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Method for producing ephedrine through imine reductase

A technology for ephedrine and reductase, applied in the biological field, can solve the problems of inability to realize industrialized production, low yield, and many impurities of ephedrine, and achieve the effects of low cost, high yield and low impurity content

Active Publication Date: 2019-12-13
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] But above-mentioned method still has the defect that yield is low and the ephedrine impurity that obtains is too much, can not realize real suitability for industrialized production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Construction of genetically engineered bacteria capable of expressing imine reductase

[0056] Chemically synthesize the genes encoding the imine reductases whose amino acid sequences are shown in SEQ ID No.1 to SEQ ID No.14 respectively, and link them with the plasmid pET-28a(+) to obtain the recombinant plasmid pET-28a(+) -ired1~pET-28a(+)-ired14, this step was completed by Nanjing GenScript Biotechnology Co., Ltd.;

[0057] Introduce the obtained recombinant plasmids pET-28a(+)-ired1~pET-28a(+)-ired14 into Escherichia coli E.coli BL21(DE3) respectively; Streak the transformed E.coli BL21(DE3) Cultivate on LB solid medium containing 50 μg / mL kanamycin at 37°C for 20-24 h; pick positive transformants and inoculate them in LB liquid medium containing 50 μg / mL kanamycin. Cultivate at 37°C and 200rpm for 12-14 hours, collect the bacteria, extract the plasmid, and perform electrophoresis verification after double digestion with restriction endonucleases EcoR I ...

Embodiment 2

[0059] Embodiment 2: the preparation of ephedrine

[0060] Escherichia coli E.coli BL21(DE3) containing the empty plasmid pET-28a(+) was used as a blank control, and (R)-1-phenyl-2-carbonyl 3-propanol (30g, 0.2mol), methylamine (62g, 2mol) and glucose (36g, 0.2mol) were substrates, and with phosphate buffer saline as solvent, the recombinant Escherichia coli E.coli BL21(DE3) / pET-28a(+)- ired1~recombinant E. coli BL21(DE3) / pET-28a(+)-ired14 whole cells and glucose dehydrogenase acted as catalysts, with NADP + As a coenzyme, dimethyl sulfoxide was used as a cosolvent to obtain a 300mL reaction system; the reaction system was reacted for 18 hours at a temperature of 25°C, a pH value of 8.5, and a rotation speed of 200rpm to obtain a reaction solution containing ephedrine ; In the reaction system, the concentration of recombinant Escherichia coli E.coli BL21(DE3) / pET-28a(+)-ired whole cells is 34g / L, NADP +The concentration of 0.4g / L, the concentration of dimethyl sulfoxide is 1...

Embodiment 3

[0062] Embodiment 3: the preparation of ephedrine

[0063] With benzaldehyde (31.8g, 0.3mol), pyruvic acid (17.6g, 0.2mol), methylamine (31g, 1mol) and glucose (45g, 0.25mol) as substrate, phosphate buffer as solvent, respectively Recombinant Escherichia coli E.coli BL21(DE3) / pET-28a(+)-ired11~recombinant Escherichia coli BL21(DE3) / pET-28a(+)-ired14 whole cells obtained in Example 1 can express acetone Genetically engineered acid decarboxylase and glucose dehydrogenase act as catalysts to convert thiamine pyrophosphate and NADP + As a coenzyme, dimethyl sulfoxide was used as a cosolvent to obtain a 300mL reaction system; the reaction system was reacted for 26 hours at a temperature of 25°C, a pH value of 7.5, and a rotation speed of 200rpm to obtain a reaction solution containing ephedrine ; In the reaction system, the concentration of genetically engineered bacteria that can express pyruvate decarboxylase is 50g / L, and the concentration of recombinant Escherichia coli E.coli...

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Abstract

The invention discloses a method for producing ephedrine through imine reductase, and belongs to the technical field of biology. The invention provides the method for producing ephedrine through iminereductase. By the method, (R)-1-phenyl-2-carbonyl-3-propanol and methylamine are used as reaction raw materials, a reaction is performed for 18h, and the yield of the ephedrine in reaction liquid canbe as high as 86.5%; by the method, benzaldehyde, pyruvate and methylamine are used as reaction raw materials, a reaction is performed for 18h, and the yield of the ephedrine in the reaction liquid can be as high as 62.7%; and therefore, the method has the advantage of being high in yield when being used for producing the ephedrine.

Description

technical field [0001] The invention relates to a method for producing ephedrine by using imine reductase, which belongs to the field of biotechnology. Background technique [0002] Traditional Chinese medicine ephedra has good medicinal value, and has the advantages of safe and reliable medication, strong tolerance, and small side effects. Its main active ingredients are ephedrine and pseudoephedrine. Among them, ephedrine belongs to adrenaline stimulant drugs, which can be used to treat bronchial asthma, various coughs, allergic reactions and hypotension embolisms; and, ephedrine also has the functions of relaxing smooth muscle, constricting blood vessels, accelerating heart rate, increasing blood pressure and stimulating the central nervous system. In addition, studies have shown that ephedrine can be used as a food additive to stimulate the central nervous system and inhibit intestinal peristalsis to control body weight. It has great application prospects in dieting and...

Claims

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Application Information

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IPC IPC(8): C12P13/00C12R1/19
CPCC12N9/0028C12P13/001
Inventor 穆晓清徐岩罗磊
Owner JIANGNAN UNIV
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