Method for preparing beta-alanine from maleic acid through multi-enzyme coupling

A technology of maleic acid isomerase and maleic acid, which is applied in the biological field, can solve the problems of low yield of repeated purification, high safety requirements of the production site, and high production cost, and achieve reduced production costs, low cost, and low substrate fully transformed effect

Inactive Publication Date: 2017-08-04
NANJING UNIV
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  • Abstract
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Problems solved by technology

Wherein the acrylonitrile method is that acrylonitrile and ammonia react in diphenylamine and tert-butanol solution to produce β-aminopropionitrile, which is then obtained by alkaline hydrolysis. The disadvantage of this method is that there are more than 40% NaCl and impurities in the product. Requires repeated purification with low yield; the β-aminopropionitrile method is to react β-aminopropionitrile with barium hydroxide to generate β-aminopropionate barium and nitrogen, and introduce CO 2 , the barium salt is precipitated to produce β-alanine, and the barium ion is removed by resin. The disadvan

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  • Method for preparing beta-alanine from maleic acid through multi-enzyme coupling

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Effect test

Embodiment 1

[0040] 1. Get Alcaligenes faecalis wet thallus 5g with maleic acid isomerase activity, Escherichia coli wet thallus 0.5g with aspartic acid-alpha-decarboxylation Add 10 g of enzyme-active Corynebacterium glutamicum wet thallus into 1000 ml of transformation solution containing 50 g / L maleic acid, 0.01 g / L pyridoxal phosphate and 0.01 g / L Tween 80, pH 6.0, 30 ℃ enzymatic reaction 15h, the reaction is terminated. After the reaction, the concentration of β-alanine in the conversion liquid was 36.1 g / L, and the molar conversion rate to maleic acid was 94%.

[0041] 2. Centrifuge the transformation solution at 4000r / min for 15min to remove the bacteria, heat the supernatant to 70-80°C, add activated carbon for decolorization, the decolorization solution is purified by ultrafiltration and nanofiltration, the purified solution is vacuum concentrated to 40ml, cooled and crystallized to room temperature , suction filtration, and drying to obtain 14 g of solid β-alanine.

[0042]3. Af...

Embodiment 2

[0044] 1. Get Alcaligenes faecalis wet thallus 5g with maleic acid isomerase activity, Escherichia coli wet thallus 0.5g with aspartic acid-alpha-decarboxylation Add 15g of enzyme-active Corynebacterium glutamicum wet cells into 1000ml of transformation solution containing 100g / L maleic acid, 0.1g / L pyridoxal phosphate and 0.1g / L TritonX 100, pH 7.0 , 37 ℃ enzymatic reaction 20h, the reaction is terminated. After the reaction, the concentration of β-alanine in the conversion solution was 72.9 g / L, and the molar conversion rate to maleic acid was 95%.

[0045] 2. Centrifuge the transformation solution at 4000r / min for 15 minutes to remove the bacteria, heat the supernatant to 70-80°C, add activated carbon for decolorization, the decolorization solution is purified by ultrafiltration and nanofiltration, the purified solution is vacuum concentrated to 80ml, cooled and crystallized to room temperature , suction filtration, and drying to obtain 29 g of solid β-alanine.

[0046] 3...

Embodiment 3

[0048] 1. Get Alcaligenes faecalis wet thallus 10g with maleic acid isomerase activity, Escherichia coli wet thallus 1.0g with aspartic acid-alpha-decarboxylation Add 20g of wet cells of Corynebacterium glutamicum with enzymatic activity to 1000ml transformation solution containing 100g / L maleic acid, 0.1g / L pyridoxal phosphate and 0.1g / L CTAB, pH 8.0 , 42 ℃ enzymatic reaction 18h, the reaction is terminated. After the reaction, the concentration of β-alanine in the conversion liquid was 73.6 g / L, and the molar conversion rate to maleic acid was 96%.

[0049] 2. Centrifuge the transformation solution at 4000r / min for 15 minutes to remove the bacteria, heat the supernatant to 70-80°C, add activated carbon for decolorization, the decolorization solution is purified by ultrafiltration and nanofiltration, the purified solution is vacuum concentrated to 80ml, cooled and crystallized to room temperature , suction filtration, and drying to obtain 29.5 g of solid β-alanine.

[0050]...

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Abstract

The invention specifically relates to a method for preparing beta-alanine from maleic acid through multi-enzyme coupling, belonging to the field of biotechnology. The method comprises the following steps: adjusting the pH value of an aqueous maleic acid solution with a certain concentration to 6 to 9 by using ammonia water; then separately adding bacterial cells respectively with maleate isomerase activity, aspartase activity and aspartate-alpha-decarboxylase activity or crude liquid of the three enzymes; carrying out enzymatic reaction at 30 to 50 DEG C; and separating and converting products by using isoelectric-point crystallization or a combination of isoelectric-point crystallization and ion exchange resin so as to obtain high-purity beta-alanine. The method provided by the invention uses maleic acid as a substrate for preparation of beta-alanine via multi-enzyme coupling and has the advantages of wide sources of raw materials, low price, simple operation, low production cost, suitability for large-scale industrial production, etc.

Description

[0001] 1. Technical field [0002] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing beta-alanine by using maleic acid as a raw material through multi-enzyme coupling. [0003] 2. Background technology [0004] β-alanine is a precursor for the synthesis of pantothenic acid, a type of B vitamin that forms part of coenzyme A and is important for the exchange of endogenous metabolic energy in various tissues. With the gradual deepening of the understanding of β-alanine, β-alanine is widely used in the fields of medicine, food, cosmetics and feed, and the market demand for β-alanine is also growing. Therefore, β-alanine Acid has a wide range of application value and development prospects. [0005] According to literature reports, the current preparation methods of β-alanine mainly include chemical synthesis and biological enzymatic methods. [0006] 1. Chemical synthesis method [0007] Chemical synthesis methods include acr...

Claims

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Application Information

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IPC IPC(8): C12P13/06C12N9/90C12N9/88
CPCC12N9/88C12N9/90C12P13/06C12Y401/01011C12Y403/01001C12Y502/01001
Inventor 焦庆才刘均忠魏宇张卫卫吴婷曹双燕余进海
Owner NANJING UNIV
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