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

Production of stabilized isotope 15N labelled L-valine Download PDF

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Publication number
CN1982466A
CN1982466A CN 200510111371 CN200510111371A CN1982466A CN 1982466 A CN1982466 A CN 1982466A CN 200510111371 CN200510111371 CN 200510111371 CN 200510111371 A CN200510111371 A CN 200510111371A CN 1982466 A CN1982466 A CN 1982466A
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fermentation
xie ansuan
corynebacterium
commercially available
stable isotope
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CN 200510111371
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梅丛笑
谭青乔
李良君
杜晓宁
宋明鸣
侯静华
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Shanghai Research Institute of Chemical Industry SRICI
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Shanghai Research Institute of Chemical Industry SRICI
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Abstract

Production of stable isotope 15N marked L-valine is carried out by selecting proper bacterium, slope preparing, culturing formula by fermentation, fermenting, separating and extracting. It can add micro-vitamin composite, improve growth and metabolism and decrease 15N utilization rate.

Description

A kind of stable isotope 15The production technique of NN label L-Xie Ansuan
Technical field
The invention belongs to stable isotope tagged compound production technical field, relate in particular to and adopt microbial fermentation and biological extraction process production stability isotropic substance 15The technology of N label L-Xie Ansuan.
Background technology
The L-Xie Ansuan- 15N is the same with the amino acid of other mark, can be used as tracer agent and is widely used in Related Research Domain such as medicine industry, foodstuffs industry and life science.The L-Xie Ansuan- 15The spike effect of N uniqueness can be used for testing and study the L-Xie Ansuan in the intravital unique physiological action of machine.To the production of L-Xie Ansuan, there are some research groups that it has been done many research work both at home and abroad, US 6737255 B2 are arranged; US4542098; US6214591B; The microorganism journal, 15 (4): P325～329,1975; The medicine biotechnology, 6 (1): P24～27,1999.But, study about biological synthesis process 15The document and the patent of the production of N stable isotope label L-Xie Ansuan are also few, the microorganism journal are arranged, 2 (2): P75～78,1994.The employing direct fermentation is produced, a large amount of enrichments of target amino acid, stable isotope 15The easy mark of N, but since in the fermentating formula organic nitrogen source be difficult to the influence of mark and natural abundance nitrogenous source, usually make the L-Xie Ansuan- 15The abundance of N descends a lot, does not reach product requirement, simultaneously 15The N utilization ratio of raw materials is also lower, thereby has increased cost, so need improve conventional fermentating formula and technology.
Summary of the invention
Purpose of the present invention is exactly to provide a kind of stable isotope for the deficiency that overcomes above-mentioned prior art existence 15The production technique of N label L-Xie Ansuan, this process using microorganism direct fermentation production L-Xie Ansuan- 15N improves growth and metabolism by adding the mcg vitamin mixture, is intended to solve 15The problem that the N abundance descends, and improve as far as possible 15The N utilization ratio is to satisfy the products production requirement.
Purpose of the present invention can be achieved through the following technical solutions: a kind of 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 2O2～20mg/L; FeSO 47H 2O2～20mg/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.
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 CGMCC1.299CGMCC 1.495 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.
The present invention utilize microorganism inclined-plane lawn directly transmit ferment produce the L-Xie Ansuan- 15, control organic nitrogen source addition directly adds vitamin H and vitamin, by improving fermentating formula and technology improves acid production rate.Can make like this 15The N raw material is utilized effectively, and abundance descends and is unlikely to influence the products production requirement.
If adopt conventional fermentation process, stable isotope 15Though N label L-Xie Ansuan output is higher relatively, 15The N abundance descends very big, often descends more than 3%, is difficult to reach the high abundance product requirement.Though adopt the full-synthetic culture medium fermentation right 15The influence of N abundance is little, and the acid amount is too low to make but produce 15The N raw material availability is not high.By changing fermentating formula and technology, directly add vitamin and vitamin H, the stable isotope that the present invention is obtained 15The L-Xie Ansuan rate ratio of N mark adopts the corresponding raising of full-synthetic culture medium fermentation and acid amount more than 10～50%, and stable isotope 15The L-Xie Ansuan of N mark 15The N abundance descends and can reduce to below 0.5%, have in addition descend hardly, improved greatly 15The N raw material availability has reduced production cost.Simultaneously, guaranteeing product 15Under the N abundance condition, simplify zymotechnique, directly from activated inclined plane switching bacterial classification, not only the production cycle shortens, and is suitable for control 15The abundance of N product, raw material is fully used simultaneously, has saved the raw material cost recovery greatly.The present invention can utilize low abundance and high abundance 15The N inorganic raw material satisfies different abundance product requirements.
Embodiment
The invention will be further described below in conjunction with specific embodiment.
Embodiment 1
The slant medium that uses: glucose 1g/L, extractum carnis 10g/L, peptone 10g/L, NaCl5g/L, agar 20g/L, fermention medium: glucose 100g/L, ( 15NH 4) 2SO 415g/L, K 2HPO 41.5g/L, MgSO 47H 2O 0.5g/L, FeSO 47H 2O 0.02g/L, MnSO 4H 2O 0.02g/L, vitamin H 50 μ g/L, VitB1 100 μ g/L, corn steep liquor 1g/L, CaCO 330g/L.
Rod bacillus CGMCC1.586 is inoculated in activated inclined plane from the preservation inclined-plane with Beijing, cultivated 24 hours in 30 ℃ of constant incubators, washing 2 inclined-plane lawns with a little sterilized water changes in the 500mL triangular flask that the 20ml fermention medium is housed, totally 10 bottles, 30 ℃ of following 200r/min cultivate in the top fermentation of patrolling shaking table and finished in 60 hours, the average product of fermentation L-Xie Ansuan- 15N can reach 18g/L.
Fermented liquid adopts 732 Zeo-karb single-columns to separate, adopt ordinary method obtain drying solid L-Xie Ansuan- 15The N product obtains product abundance 98.28% through mass spectroscopy, and abundance descends 0.427%.
If change corn steep liquor in the fermention medium into 10g/L, zymotechnique is constant, the average product of fermentation L-Xie Ansuan- 15N can reach 33g/L, but abundance has only 95.34%, and abundance descends 3.6%.If do not add corn steep liquor in the fermention medium, the average product of fermentation L-Xie Ansuan- 15N has only 5g/L, abundance 98.71%, and abundance descends 0.232%.If do not add VITAMIN mixtures such as vitamin H, VitB1 in the above-mentioned fermention medium, the average product of fermentation L-Xie Ansuan- 15N has only 2g/L.Take all factors into consideration, this invention effect is remarkable.
Embodiment 2
The slant medium that uses: glucose 1g/L, extractum carnis 10g/L, peptone 10g/L, NaCl5g/L, agar 20g/L, fermention medium: glucose 120g/L, ( 15NH 4) 2SO 430g/L, K 2HPO 41g/L, MgSO 47H 2O 0.8g/L, FeSO 47H 2O 0.05g/L, MnSO 4H 2O 0.02g/L, vitamin H 50 μ g/L, VitB1 100 μ g/L, thalline hydrolyzed solution 5g/L, CaCO 340g/L.
Rod bacillus CGMCC1.1001 is inoculated in activated inclined plane from the preservation inclined-plane with Beijing, cultivated 18 hours in 30 ℃ of constant incubators, washing 2 inclined-plane lawns with a little sterilized water changes in the triangular flask of the above-mentioned 500mL that fermention medium 20mL is housed, 30 ℃ of following 200rpm cultivate in the top fermentation of patrolling shaking table and finished in 72 hours, the average product of fermentation L-Xie Ansuan- 15N can reach 25g/L.
Fermented liquid adopts 1500H Zeo-karb single-column to separate, adopt ordinary method obtain drying solid L-Xie Ansuan- 15The N product obtains product abundance 98.83% through mass spectroscopy, and abundance descends 0.131%.
Embodiment 3
The slant medium that uses: glucose 1g/L, extractum carnis 10g/L, peptone 10g/L, NaCl5g/L, agar 20g/L, fermention medium: sucrose 140g/L, ( 15NH 4) 2SO 440g/L, K 2HPO 43g/L, MgSO 47H 2O 0.2g/L, FeSO 47H 2O 0.02g/L, MnSO 4H 2O 0.08g/L, vitamin H 100 μ g/L, VitB1 50 μ g/L, corn steep liquor 2g/L, CaCO 330g/L.
Brevibacterium flavum ATCC14067 is inoculated in activated inclined plane from the preservation inclined-plane, cultivated 15 hours in 30 ℃ of constant incubators, washing 2 inclined-plane lawns with a little sterilized water changes in the 500mL triangular flask that the 20mL fermented liquid is housed, 30 ℃ of following 200r/min cultivate in the top fermentation of patrolling shaking table and finished in 96 hours, the average product of fermentation L-Xie Ansuan- 15N can reach 21g/L.
Fermented liquid adopts FMC11Na Zeo-karb single-column to separate, adopt ordinary method obtain drying solid L-Xie Ansuan- 15The N product obtains product abundance 98.66% through mass spectroscopy, and abundance descends 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.
CN 200510111371 2005-12-12 2005-12-12 Production of stabilized isotope 15N labelled L-valine Pending CN1982466A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101962664A (en) * 2010-11-02 2011-02-02 天津科技大学 Fermentation process for producing L-valine efficiently
CN103642766A (en) * 2013-07-02 2014-03-19 廊坊梅花生物技术开发有限公司 Protein, DNA molecule, conversion host containing DNA and method for production of L-valine by utilization of conversion host
CN105274182A (en) * 2015-10-28 2016-01-27 新疆阜丰生物科技有限公司 Process for efficiently extracting L-valine from fermentation liquor
CN117965654A (en) * 2024-04-02 2024-05-03 东晓生物科技股份有限公司 Method for improving fermentation conversion rate of L-valine

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101962664A (en) * 2010-11-02 2011-02-02 天津科技大学 Fermentation process for producing L-valine efficiently
CN103642766A (en) * 2013-07-02 2014-03-19 廊坊梅花生物技术开发有限公司 Protein, DNA molecule, conversion host containing DNA and method for production of L-valine by utilization of conversion host
CN103642766B (en) * 2013-07-02 2016-12-28 廊坊梅花生物技术开发有限公司 Albumen, DNA molecular, convert host and this conversion host for the method producing L valine containing this DNA
CN105274182A (en) * 2015-10-28 2016-01-27 新疆阜丰生物科技有限公司 Process for efficiently extracting L-valine from fermentation liquor
CN105274182B (en) * 2015-10-28 2018-09-14 新疆阜丰生物科技有限公司 The technique of high efficiency extraction Valine from zymotic fluid
CN117965654A (en) * 2024-04-02 2024-05-03 东晓生物科技股份有限公司 Method for improving fermentation conversion rate of L-valine
CN117965654B (en) * 2024-04-02 2024-07-02 东晓生物科技股份有限公司 Method for shortening fermentation period of L-valine

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