOPA is chosen as substrate, 0.1-1mM phosphopyridoxal pyridoxal phosphate is added, then is added buffer solution and adjusted reaction system PH to 5-9, is eventually adding DOP Adecarboxylase crude enzyme liquid 0.5-2g/l, and 40-45 DEG C of reaction 0.5h-3h obtains product dopamine, instead The final concentration of substrate DOPA in system is answered to be no more than 6g/l.

2. a kind of method for being catalyzed production dopamine based on DOP Adecarboxylase according to claim 1, which is characterized in that step The method of the genetic engineering bacterium of the DOP Adecarboxylase of building expression described in 1 is as follows: according to the harmonia axyridia source DOPA reported After the amino acid sequence (GenBank:AMQ13055.1) of decarboxylase (DDC) carries out codon optimization, full genome synthesizes the sequence Column, are subcloned on carrier pET28a, obtain recombinant plasmid pET28a-DDC, and the recombinant plasmid pET28a-DDC built is used Calcium Chloride Method conversion enters Bacillus coli expression host BL21(DE3), obtain DOP Adecarboxylase expression strain BL21(DE3)/ pET28a-DDC。

3. a kind of method for being catalyzed production dopamine based on DOP Adecarboxylase according to claim 1, which is characterized in that step Genetic engineering bacterium BL21(DE3 in 2)/pET28a-DDC cultural method specifically: the genetic engineering of picking expression DOP Adecarboxylase Bacterium BL21(DE3)/pET28a-DDC single colonie, be inoculated in LB culture medium, 37 DEG C of culture to OD values reach 0.6 or more, then plus Enter the IPTG of 0.5mM, 18 DEG C culture 10-12 hours, the genetic engineering bacterium BL21(DE3 of expression DOP Adecarboxylase is collected by centrifugation)/ pET28a-DDC。

4. a kind of method for being catalyzed production dopamine based on DOP Adecarboxylase according to claim 1, which is characterized in that step The final concentration amount of DOPA is 5g/l in 4, and the additive amount of phosphopyridoxal pyridoxal phosphate is 0.4mM, and the pH of reaction system is 6.8-7.0, reaction Temperature is 42 DEG C, and addition DOP Adecarboxylase crude enzyme liquid concentration is 1g/l, reaction time 1h.

5. according to right to go 1 described in it is a kind of based on DOP Adecarboxylase be catalyzed produce dopamine method, which is characterized in that step Buffer described in 4 is a kind of in phosphate buffer, PBS buffer solution or Tris- hydrochloride buffer.

6. a kind of method for being catalyzed production dopamine based on DOP Adecarboxylase according to claim 1, which is characterized in that step Reaction system is added as substrate, initial concentration 2g/l in DOPA by several times in 4, and final concentration is no more than 6g/l.

7. a kind of method for being catalyzed production dopamine based on DOP Adecarboxylase according to claim 1, which is characterized in that described The power of Ultrasonic Cell Disruptor is 300W in step 3, and shattering process is intermittent broken.