Prepn of isotopically labeled 15 N-L-aspartic acid and 15 N-L-alanine

A technology of isotope labeling and aspartic acid, applied in the direction of fermentation, etc., can solve problems that have not been seen yet, and achieve the effect of large-scale industrialization implementation prospects

Inactive Publication Date: 2004-04-07
上海新立工业微生物科技有限公司
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  • Summary
  • Abstract
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  • Application Information

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Problems solved by technology

However, no isotopic labels produced by enzymatic methods have b

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] The strain Escherichia coli Asp-4 was cultured in a slant medium at 37°C for 24 to mature, then inserted into the aspartase enzyme liquid medium, filled with 50ml of medium in a 250ml shaker flask, and cultivated on a rotary shaker at 37°C at 220rpm for 24 hours. After the cultivation is completed, the culture solution is placed in a sterile centrifuge tube and centrifuged at 4000rpm for 15 minutes, and 0.5 g of the wet bacterial cells after centrifugation is added to a solution containing 5% fumaric acid, 8% 15 In 200 ml of N-ammonium sulfate and 0.0125% magnesium sulfate enzyme reaction solution, adjust the pH to 8.2-8.8 with NaOH, and react in a constant temperature incubator at 42° C. for 24 hours. After the reaction, the results are shown in Table 1:

[0067] Table 1

[0068] Item No.1 No.2 No.3 Average

[0069] 15 N-(NH 4 ) 2 SO 4 Amount added (g) 40 40 40

[0070] Amount of fumaric acid substrate added (g) 10 10 10

[...

Embodiment 2

[0078] Add 0.5 g of mature wet thalline cells containing 5% fumaric acid, 0.0125% magnesium sulfate and 3% 15 N-ammonium sulfate, 3% 15 In the mixed nitrogen source reaction liquid 200ml of N-ammonium chloride, use 15 Adjust the pH to 8.2-8.8 with N-ammonia water, and react in a constant temperature incubator at 42°C for 24 hours. Results The amount of aspartic acid produced was 52.0 g, the conversion rate of fumaric acid was 96.8%, and the molar conversion rate of nitrogen source was 62.4%.

Embodiment 3

[0080] The reaction solution completed in Example 1 was heated to 85-90° C., 0.5% activated carbon was added, stirred and kept for 45 minutes, vacuum filtered, and the filter cake was washed with distilled water. Combine the filtrates and concentrate the filtrate to 20% in vacuum at 65°C and 680mmHg, heat to 80°C, slowly add concentrated sulfuric acid while stirring, when the pH is 3.0, fine crystals are precipitated, stir and cool in a 100rpm constant speed stirrer to crystallize. When cooled to room temperature, refrigerate overnight at 5°C. The crystallization solution was vacuum filtered and crystal washed to obtain the finished wet crystal, which was dried at 60°C overnight. The results are shown in Table 2:

[0081] Table 2

[0082] Item No.1 No.2 No.3 Average

[0083] Reaction volume (ml) 625 630 618

[0084] Aspartic acid content (%) 7.93 8.14 8.25 8.11

[0085] Obtained product (g) 42.5 43.1 43.9

[0086] Total yield (%) 85.8 84....

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Abstract

The present invention is preparation process of isotopically labeled 15N-L-aspartic acid and 15N-L-alanine. Aspartase is proudced through microbial fermentation; inorganic material containing isotopic label 15N and fumaric acid are synthesized into 15N-L-aspartic acid via enzyme process. In the same time, 15N-L-aspartic acid is enzyme transformed into 15N-L-alanine. The said process is superior to available chemical synthesis process, which has low utilization of inorganic material containing isotopic label 15N, and to pure fermentation process, which has nore amino acids as side product and difficulty in separation. The present invention has 15N-L-aspartic acid concentration in ultimate reaction liquid over8%, molar conversion rate of inorganic material containing isotopic label 15N over 63 %, purity of product over 99 % and product abundance over 98.5 %.

Description

technical field [0001] The present invention relates to a 15 N-L-Aspartic Acid and 15 The preparation method of N-L-alanine, specifically relates to the preparation of isotope labeling by microbial enzyme reaction method 15 N-L-Aspartic Acid and 15 N-L-alanine method. technical background [0002] Nitrogen-15 (referred to as 15 N, the same below) is a stable isotope of nitrogen. As a tracer, it is widely used in organic chemistry, biochemistry, medicine, pharmacy, agriculture and other fields, especially for the research of life science. Especially with the completion of the determination and drawing of the human genome sequence map, the artificial synthesis of proteins is becoming more and more important to promote life science research in the world, and 15 N-amino acid plays an irreplaceable and unique tracer role in this process, its application is more and more extensive, and its market prospect is good. [0003] Due to the small yield and high requirements for pur...

Claims

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Application Information

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IPC IPC(8): C12P13/06C12P13/20
Inventor 毛展伟劳含章钱志良
Owner 上海新立工业微生物科技有限公司
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