A method for preparing dl-alanine with fumaric acid as raw material multi-enzyme coupling

A technology of alanine and fumaric acid, applied in the biological field, can solve the problems of complex fermentation broth, low sugar-acid conversion efficiency and high extraction cost, and achieve the effects of reducing equipment investment, low cost and shortening reaction time.

Active Publication Date: 2018-04-24
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The raw material used to produce DL-alanine by direct fermentation is glucose. According to the reported fermentation level, the conversion efficiency of sugar and acid is low, and the composition of the fermentation broth is complex and the extraction cost is high.
The production cost of this method is much higher than that of the chemical synthesis method. At present, there is no report on large-scale industrial production of DL-alanine by fermentation method in China.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Take 0.5g of Escherichia coli wet thallus with aspartase activity, 5g of Pseudomonas dacunhae wet thallus with aspartate-β-decarboxylase activity and Corynebacterium glutamicum wet bacterium with alanine racemase activity 10g of the body was added to 1000ml of transformation liquid, the transformation liquid contained 50g / L fumaric acid, 0.01g / L pyridoxal phosphate and 0.01g / L Tween 80, pH 6.0, 25 ℃ enzymatic reaction for 10h, the reaction liquid Optical rotation is zero. After the reaction, the concentration of DL-alanine in the conversion liquid was 36.4 g / L, and the molar conversion rate to fumaric acid was 95%.

[0037]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 , su...

Embodiment 2

[0040] 1. Take 0.5 g of Escherichia coli wet thallus with aspartate activity, 10 g of Pseudomonas dacunhae wet thallus with aspartate-β-decarboxylase activity and Corynebacterium glutamicum wet bacterium with alanine racemase activity Add 10g of the body to 1000ml conversion solution, the conversion solution contains 100g / L fumaric acid, 0.1g / L pyridoxal phosphate and 0.1g / L TritonX 100, pH 7.0, enzymatic reaction at 40°C for 18h, the reaction solution is optically active to zero. After the reaction, the concentration of DL-alanine in the conversion liquid was 73.6 g / L, and the molar conversion rate to fumaric acid was 96%.

[0041] 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 150ml, cooled and crystallized to room temperature , s...

Embodiment 3

[0044] 1. Take 1.0 g of Escherichia coli wet bacteria with aspartase activity, 15 g of Pseudomonas dacunhae wet bacteria with aspartate-β-decarboxylase activity and Corynebacterium glutamicum wet bacteria with alanine racemase activity Add 15g of the body to 1000ml of transformation solution, the conversion solution contains 300g / L fumaric acid, 0.5g / L pyridoxal phosphate and 0.5g / L CTAB, pH 8.0, 45 ℃ enzymatic reaction for 30h, the optical rotation of the reaction solution is zero. After the reaction, the concentration of DL-alanine in the conversion liquid was 218.6 g / L, and the molar conversion rate to fumaric 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 400ml, cooled and crystallized to room temperature ...

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Abstract

The invention belongs to the field of biotechnology, and in particular relates to a method for preparing DL-alanine by using fumaric acid as a raw material through multi-enzyme coupling. The preparation method uses fumaric acid as a raw material, and combines bacterial cells containing aspartase, aspartate-β-decarboxylase and alanine racemase or the crude enzyme liquid of the three enzymes with a certain Concentrated ammonium fumarate aqueous solution is mixed, and the enzymatic reaction is carried out at 25 ℃ ~ 55 ℃, pH 6 ~ 8, and the conversion product is separated by isoelectric point crystallization or isoelectric point crystallization and ion exchange resin to obtain high purity DL‑Alanine. The method adopts multi-enzyme coupling biotransformation technology to efficiently prepare DL-alanine, and has the advantages of wide source of raw materials, simple and convenient production process, short enzymatic conversion time and low production cost.

Description

1. Technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing DL-alanine by using fumaric acid as a raw material through multi-enzyme coupling. 2. Background technology [0002] DL-alanine is one of the non-essential amino acids for the human body. It can be used in the fields of medicine, food, cosmetics and feed additives, and has a very wide range of applications. [0003] At present, according to literature reports, the preparation methods of DL-alanine mainly include chemical synthesis, direct fermentation and biological enzyme method. [0004] 1. Chemical synthesis method [0005] Chemical synthesis methods include Strecker method, Buchere method, α-halogenated carboxylic acid ammoniation method, phase transfer catalytic synthesis method and L-alanine chemical racemization method. Among them, the Strecker and Buchere methods have cheap raw materials, but the separation of intermediate products after...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P39/00C12P13/06C12R1/19C12R1/38C12R1/15
Inventor 焦庆才刘均忠吴四平陆阳高亮刘茜
Owner NANJING UNIV
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