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Method for synthesizing noradrenaline through double-enzyme coupling

A technology of norepinephrine and crude enzyme solution, which is applied in the field of double-enzyme coupling synthesis of norepinephrine, which can solve the problems of many by-products and high cost of disassembly, and achieve the effects of low production cost, environmental friendliness and mild reaction conditions

Pending Publication Date: 2021-06-15
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Aiming at the deficiencies in the prior art, the present invention provides a method for synthesizing norepinephrine through coupling of two enzymes. The method has mild reaction conditions, is easy to control, and has low cost, avoiding many by-products and high cost of splitting in the chemical synthesis process. problem, reduce its preparation cost

Method used

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  • Method for synthesizing noradrenaline through double-enzyme coupling
  • Method for synthesizing noradrenaline through double-enzyme coupling
  • Method for synthesizing noradrenaline through double-enzyme coupling

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Experimental program
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Effect test

Embodiment 1

[0035] Step 1, construct the genetically engineered bacteria BL21(DE3) / pGEX-6p-1-S-TA expressing S-TA

[0036] After codon optimization based on the reported amino acid sequence of L-threonine aldolase from black bears, the sequence is shown in SEQ NO.1, and the optimized sequence was synthesized by entrusting Qingke Biotechnology Co., Ltd. (Nanjing), Subcloned to the vector pGEX-6p-1 to obtain the recombinant plasmid pGEX-6p-1-S-TA, and transformed the constructed recombinant plasmid pGEX-6p-1-S-TA into the E. coli expression host by the calcium chloride method BL21(DE3), the strain BL21(DE3) / pGEX-6p-1-S-TA expressing L-threonine aldolase was obtained;

[0037] Step 2, construct the genetically engineered bacteria BL21(DE3) / pCDF-duet-TDC expressing TDC

[0038] After codon optimization based on the reported amino acid sequence of tyrosine decarboxylase derived from Lactobacillus brevis, the sequence is shown as SEQ NO.2, and the optimized sequence was entrusted to Qingke Bio...

Embodiment 2

[0048] To catalyze the production of norepinephrine, the specific operation steps are as follows: the reaction system is 1ml, the final concentration of 3,4-dihydroxybenzaldehyde is 60mM / L, the final concentration of glycine is 1M / L, and the final concentration of pyridoxal 5-phosphate is 20μm / L, then add 1g / L S-TA, 6g / L TDC, adjust the system to 1ml with PBS buffer, pH 7.5, and react at 30°C for 2-12h.

[0049] After the reaction, utilize high-performance liquid chromatography, XSelect HSS T3 type chromatographic column (4.6 * 250mm, 5 μ m), mobile phase is 23.3g sodium chloride (Shanghai Merrill Chemical Technology Co., Ltd.), 1ml glacial acetic acid, dilute with water to 1 L, mixed with 2% pure acetonitrile (analytical grade) solution, the column temperature was 30°C, the ultraviolet detection wavelength was 280 nm, and the flow rate was 5ml / min.

[0050] Take the gradient elution method:

[0051]

[0052] After detection by high performance liquid chromatography, the ...

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Abstract

The invention discloses a method for synthesizing noradrenaline through double-enzyme coupling, which comprises the following steps: by taking BL21DE3 as an original strain, respectively overexpressing an S-TA gene for coding L-threonine aldolase from black bear and a TDC gene for coding tyrosine decarboxylase from lactobacillus brevis on the basis of the original strain, so as to obtain two engineering bacteria, performing the reaction of a crude enzyme liquid obtained by cell disruption with 3, 4-dihydroxybenzaldehyde as a substrate, glycine as an auxiliary substrate and pyridoxal 5-phosphate as a cofactor to obtain noradrenaline of which the yield is 6.3 g / L. Compared with the prior art, the biosynthesis method disclosed by the invention has the advantages of greenness, high yield, mild conditions and the like, particularly has few byproducts and high environmental friendliness, and the cost of required production equipment is greatly reduced.

Description

technical field [0001] The invention belongs to the technical field of preparation of norepinephrine, and in particular relates to a method for synthesizing norepinephrine through double-enzyme coupling. Background technique [0002] Norepinephrine (NE) is a neurotransmitter mainly synthesized and secreted by sympathetic postganglionic neurons and adrenergic nerve endings in the brain. NE in the blood mainly comes from the adrenal medulla. Synthetic NE is a vasopressor drug that can be effectively used to support circulatory function in a variety of critically ill patients. At present, NE is often used as the first-line treatment drug for maintaining blood pressure in patients with septic shock. The clinical drug form is L-isomer, and its bitartrate salt is commonly used, that is, norepinephrine bitartrate. It is mainly used to treat shock caused by acute hypotension and peripheral vasodilation. [0003] In the synthetic routes reported in the literature at present, the ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/60C12N9/88C12P13/00
CPCC12N15/70C12N9/88C12P13/001C12Y401/02005C12Y401/01025C12N2800/22
Inventor 王昕乔荣梅陈可泉张琨冯娇麦丹丹马琛陆佳晨
Owner NANJING UNIV OF TECH
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