阅读说明：本技术 一种基于苯乙醇胺-n-甲基转移酶催化产肾上腺素的方法 (Method for producing epinephrine based on catalysis of phenylethanolamine-N-methyltransferase ) 是由 陈可泉 程莎莎 王昕� 冯娇 高思远 何雪峰 王思源 陆秋豪 许晟 张阿磊 欧阳 于 2020-06-23 设计创作，主要内容包括：本发明公开了一种基于苯乙醇胺-N-甲基转移酶催化产肾上腺素的方法,该方法具体包括以下步骤：生成表达苯乙醇胺-N-甲基转移酶(PNMT)的基因工程菌BL21(DE3)/pET28a-PNMT；培养基因工程菌BL21(DE3)/pET28a-PNMT和诱导表达PNMT；收集上述菌体,用缓冲液重悬菌体后,匀浆破碎,离心消除杂质,得PNMT的粗酶液；以去甲肾上腺素和S-腺苷-蛋氨酸(SAM)为底物,加入PNMT粗酶液反应得产物肾上腺素。本发明方法是首次系统的做出从去甲肾上腺素到肾上腺素的酶催化生产工艺,催化效率高达2g/l,转化率高达95％以上,环境友好,原料易得,生产成本低,工艺简单,反应条件温和,具有很好的工业化生产前景。(The invention discloses a method for producing epinephrine based on the catalysis of phenylethanolamine-N-methyltransferase, which specifically comprises the following steps: generating genetically engineered bacteria BL21 (DE 3)/pET 28a-PNMT for expressing phenylethanolamine-N-methyltransferase (PNMT); culturing genetically engineered bacteria BL21 (DE 3)/pET 28a-PNMT and inducing expression PNMT; collecting the thalli, re-suspending the thalli by using a buffer solution, homogenizing and crushing, and centrifuging to remove impurities to obtain a crude enzyme solution of PNMT; noradrenaline and S-adenosine-methionine (SAM) are used as substrates, and crude PNMT enzyme solution is added for reaction to obtain the product adrenaline. The method of the invention is a first-time systematic enzyme catalysis production process from noradrenaline to adrenaline, the catalysis efficiency is as high as 2g/l, the conversion rate is as high as more than 95%, the method is environment-friendly, the raw materials are easy to obtain, the production cost is low, the process is simple, the reaction condition is mild, and the method has good industrial production prospect.)

1. A method for producing epinephrine based on the catalysis of phenylethanolamine-N-methyltransferase is characterized by comprising the following steps:

step 1, constructing gene engineering bacteria BL21 (DE 3)/pET 28a-PNMT for expressing PNMT

After codon optimization is carried out according to the reported amino acid sequence of the human phenylethanolamine-N-methyltransferase, the sequence after full gene synthesis optimization is subcloned to a vector pET28a to obtain a recombinant plasmid pET28a-PNMT, the constructed recombinant plasmid pET28a-PNMT is transformed into an escherichia coli expression host BL21 (DE 3) by a calcium chloride method to obtain a phenylethanolamine-N-methyltransferase expression strain BL21 (DE 3)/pET 28 a-PNMT;

step 2, culturing genetically engineered bacteria BL21 (DE 3)/pET 28a-PNMT of PNMT

Selecting a single colony of genetically engineered bacteria BL21 (DE 3)/pET 28a-PNMT for expressing PNMT, inoculating the single colony to an LB culture medium, culturing at 37 ℃ until the OD value reaches above 0.6, then adding 0.25mM-1mM IPTG, culturing at 15-30 ℃ for 10-18 hours, and centrifugally collecting genetically engineered bacteria BL21 (DE 3)/pET 28a-PNMT for expressing phenylethanolamine-N-methyltransferase;

step 3, collecting genetically engineered bacteria BL21 (DE 3)/pET 28a-PNMT for expressing phenylethanolamine-N-methyltransferase, after resuspending the bacteria with a buffer solution with pH2-9, using a homogenate crusher, after crushing, centrifuging the crushed bacteria at the temperature of 4000-8000rpm and 4 ℃ for 8-10min to obtain a PNMT crude enzyme solution, and determining the protein concentration by using a microplate reader;

and 4, selecting noradrenaline and SAM as substrates, adding a buffer solution to adjust the pH value of the reaction system to 2-9, finally adding 0.5-20g/l of crude PNMT enzyme solution, and reacting at 20-60 ℃ for 0.5-24 h to obtain the product adrenaline.

2. The phenylethanolamine-N-methyltransferase-based method for producing epinephrine according to claim 1, wherein the enzyme production conditions in step 2 are as follows: IPTG was added in an amount of 0.75mM at an induction temperature of 25 ℃.

3. The phenylethanolamine-N-methyltransferase-based method for producing epinephrine according to claim 1, wherein the catalytic conditions in step 4 are as follows: temperature 50 ℃, pH = 5; the noradrenaline: the molar ratio of SAM was 1: 3.

Technical Field

The invention belongs to the technical field of preparation of epinephrine, and particularly relates to a method for producing epinephrine based on catalysis of phenylethanolamine-N-methyltransferase.

Background

Adrenaline is released from the adrenal gland of the central nervous system, and when the external environment makes the human body in states of fear, tension and the like, the body can quickly secrete adrenaline. It can make the body breathe tightly and the pupil expand, and provide a great deal of energy for the body movement. In addition, epinephrine also causes vasodilation of the heart, liver; the blood vessels of the skin and the mucosa shrink to relieve the pain of the patients with diseases and dangers, and the hope of bringing them for growth is provided. In organisms, the synthesis speed of adrenaline is high, namely, noradrenaline is generated in adrenal medulla firstly, and then the noradrenaline is generated through methylation under the action of PNMT.

Adrenalin has important application value as a critical medicine, so research on the action mechanism and the synthetic pathway of adrenalin must be enhanced, and a relatively safe, cheap and efficient adrenalin synthetic pathway is searched. At present, the common chemical synthesis method of adrenaline has violent reaction conditions, is difficult to control, and has lower adrenaline yield.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a method for producing epinephrine based on the catalysis of phenylethanolamine-N-methyltransferase, which has the advantages of mild reaction conditions, easy control, low cost and high yield, avoids using organic reagents which are toxic and harmful to the environment or human bodies, solves the pollution problem generated by chemical methods, and is environment-friendly.

A method for producing epinephrine based on the catalysis of phenylethanolamine-N-methyltransferase comprises the following steps:

step 1, constructing gene engineering bacteria BL21 (DE 3)/pET 28a-PNMT for expressing PNMT

After codon optimization is carried out according to the reported amino acid sequence of the human phenylethanolamine-N-methyltransferase, the sequence after full gene synthesis optimization is subcloned to a vector pET28a to obtain a recombinant plasmid pET28a-PNMT, the constructed recombinant plasmid pET28a-PNMT is transformed into an escherichia coli expression host BL21 (DE 3) by a calcium chloride method to obtain a phenylethanolamine-N-methyltransferase expression strain BL21 (DE 3)/pET 28 a-PNMT;

step 2, culturing genetically engineered bacteria BL21 (DE 3)/pET 28a-PNMT of PNMT

Selecting a single colony of genetically engineered bacteria BL21 (DE 3)/pET 28a-PNMT for expressing PNMT, inoculating the single colony to an LB culture medium, culturing at 37 ℃ until the OD value reaches above 0.6, then adding 0.25mM-1mM IPTG, culturing at 15-30 ℃ for 10-18 hours, and centrifugally collecting genetically engineered bacteria BL21 (DE 3)/pET 28a-PNMT for expressing phenylethanolamine-N-methyltransferase;

step 3, collecting genetically engineered bacteria BL21 (DE 3)/pET 28a-PNMT for expressing phenylethanolamine-N-methyltransferase, after resuspending the bacteria with a buffer solution with pH2-9, using a homogenate crusher, after crushing, centrifuging the crushed bacteria at the temperature of 4000-8000rpm and 4 ℃ for 8-10min to obtain a PNMT crude enzyme solution, and determining the protein concentration by using a microplate reader;

and 4, selecting noradrenaline and SAM as substrates, adding a buffer solution to adjust the pH value of the reaction system to 2-9, finally adding 0.5-20g/l of crude PNMT enzyme solution, and reacting at 20-60 ℃ for 0.5-24 h to obtain the product adrenaline.

As an improvement, the enzyme production conditions in the step 2 are as follows: IPTG was added in an amount of 0.75mM at an induction temperature of 25 ℃.

As a refinement, the catalytic conditions in step 4 are: temperature 50 ℃, pH = 5; the noradrenaline: the molar ratio of SAM was 1: 3.

Has the advantages that:

compared with the prior art, the method for producing epinephrine based on the catalysis of phenylethanolamine-N-methyltransferase has the following advantages: the method is a first-time systematic enzyme catalysis production process from noradrenaline to adrenaline, the catalysis efficiency is as high as 2g/l, the conversion rate is as high as more than 95%, the method is environment-friendly, the raw materials are easy to obtain, the production cost is low, the process is simple, the reaction condition is mild, and the method has a good industrial production prospect.

Drawings

FIG. 1 is a liquid chromatogram of the catalytic product of the present invention, wherein 1-noradrenaline and 2-adrenaline.

Detailed Description