Method for preparing DL-alanine with fumaric acid as raw material through multi-enzyme coupling

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

Active Publication Date: 2015-04-22
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 comp...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] 1. Get 0.5 g of Escherichia coli wet thallus with aspartase activity, 5 g of Pseudomonas dacunhae wet thallus with aspartate-β-decarboxylase activity and Corynebacterium glutamicum wet with alanine racemase activity Add 10g of bacteria to 1000ml of transformation solution, which contains 50g / L fumaric acid, 0.01g / L pyridoxal phosphate and 0.01g / L Tween 80, pH 6.0, 25℃ for 10h, 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 , suction filtration, and drying to obtain 22 g of solid DL-al...

Embodiment 2

[0040] 1. Get 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 with alanine racemase activity Add 10g of bacteria to 1000ml transformation solution, which contains 100g / L fumaric acid, 0.1g / L pyridoxal phosphate and 0.1g / L TritonX 100, pH 7.0, 40℃ for 18h, the reaction solution Optical rotation is 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 , suction filtration, and drying to obtain 46 g of sol...

Embodiment 3

[0044] 1. Get 1.0 g of Escherichia coli wet thallus with aspartate activity, 15 g of Pseudomonas dacunhae wet thallus with aspartate-β-decarboxylase activity and Corynebacterium glutamicum wet with alanine racemase activity Add 15g of bacteria to 1000ml of transformation solution, which contains 300g / L fumaric acid, 0.5g / L pyridoxal phosphate and 0.5g / L CTAB, pH 8.0, enzymatic reaction at 45°C for 30h, and the reaction solution is optically active to 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 , suction filtration and dr...

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Abstract

The invention belongs to the technical field of biology and particularly relates to a method for preparing DL-alanine with fumaric acid as raw material through multi-enzyme coupling. The preparation method uses the fumaric acid as raw materials, bacteria cells containing aspartase, aspartic acid-beta-decarboxylase and alanine racemase or crude enzyme of the aspartase, the aspartic acid-beta-decarboxylase and the alanine racemase is mixed with a fumaric acid water solution of a certain concentration, enzymatic reaction is carried out at the temperature of 25-55 DEG C, under the pH 6-8, converted products are separated though an isoelectric point crystallization method or a method combining isoelectric point crystallization and ion exchange resin, and high-purity DL-alanine is obtained. According to the method, the DL-alanine is prepared efficiently by adopting the multi-enzyme coupling technology, the method has the advantages of being wide in raw material source, simple in operation of production process, short in enzymatic conversion time, low in production cost and the like.

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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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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