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Preparation method of 13C and 15N double labeled L-lysine hydrochloride

A lysine hydrochloride, double-labeling technology is applied in the field of production and preparation of stable isotope-labeled compounds, which can solve problems such as failure to meet product quality requirements, decline in isotopic abundance, lack of patents and literature reports, etc., and achieve results. Significant, lower production costs, effective use of the effect

Active Publication Date: 2011-09-07
SHANGHAI RES INST OF CHEM IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the production of L-lysine mostly adopts fermentation method, and there are many research reports on the production of L-lysine by fermentation method, but in the research on biosynthesis 13 C, 15 There are no patents and literature reports in the production field of N stable isotope-labeled L-lysine hydrochloride
It is prepared by direct fermentation, and the target amino acid is enriched in a large amount, and the separation is relatively simple. However, due to the presence of organic carbon and nitrogen sources in the seeds and fermentation formula, the abundance of stable isotopes will be diluted. If the process is not optimized and controlled, the isotope of the product is often The abundance is greatly reduced, so that the quality requirements of the product cannot be met

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Brevibacterium flavum (Brevibacterium flavum) ATCC14067 was used as the starting strain, and the culture medium used included slant preservation medium, slant activation medium, and abundance shake flask fermentation medium. The slant preservation medium and slant activation medium are the same as those for conventional fermentation, and the formula is as follows:

[0026] The slant preservation medium (g / L) is: peptone 10, beef extract 10, NaCl 5.0, agar 20, pH 7.0-7.2.

[0027] The slant activation medium (g / L) is: glucose 5.0, peptone 10, beef extract 10, NaCl 5.0, agar 20, pH 7.0-7.2.

[0028] The pH of the above medium was adjusted with NaOH at a concentration of 2 mol / L, and sterilized at 121°C for 20 minutes.

[0029] The low-abundance fermentation medium formula (g / L) is as follows:

[0030] 13 C-glucose 150, 15 N-Ammonium sulfate 40, MgSO 4 0.35, KH 2 PO 4 1.0, biotin 300μg / L, vitamin B 1 400μg / L, homoserine 0.25, corn steep liquor 4.5mL / L, MnSO 4 4H...

Embodiment 2

[0034] Using Corynebacterium glutamicium AS 1.563 as the starting strain, the culture medium used includes slant preservation medium, slant activation medium, and abundance shake flask fermentation medium. The slant preservation medium and slant activation medium are the same as those for conventional fermentation, and the formula is as follows:

[0035] The slant preservation medium (g / L) is: peptone 10, beef extract 10, NaCl 5.0, agar 20, pH 7.0-7.2.

[0036] The slant activation medium (g / L) is: glucose 5.0, peptone 10, beef extract 10, NaCl 5.0, agar 20, pH 7.0-7.2.

[0037] The pH of the above medium was adjusted with NaOH at a concentration of 2 mol / L, and sterilized at 121°C for 20 minutes.

[0038] The high-abundance fermentation medium formula (g / L) is as follows:

[0039] 13 C-glucose 145, 15 N-Ammonium sulfate 45, MgSO 4 0.25,K 2 HPO 4 1.0, biotin 400μg / L, vitamin B 1 600μg / L, homoserine 0.15g, corn steep liquor 7.5mL / L, MnSO 4 4H 2 O 0.02, FeSO 4 ·7H ...

Embodiment 3

[0043] Corynebacterium glutamicium (Corynebacterium glutamicium) ATCC13869 was used as the starting strain, and the culture medium used included slant preservation medium, slant activation medium, and abundance shake flask fermentation medium. The slant preservation medium and slant activation medium are the same as those for conventional fermentation, and the formula is as follows:

[0044] The slant preservation medium (g / L) is: peptone 10, beef extract 10, NaCl 5.0, agar 20, pH 7.0-7.2.

[0045] The slant activation medium (g / L) is: glucose 5.0, peptone 10, beef extract 10, NaCl 5.0, agar 20, pH 7.0-7.2.

[0046] The pH of the above medium was adjusted with NaOH at a concentration of 2 mol / L, and sterilized at 121°C for 20 minutes.

[0047] The high-abundance fermentation medium formula (g / L) is as follows:

[0048] 13 C-glucose 130, 15 N-Ammonium sulfate 35, MgSO 4 0.25,K 2 HPO 4 1.0, biotin 600μg / L, vitamin B 1 700μg / L, homoserine 0.20g, bean cake hydrolyzate 4....

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Abstract

The invention relates to a preparation method of 13C and 15N double labeled L-lysine hydrochloride, comprising the following steps: (1) fermenting strain selection; (2) fermenting culture formulation; (3) fermentation process; and (4) separation and extraction. The fermentation process adopted by the invention is suitable for preparing the 13C and 15N double labeled L-lysine hydrochloride; the acid yield under the process condition is improved by 40-60% compared with the acid yield by adopting a fully synthetic culture medium, and the effect is obvious; the substances, such as homoserine, biotin and vitamin B1 and the like are added through controlling the additive amount of carbon source glucose, and inorganic nitrogen sources, such as ammonium sulfate and the like, as well as organic nitrogen sources, such as corn steep liquor and the like, thus the fermenting formulation is improved, the isotope abundance is controlled, the risk of abundance reduction is reduced, the acid yield is ensured, the isotope raw material is effectively utilized, the production cost is lowered, and the raw materials with different abundance specifications can be utilized to prepare the products with different abundances, thus satisfying various abundance requirements.

Description

technical field [0001] The invention belongs to the field of production and preparation of stable isotope-labeled compounds, relates to the field of microbial fermentation technology and biological downstream separation, especially relates to a 13 C. 15 The preparation method of N double-labeled-L-lysine hydrochloride. Background technique [0002] Traditional methods for producing stable isotope-labeled L-amino acids can be prepared by decomposing and separating labeled proteins, organic synthesis, enzymatic methods, and microbial fermentation methods. The method of decomposing and separating labeled proteins is often used to prepare labeled compound amino acids. It is very difficult to separate and obtain labeled single amino acids. Due to the limitation of raw materials, it is rarely used nowadays. The organic synthesis method is relatively simple, but the preparation of L-amino acid requires optical resolution. 13 C. 15 The raw material utilization rate of N stable i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P13/08C12R1/15C12R1/13
Inventor 侯静华孙建春刘占锋杜晓宁李良君
Owner SHANGHAI RES INST OF CHEM IND
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