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Technology for preparing 2, 4-diaminobutyric acid by enzyme catalysis method

A technology of diaminobutyric acid and enzymatic catalysis is applied in the field of preparing 2,4-diaminobutyric acid by enzymatic catalysis, which can solve the problems of difficult separation and purification, small molecular weight, high safety risk, etc. Feasibility of industrial application and high product conversion rate

Active Publication Date: 2020-02-18
SHENZHEN READLINE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although 2,4-diaminobutyric acid is widely distributed in nature, its separation and purification are very difficult due to its low abundance, small molecular weight, and high water solubility; most of the 2,4-diaminobutyric acid sold on the market It is prepared by chemical synthesis. The more classic preparation process utilizes L-aspartic acid to be directly converted into 2,4-diaminobutyric acid under the conditions of sodium azide, concentrated sulfuric acid and chloroform (the highest yield can reach 90%, G.I.Tesser and J.W.Van Nispen, "NOTE ON THE PREPARATION Of L-2,4-DIAMINOBUTYRIC ACID, Synthetic communication, 1971, 1, 285-287), the process continues to this day, however, because the reaction involves multiple toxic, easily Explosive chemicals, so the safety risk of its large-scale production is very large

Method used

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  • Technology for preparing 2, 4-diaminobutyric acid by enzyme catalysis method
  • Technology for preparing 2, 4-diaminobutyric acid by enzyme catalysis method
  • Technology for preparing 2, 4-diaminobutyric acid by enzyme catalysis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Catalytic production of 2,4-diaminobutyric acid by wet cell one-pot method

[0040] Add 6.65 g of L-aspartic acid (50 mM), 1.1 g of adenosine triphosphate disodium salt ATP (2 mM), and 5.7 g of polyphosphoric acid in 1L of 100 mM pH 8.0 tris-hydrochloride (Tris.HCl) solution. (Sigma, 25 poly, 55mM monophosphate), 0.9g magnesium chloride (10mM), 0.75g potassium chloride (10mM), reduced nicotinamide adenine dinucleotide phosphate NADPH monosodium salt 0.76g (1.0mM), reduced Prototype coenzyme nicotinamide adenine dinucleotide NADH disodium salt 0.71g (1.0mM), sodium phosphite 6.8g (55mM), ammonium formate 3.8g (60mM), alanine 89mg (1mM), pyridoxine phosphate Aldehyde 0.7 mg (0.0025mM), after the pH value was adjusted to 8.0, the overexpressed ASK, ASADH, AMT, PPK, Wet cells for PDH, ADH and FDH enzymes. During the reaction process, the pH of the reaction system was maintained between 6.0-9.0 by adding low-concentration HCl and NaOH aqueous solution, stirred g...

Embodiment 2

[0044] Example 2: Catalytic production of 2,4-diaminobutyric acid by liquid enzyme one-pot method

[0045] Collected cell mixture containing aspartokinase ASK, aspartate semialdehyde oxidase ASADH, transaminase AMT, ATP regenerating enzyme PPK, NADP regenerating enzyme PDH, alanine dehydrogenase ADH and formate dehydrogenase FDH In 20 times the volume of 25mM Tris pH 8.0 buffer (buffer A), and then through high-pressure crushing, high-speed centrifugation (16000rpm, 45min) to collect the supernatant containing crude protein, the enzyme crude solution directly for the next reaction to prepare 2 ,4-Diaminobutyric acid.

[0046] Similar to Example 1, 26.6 grams of L-aspartic acid (200 mM), 1.1 grams of adenosine triphosphate disodium salt ATP (2 mM) were successively added to 1 L of 100 mM pH 8.0 trishydrochloride (Tris.HCl) solution , 22.8 g polyphosphoric acid (Sigma, 25 poly, 220 mM monophosphate), 0.9 g magnesium chloride (10 mM), 0.75 g potassium chloride (10 mM), reduced n...

Embodiment 3

[0050] Example 3: Catalytic production of 2,4-diaminobutyric acid by immobilized enzyme in one pot

[0051] Collected cell mixture containing aspartokinase ASK, aspartate semialdehyde oxidase ASADH, transaminase AMT, ATP regenerating enzyme PPK, NADP regenerating enzyme PDH, alanine dehydrogenase ADH and formate dehydrogenase FDH In 20 times the volume of 25mM Tris pH 8.0 buffer (buffer A), then through high-pressure crushing, high-speed centrifugation (16000rpm, 45min) to collect the supernatant containing crude protein, add ammonium sulfate solids to the solution until Wash out (35%-55%, w / v ammonium sulfate / buffer). The enzyme solid was collected by centrifugation (10000rpm, 12min) and then slowly dissolved into 10 times the volume of buffer A, passed through a G25 size exclusion chromatography column (purchased from Sigma) for desalination, and was exchanged with DEAE Seplite FF (Xi'an Lanxiao Company) for anion exchange. After column separation, the primary purified enzy...

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Abstract

The invention relates to the technical field of biochemistry, and discloses a technology for preparing a 2, 4-diaminobutyric acid by an enzyme catalysis method. An L-aspartic acid is taken as an origin, an aspartic acid-4-phosphoric acid and aspartic acid semialdehyde intermediate is generated through one series of enzymatic reaction, and finally, the intermediate is converted into a target product, i.e., the 2, 4-diaminobutyric acid. The whole reaction system can increase four types of coenzyme regeneration systems, the use amounts of ATP (adenosine triphosphate), NADPH (Nicotinamide AdenineDinucleotide Phosphate), NADH (Nicotinamide adenine dinucleotide) and alanine are lowered, and reaction is effectively pushed to a high conversion rate. Various types of enzymatic reaction of a reaction system do not mutually interfere, the technology is convenient in a reaction operation, a final product has a high conversion rate, and industrial application feasibility can be further improved byan immobilized enzyme way.

Description

technical field [0001] The invention relates to the field of biochemical technology, in particular to a process for preparing 2,4-diaminobutyric acid by an enzyme-catalyzed method. Background technique [0002] 2,4-Diaminobutyric acid is an unnatural amino acid that exists in nature. It is a diamino amino acid similar to lysine and ornithine; its molecular formula is C 4 h 10 N 2 o 2 , with a molecular weight of 118, CAS No: 305-62-4. 2,4-Diaminobutyric acid is widely distributed in plants, flowers, yeast and some bacteria. Although it is not the basic composition of proteins, it is widely involved in various natural Biosynthesis of polypeptide antibiotics, such as polypeptin, Comirin, Polymyxin, etc. 2,4-Diaminobutyric acid also has high industrial application value. For example, it is the basic raw material for the production of snake venom (snake venom is a substance secreted by the snake gland of venomous snakes, which has the functions of analgesia, hemostasis, inhi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/04C07C227/40C07C229/26
CPCC12P13/04C07C227/40C07C229/26
Inventor 于铁妹樊卫林立峰何平潘俊锋刘建
Owner SHENZHEN READLINE BIOTECH CO LTD
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