CN1982466A - Production of stabilized isotope 15N labelled L-valine - Google Patents

Abstract

The invention relates to a production process for labeling L-valine with stable isotope ¹⁵ N. The production process comprises the following steps: (1) selecting suitable strains, (2) preparing inclined planes, (3) fermenting and cultivating formula, (4) ) fermentation process, (5) separation and extraction. The present invention adopts microbial direct fermentation method to produce L-valine- ^15N , improves growth and metabolism by adding trace vitamin complexes, aims to solve the problem of ^15N abundance decline, and maximize ^15N utilization rate to meet product Production requirements.

Description

A production process of stable isotope 15NN labeling L-valine

technical field

The invention belongs to the technical field of production of stable isotope-labeled compounds, in particular to a process for producing stable isotope ¹⁵ N-labeled L-valine by means of microbial fermentation and biological extraction.

Background technique

Like other labeled amino acids, L-valine- ¹⁵ N can be widely used as a tracer in related research fields such as pharmaceutical industry, food industry and life science. The unique tracer effect of L-valine- ¹⁵ N can be used to experiment and study the unique physiological role of L-valine in the body. To the production of L-valine, some research groups have done a lot of research work to it at home and abroad, have US 6737255 B2; US4542098; US6214591B; 6(1): P24-27, 1999. However, there are not many literatures and patents on the production of ¹⁵ N stable isotope-labeled L-valine by biosynthesis. Produced by direct fermentation, the target amino acid is enriched in large quantities, and the stable isotope ¹⁵ N is easy to label. However, due to the difficulty of labeling the organic nitrogen source in the fermentation formula and the influence of the natural abundant nitrogen source, L-valine- ¹⁵ N is often used The abundance of 15 N has dropped a lot, and the product requirements cannot be met. At the same time, the utilization rate of ¹⁵ N raw materials is also low, thereby increasing the cost. Therefore, it is necessary to improve the conventional fermentation formula and process.

Contents of the invention

The object of the present invention is exactly to provide a kind of production technique of stable isotope ¹⁵ N labeling L-valine in order to overcome the deficiency that above-mentioned prior art exists, and this technique adopts microbial direct fermentation method to produce L-valine- ^15N , The growth and metabolism are improved by adding trace amounts of vitamin complexes, aiming to solve the problem of decreased abundance of ¹⁵ N, and to increase the utilization rate of ¹⁵ N as much as possible to meet the production requirements of the product.

The object of the present invention can be achieved through the following technical solutions: a production process for stable isotope ¹⁵ N-labeled L-valine, characterized in that the production process comprises the following steps:

(1) Select applicable strains

Selection of Brevobacterium flavum, Corynebacterium pekinense, Corynebacterium crenatum, Escherichia coli, Brevibacterium lactofermentum for L-valine production 1. One of Corynebacterium glutamicum;

(2) Bevel preparation

Inoculate the slant with the above fermentation strains, culture at 28-35°C for 8-24 hours, and refrigerate the slant for later use;

(3) Fermentation culture formula

Using fully synthetic medium as the basic medium, adding a small amount of organic nitrogen source, the main formula is as follows:

Glucose 60～140g/L or sucrose 120～250g/L; ammonium chloride 15～50g/L or ammonium sulfate 15～30g/L; corn steep liquor or cell hydrolyzate 0～5g/L; K ₂ HPO ₄ ·3H ₂ O 1～4g/L; MgSO ₄ ·7H ₂ O0.2～0.8g/L; MnSO ₄ ·H ₂ O2～20mg/L; FeSO ₄ ·7H ₂ O2～20mg/L; ZnSO ₄ ·7H ₂ O0.5～10mg/L; biotin 0～100μg/L; thiamine hydrochloride 20～100μg/L; CaCO ₃ 20～60g/L;

(4) Fermentation process

Wash the bacterial lawn cultivated in the activated slant with a small amount of sterile water and inoculate it into a sterilized fermentation shaker flask or fermenter containing the above-mentioned fermentation medium to start fermentation. The control condition of the shaker fermentation is: initial pH6. 4-7.2, the rotating speed of the shaker is 180-220r/min, and the fermentation time is 60-96 hours. The control conditions of the fermenter are: the initial fermentation temperature is 30-33°C, the ventilation rate is 0.5-1.5VVM, the tank pressure is 0.02-0.05Mpa, the dissolved oxygen is 0.5%-90% saturation, and it is defoamed by polyether or silicone defoamer. soak; ferment for 18-32 hours, increase the speed and ventilation to ensure that the dissolved oxygen is at 30-50% saturation; control the pH in the whole fermentation process to be slightly acidic, adjust the pH to 6-7 by adding ¹⁵ N labeled urea, ammonia water or liquid ammonia, and flow Add 50% glucose to control the glucose concentration in the fermentation broth to 2% to 5%, and the fermentation time is 48 to 72 hours;

(5) Separation and extraction

The separation of stable isotope ¹⁵ N-labeled L-valine in the fermentation broth is separated and extracted by ion exchange method to obtain stable isotope ¹⁵ N-labeled L-valine, and the product is obtained by vacuum concentration, catching ammonia, low temperature crystallization with ethanol and vacuum drying .

In the described step (1), Brevobacterium flavum (Brevobacterium flavum) is selected from one or more of commercially available ATCC14067, ATCC14020, ATCC13869, ATCC14066, and Beijing Corynebacterium (Corynebacterium pekinense) is selected from commercially available CGMCC1.586, CGMCC1 One or more of .299CGMCC 1.495, Corynebacterium crenatum is selected from one or more of commercially available CGMCC1.452, CGMCC1.1001, CGMCC1.1007, Escherichia coli is selected from Commercially available ATCC13005, Brevibacterium lactofermentum are selected from commercially available ATCC13058, Corynebacterium glutamicum are selected from commercially available ATCC2256, ATCC13032, ATCC14751 one or more.

The invention utilizes the microbial slant lawn to directly ferment and produce L-valine- ¹⁵ , controls the addition amount of organic nitrogen source, directly adds biotin and thiamine hydrochloride, and improves the acid production rate by improving the fermentation formula and process. In this way, ^{the 15} N raw material can be effectively used, and the decrease in abundance will not affect the production requirements of the product.

If the conventional fermentation method is adopted, although the yield of stable isotope ¹⁵ N-labeled L-valine is relatively high, the abundance of ¹⁵ N decreases greatly, often by more than 3%, and it is difficult to meet the requirements of high-abundance products. Although the use of fully synthetic medium for fermentation had little effect on the abundance of ¹⁵ N, the acid production was so low that the utilization rate of ¹⁵ N raw materials was not high. By changing the fermentation formula and process, directly adding thiamine hydrochloride and biotin, the yield of stable isotope ¹⁵ N-labeled L-valine obtained in the present invention is correspondingly increased by 10 to 50% compared to the amount of acid produced by fermentation using a fully synthetic medium. %, while the stable isotope ¹⁵ N-labeled L-valine ¹⁵ N abundance can be reduced to less than 0.5%, and some even hardly decrease, which greatly improves the utilization rate of ¹⁵ N raw materials and reduces production costs. At the same time, under the condition that ^{the 15} N abundance of the product is guaranteed, the fermentation process is simplified and the strains are directly transferred from the activated slope, which not only shortens the production cycle, but also is suitable for controlling the abundance of ^{the 15} N product. At the same time, the raw materials are fully utilized, which greatly saves Raw material recovery costs. The invention can utilize low-abundance and high-abundance ¹⁵ N inorganic raw materials to meet the requirements of products with different abundances.

Detailed ways

The present invention will be further described below in conjunction with specific examples.

Example 1

The slant medium used: glucose 1g/L, beef extract 10g/L, peptone 10g/L, NaCl 5g/L, agar 20g/L, fermentation medium: glucose 100g/L, ( ¹⁵ NH ₄ ) ₂ SO ₄ 15g/L L, K ₂ HPO ₄ 1.5g/L, MgSO ₄ 7H ₂ O 0.5g/L, FeSO ₄ 7H ₂ O 0.02g/L, MnSO ₄ H ₂ O 0.02g/L, Biotin 50μg/L, Thiamine 100μg/L, corn steep liquor 1g/L, CaCO ₃ 30g/L.

Corynebacterium Beijing CGMCC1.586 was inoculated from the preserved slant to the activated slant, cultured in a 30°C constant temperature incubator for 24 hours, washed with a little sterile water, and transferred to a 500mL Erlenmeyer flask containing 20ml of fermentation medium. A total of 10 bottles were fermented and cultured on a revolving shaker at 200 r/min at 30°C for 60 hours, and the average production of L-valine- ¹⁵ N by fermentation could reach 18 g/L.

The fermentation broth was separated by a single column of 732 cation exchange resin, and the dry solid L-valine- ^15N product was obtained by conventional methods. The product abundance was 98.28% by mass spectrometry analysis, and the abundance decreased by 0.427%.

If the corn steep liquor in the fermentation medium is changed to 10g/L, the fermentation process remains unchanged, and the average L-valine- ^15N produced by fermentation can reach 33g/L, but the abundance is only 95.34%, and the abundance decreases by 3.6%. If corn steep liquor is not added to the fermentation medium, the average L-valine- ^15N produced by fermentation is only 5g/L, the abundance is 98.71%, and the abundance decreases by 0.232%. If vitamin complexes such as biotin and thiamine are not added to the above-mentioned fermentation medium, the average L-valine- ^15N produced by fermentation is only 2g/L. Considered comprehensively, this invention effect is remarkable.

Example 2

The slant medium used: glucose 1g/L, beef extract 10g/L, peptone 10g/L, NaCl 5g/L, agar 20g/L, fermentation medium: glucose 120g/L, ( ¹⁵ NH ₄ ) ₂ SO ₄ 30g/L L, K ₂ HPO ₄ 1g/L, MgSO ₄ 7H ₂ O 0.8g/L, FeSO ₄ 7H ₂ O 0.05g/L, MnSO ₄ H ₂ O 0.02g/L, Biotin 50μg/L, Sulfur Amine 100μg/L, cell hydrolyzate 5g/L, CaCO ₃ 40g/L.

Inoculate Corynebacterium Beijing CGMCC1.1001 from the preserved slant to the activated slant, cultivate it in a 30°C constant temperature incubator for 18 hours, wash the two slant lawns with a little sterile water, and transfer them to the above-mentioned 500mL Erlenmeyer flask containing 20mL of fermentation medium At 30°C and 200rpm, the fermentation was completed on a revolving shaker for 72 hours, and the average production of L-valine- ^15N in the fermentation could reach 25g/L.

The fermentation broth was separated by a single column of 1500H cation exchange resin, and the dry solid L-valine- ^15N product was obtained by conventional methods. The abundance of the product was 98.83% by mass spectrometry analysis, and the abundance decreased by 0.131%.

Example 3

The slant medium used: glucose 1g/L, beef extract 10g/L, peptone 10g/L, NaCl 5g/L, agar 20g/L, fermentation medium: sucrose 140g/L, ( ¹⁵ NH ₄ ) ₂ SO ₄ 40g/L L, K ₂ HPO ₄ 3g/L, MgSO ₄ 7H ₂ O 0.2g/L, FeSO ₄ 7H ₂ O 0.02g/L, MnSO ₄ H ₂ O 0.08g/L, Biotin 100μg/L, Sulfur Amine 50μg/L, corn steep liquor 2g/L, CaCO ₃ 30g/L.

Inoculate Brevibacterium flavum ATCC14067 from the preserved slant to the activated slant, and cultivate it in a constant temperature incubator at 30°C for 15 hours, wash the two slant lawns with a little sterile water and transfer them to a 500mL Erlenmeyer flask containing 20mL of fermentation broth, at 30°C 200r/min on a revolving shaker for 96 hours of fermentation and cultivation, and the average production of L-valine- ^15N in fermentation can reach 21g/L.

The fermentation broth was separated by a single column of FMC11Na cation exchange resin, and the dry solid L-valine- ^15N product was obtained by conventional methods. The abundance of the product was 98.66% by mass spectrometry analysis, and the abundance decreased by 0.283%.

Claims (2)

1. stable isotope ¹⁵The production technique of N label L-Xie Ansuan is characterized in that, this production technique may further comprise the steps:

(1) selects to be suitable for bacterial classification

Selection is applicable to a kind of in brevibacterium flavum (Brevobacterium flavum) that the L-Xie Ansuan produces, Beijing rod bacillus (Corynebacterium pekinense), Corynebacterium crenatum (Corynebacterium crenatum), intestinal bacteria (Escherichia coli), brevibacterium lactofermentum (Brevibacterium lactofermentum), the Corynebacterium glutamicum (Corynebacterium glutamicum);

(2) inclined-plane preparation

With above-mentioned fermentation strain inoculation inclined-plane, to cultivate 8～24 hours for 28～35 ℃, inclined-plane refrigeration is stand-by;

(3) fermentation culture prescription

With the full-synthetic culture medium is minimum medium, adds a small amount of organic nitrogen source, and it is as follows mainly to fill a prescription:

Glucose 60～140g/L or sucrose 120～250g/L; Ammonium chloride 15～50g/L or ammonium sulfate 15～30g/L; Corn steep liquor or thalline hydrolyzed solution 0～5g/L; K ₂HPO ₄3H ₂O 1～4g/L; MgSO ₄7H ₂O0.2～0.8g/L; MnSO ₄H ₂O 2～20mg/L; FeSO ₄7H ₂O 2～20mg/L; Zn SO ₄7H ₂O0.5～10mg/L; Vitamin H 0～100 μ g/L; Vitamin 20～100 μ g/L; CaCO ₃20～60g/L;

(4) zymotechnique

Be inoculated in after will cultured lawn washes with a small amount of sterilized water in activated inclined plane in sterilized fermentation shake flask that above-mentioned fermention medium is housed or the fermentor tank and begin fermentation, shaker fermentation control condition is: initial pH6.4～7.2, shaking speed 180～220r/min, fermentation time 60～96 hours.Fermentor tank control condition is: 30～33 ℃ of fermentation initial temperatures, and air flow 0.5～1.5VVM, tank pressure 0.02～0.05Mpa, dissolved oxygen 0.5%～90% saturation ratio is by polyethers or silicone defoamer froth breaking; Fermented 18～32 hours, and improved rotating speed and air flow and guarantee that dissolved oxygen is in 30～50% saturation ratios; PH is in subacidity for the fermentation Whole Process Control, by adding ¹⁵N labeled urea, ammoniacal liquor or liquefied ammonia are regulated pH6～7, and stream adds 50% glucose control fermented liquid glucose concn 2%～5%, fermentation time 48～72 hours;

(5) separation and Extraction

Stable isotope in the fermented liquid ¹⁵The separation of N label L-Xie Ansuan adopts the ion exchange method separation and Extraction to obtain stable isotope ¹⁵N label L-Xie Ansuan is caught up with ammonia and is adopted ethanol low temperature crystallization and vacuum-drying to obtain product by vacuum concentration.

2. a kind of stable isotope according to claim 1 ¹⁵The production technique of N label L-Xie Ansuan, it is characterized in that, brevibacterium flavum (Brevobacterium flavum) is selected from commercially available ATCC14067 in the described step (1), ATCC14020, ATCC13869, among the ATCC14066 one or more, Beijing rod bacillus (Corynebacterium pekinense) is selected from commercially available CGMCC1.586, among the CGMCC1.299CGMCC1.495 one or more, Corynebacterium crenatum (Corynebacterium crenatum) is selected from commercially available CGMCC1.452, CGMCC1.1001, among the CGMCC1.1007 one or more, intestinal bacteria (Escherichia coli) are selected from commercially available ATCC13005, brevibacterium lactofermentum (Brevibacteriumlactofermentum) is selected from commercially available ATCC13058, and Corynebacterium glutamicum (Corynebacteriumglutamicum) is selected from commercially available ATCC2256, ATCC13032, among the ATCC14751 one or more.