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D-aminosuccinic acid and L-alanine preparation method

A technology of aspartic acid and alanine, which is applied in the field of enzyme engineering, can solve the problems of high production cost and achieve the effects of simple operation, lower production cost and simplified process flow

Inactive Publication Date: 2003-03-26
NANJING UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

What these patents have in common is that they focus on the research on the immobilization method and the production of L-alanine. In the process of the mentioned production of D-aspartic acid, it is necessary to add expensive coenzyme pyridoxal phosphate and metal ion Co 2+ , resulting in high production costs

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Pseudomonas strain NX-1 was cultured in the following 200ml culture medium:

[0019] Sodium Fumarate 0.5%, Ammonium Fumarate 1.0%, Corn Steep Steep 2.0%, Peptone 0.8%, K 2 HPO 4 0.05%, MgSO 4 .7H 2 O0.01%, pH7.0, 30 ± 1 ℃ shaking flask culture 24h, centrifuge wet cells, L-aspartic acid-β decarboxylase activity per gram of cells is 1.42 × 10 4 U. The cells were added to 200 mL of 10% DL-ammonium aspartate solution, and reacted at 37° C. for 10 h. The concentration of D-aspartic acid in the reaction solution is 5%, the concentration of L-alanine is 3.345%, and the molar conversion rate of L-aspartic acid is 100%.

[0020] The reaction solution was decolorized with 0.5% activated carbon, the filtrate was adjusted to pH 3.0, cooled to 5°C, and the precipitated crystals were washed with cold water to obtain 7.2 g of D-aspartic acid. The remaining mother liquor flows through 001×7 type resin (H + type), washed with water and eluted with 1% ammonia solution, and the fil...

Embodiment 2

[0024] Pseudomonas strain NX-1 was cultured in the following 200ml culture medium:

[0025] Sodium Glutamate 2%, Corn Steep Steep 2.0%, Peptone 0.8%, K 2 HPO 4 0.05%, MgSO 4 .7H 2 O0.01%, pH7.0, shake the flask at 30°C±1°C for 24h. The activity of L-aspartic acid-β-decarboxylase per mL of fermentation broth was measured to convert 1000 μmol L-aspartic acid per hour. The cells were centrifuged, mixed with 200mL 20% DL-ammonium aspartate solution, and reacted at 32°C for 20h. The concentration of D-aspartic acid in the reaction solution is 10%, the concentration of L-alanine is 6.69%, and the molar conversion rate of L-aspartic acid is 100%.

[0026] The reaction solution was decolorized with activated carbon, the filtrate was adjusted to pH 3.0, concentrated in vacuo to 100 mL, cooled to 5°C and left overnight. Precipitate crystals, wash with cold water to obtain 18.6g D-aspartic acid, [α] D 25 =-25.5° (C=1,5NHCL). Mother liquor flows through 001×7 type resin (H + ) t...

Embodiment 3

[0028] With the formula of the medium in Example 2, put 1L medium in a 1.5L fermenter, insert Pseudomonas strain NX-1 according to 0.5% inoculum, stirring speed 500r / min, ventilation ratio 1: 0.3, 30+1 After culturing at ℃ for 18 hours, the activity of L-aspartic acid-β decarboxylase per mL of culture medium can convert 1500 μmol L-aspartic acid per hour. The cells were obtained by centrifugation, mixed with 1000 mL of 10% DL-ammonium aspartate solution, and reacted at 40°C for 8 hours. The concentration of D-aspartic acid in the reaction solution was 5%. The concentration of L-alanine was 3.345%, and the molar conversion rate to L-aspartic acid was 100%.

[0029] After the reaction, the reaction solution was centrifuged, and the cells were taken out, and added to the substrate solution for reaction as above, and repeated 5 times. The results are shown in Table 1:

[0030] Table 1 Results of D-aspartic acid production by cells recycling

[0031] Reaction time...

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Abstract

The invention provides a method for preparing D-asparagus fern ammoniac acid and alpha-alanine. The method is as follows: cultivate false single-cell bacterium microbe to produce high-activity L-asparagus fern ammoniac acid-beta decarboxyl; adopt dissociative-cell method to mix cells having enzyme or culture solution with DL-asparagus fern ammoniac acid or DL-asparagus fern ammoniac acid salt solution and make enzyme reaction under 32-45 degree C, and then separate the reacting production by using the method of equi-electrical point crystal combined with ion exchange resin to obtain the invention.

Description

technical field [0001] The invention relates to a preparation method of D-aspartic acid and L-alanine, belonging to the field of enzyme engineering. Background technique [0002] The preparation techniques of D-aspartic acid include chemical asymmetric resolution (Japanese Patent Publication No. 52-54190) and preferential crystallization (Japanese Patent Publication No. 54-25006). However, these methods are complicated in process, and the optical purity of the product is low, so there is no great practical value. [0003] In the reported enzymatic production of D-aspartic acid technology, microorganisms such as Candida genus are used to eliminate L-aspartic acid in DL-aspartic acid to obtain D-aspartic acid (Show 56 -20753); use genetic improvement to obtain L-aspartic acid-α-decarboxylase producing bacteria, and simultaneously prepare D-aspartic acid and β-alanine from DL-aspartic acid (CN1242054A), and Simultaneous preparation of D-aspartic acid and L-malic acid from DL-...

Claims

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Application Information

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IPC IPC(8): C12P13/06C12P13/20
Inventor 欧阳平凯徐虹代书玲韦萍
Owner NANJING UNIV OF TECH
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