CN1487088A - Prepn of isotopically labeled 15 N-L-threonine - Google Patents

Prepn of isotopically labeled 15 N-L-threonine Download PDF

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Publication number
CN1487088A
CN1487088A CNA031419941A CN03141994A CN1487088A CN 1487088 A CN1487088 A CN 1487088A CN A031419941 A CNA031419941 A CN A031419941A CN 03141994 A CN03141994 A CN 03141994A CN 1487088 A CN1487088 A CN 1487088A
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threonine
substratum
material containing
inorganic material
ammonium
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CNA031419941A
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CN1272439C (en
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健 章
章健
项泽宇
孙建荣
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Shanghai Xinli Industrial Microbe Technology Co Ltd
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Shanghai Xinli Industrial Microbe Technology Co Ltd
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Abstract

The present invention is preparation process of isotopically labeled 15N-L-threonine. Inorganic material containing isotopic label 15N is synthesized into 15N-L-threonine through microbial fermentation and biosynthesis. In the same time, 15N-L-threonine is purified via biological separation. The said process is superior to available chemical synthesis process, which has low utilization of inorganic material containing isotopic label 15 N, and has low production cost of 15N-L-threonine. The presnet invention has 15N-L-threonine concentration in ultimate reaction liquid over2%, molar conversion rate of inorganic material containing isotopic lable 15N over 50 %, purity of product over 99 % and product abundance over 98.5 %.

Description

The specification sheets isotopic labeling 15The preparation method of N-L-Threonine
Technical field
The present invention relates to a kind of isotopic labeling 15The preparation method of N-L-Threonine is specifically related to a kind of microbial fermentation preparation that utilizes 15The method of N-L-Threonine, particularly a kind of fermentative preparation 15The microorganism of N-L-Threonine.
Background technology
Nitrogen-15 (is called for short 15N, down together) be the stable isotope of nitrogen element, as tracer agent, it is widely used in fields such as organic chemistry, biological chemistry, medical science, pharmacology, agricultural section, and especially the research to life science has very important meaning.Particularly along with the mensuration of human genomic sequence collection of illustrative plates with complete, life science is more and more important in the world to promoting for proteinic synthetic, and 15N-amino acid plays irreplaceable unique spike effect in this process, it is used more and more widely, good market prospects.
Because this series products output is little, purity and abundance require high, produce with chemical synthesis usually.Usually, the amino acid that chemical synthesis is produced is DL type amino acid, splits through enzyme process and obtains L type amino acid.What often biological field needed is L type amino acid, and D type amino acid is mostly discarded, causes 15The utilization ratio of N-inorganic raw material is extremely low, and production cost is high, and selling price is high.
Fermentative Production amino acid generally adopts at world wide, but does not find to use fermentative Production so far 15The patent report of N-L-Threonine.Have following reason: (1) fermentation method biosynthesizing amino acid is because its pathways metabolism is very complicated, and by-product amino acid is more, and separation and purification is brought certain difficulty; (2) also be because the amino acid whose complicacy of biosynthesizing, often need to add some natural organic raw material during the fermentation, as: corn steep liquor, hair hydrolysis liquid, yeast extract paste, soybean hydrolyzed solution or the like, the interpolation of these materials will directly cause tunning the abundance value ( 15N/N) descend, do not accepted by market.
Summary of the invention
One of technical issues that need to address of the present invention are to disclose a kind of energy fermentative production 15The bacteria culture of N-L-Threonine, the i.e. intestinal bacteria of genetically engineered structure.
Two of the technical issues that need to address of the present invention are to disclose a kind of fermentative Production of using 15The method of N-L-Threonine is to obtain high purity, abundant 15N-L-Threonine product overcomes the defective of prior art.
The microorganism of using:
The present invention is used for producing 15The microorganism of N-L-Threonine is a kind of intestinal bacteria (E.coli) that make up with gene engineering method [(pTH08+prh-T04)/VT418], this bacterial classification on April 21st, 2003 in China Committee for Culture Collection of Microorganisms's preservation, preserving number is: CGMCC No.0923.
Above-mentioned bacterial classification has following character:
1. form physiological and biochemical property:
Morphological specificity: direct rod shape, 1.1-1.5 μ m * 2.0-6.0 μ, Gram-negative, single existence in the fermented liquid, bacterium colony is smooth, moistening on nutrient agar, the surface is glossy.
Physiological and biochemical property: AHV r, Met -, Ile -' Dap -
2. the feature on various substratum:
Solid inclined-plane isolation medium: sucrose 2.0g, NH 4Cl1.0g, KH 2PO 41.5g, Na 2HPO 43.5g MgSO 47H 2O20.0g, agar 20g, pH7.0～7.2 are transferred in the dissolving back, adding distil water dissolving and constant volume 1000ml, 0.8Kg/cm 2Sterilized 30 minutes, and added penbritin solution when being cooled to 50 ℃, making ultimate density is 50 μ g/ml.
Liquid proliferated culture medium: sucrose 40.0g, (NH 4) 2SO 410.0g, KH 2PO 41.0g, MgSO 47H 2O 0.5g, yeast extract 2.0g, CaCO 315.0g, adding distil water dissolving and constant volume 1000ml, 0.8Kg/cm 2Sterilized 30 minutes, and added penbritin solution when being cooled to 50 ℃, making ultimate density is 50 μ g/ml.
Fermention medium:
Carbon source: glucose, sucrose, fructose etc. one or more.
Nitrogenous source: 15N-ammonium sulfate, 15N-ammonium chloride, 15N-urea, 15N-ammonium nitrate etc. one or more.
Nutritive ingredient: dipotassium hydrogen phosphate, potassium primary phosphate, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, sal epsom, ferric sulfate, manganous sulfate, zinc sulfate, copper sulfate etc. one or more.
3. culture condition:
Culture temperature: 30-40 ℃, temperature preferably: 36-38 ℃.
Training method: ventilate and stir the oxygen consumption fermentation.
Control dissolved oxygen concentration: 10-60%, preferably in the fermenting process at 20-30%.
Control pH value: 5.5-7.0 is preferably at 5.8-6.2 in the fermenting process.
Adopt neutralizing agent in the fermenting process: sodium hydroxide, potassium hydroxide, calcium hydroxide etc.
The source of the used bacterial classification of the present invention:
Genetically engineered that the present invention adopts makes up bacterial classification [E.coli (pTH08+prh-T04)/VT418], and concrete building mode without limitation can following mode carry out:
Selecting intestinal bacteria wild type strain E.coli MC4100 is starting strain, by uv, nitrosoguanidine (NTG) and ethyl sulfate (EDS) is mutagenic compound, has removed Threonine biosynthesizing feedback regulation mutant with screening Threonine superior strain VNBKB-3507 (AHV by mutagenic and breeding r, Met 1, DAP -, Ile +) and F-strain VT418 (Thr -, AHV r, Met 1, DAP -, Ile +).Three key enzyme AKI-HDI, the HKI of the coding Threonine pathways metabolism in this starting strain and genetically manipulated (thrA, thrB, thrC) of threonine synthetase are cloned under the T7 phage promoter of plasmid pET11a, make up plasmid pET11a-08.Be connected to structure secretory gene plasmid prh-T04 on the escherichia coli plasmid of being with the T7 phage promoter with being responsible for homoserine and homoserine lactone excretory rhtB gene and responsible Threonine excretory rhtC gene, and in F-strain intestinal bacteria VT418, express.
Adopt " common bacteria system identification handbook " (eastern elegant pearl, Cai Miaoying write) described discrimination method and above-mentioned testing data to show, the bacterial classification of addressing of the present invention [(pTH08+prh-T04)/VT418] belongs to intestinal bacteria, but with intestinal bacteria tangible difference is arranged again, its main difference point is that this bacterial classification carries the threonine operon gene with strong promoter and helps Threonine excretory gene plasmid, has high yield Threonine feature.
Analytical procedure of the present invention:
Adopt the Fei Linshi method to measure reducing sugar;
Adopt the HPLC method to measure threonine content;
Adopt micro-example conversion processes sample, detect by the gas isotope mass spectrograph 15N-Threonine abundance.
Adopt above-mentioned strain fermentation production 15The method of N-L-Threonine comprises the steps:
With the 35-38 ℃ of amplification after 18-24 hour in substratum of said bacterial classification, under aseptic condition washing and collect thalline insert contain carbon source, 15The stirring fermentation culture of ventilating in the substratum of N-nitrogenous source and other nutritive ingredient, culture temperature is 30-40 ℃, temperature preferably is 36-38 ℃, the control dissolved oxygen concentration is 10-60% in the fermenting process, preferably at 20-30%, one or more adjusting fermented liquids pH value with alkaline matters such as sodium hydroxide, potassium hydroxide, calcium hydroxides is 5.5-7.0, and preferably at 5.8-6.2, the cultivation time is 36-48 hour.
The preferred above-mentioned liquid proliferated culture medium of said substratum.
Said carbon source comprises in the saccharine materials such as glucose, sucrose, fructose or lactose one or more.
Said 15The N-nitrogenous source comprises 15N-ammonium sulfate, 15N-ammonium chloride, 15N-urea, 15In the N-ammonium nitrate etc. one or more.
Addressed 15The N-nitrogenous source can adopt Shanghai Chemical Research Inst to produce and products such as the ammonium sulfate of public offering, ammonium chloride.Said nutritive ingredient comprises one or more in the inorganic salt such as dipotassium hydrogen phosphate, potassium primary phosphate, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, sal epsom, ferric sulfate, manganous sulfate, zinc sulfate, copper sulfate.
The invention still further relates to and from fermented liquid, extract purifying 15The method of N-L-Threonine, this method comprises the steps:
The fermented liquid of fermenting-ripening is regulated pH value 1-3 with acidic substance, being heated to 80-100 ℃ kept 10-30 minute, remove thalline with centrifugal or modes such as filtration or micro-filtration, supernatant liquor is entered strong acidic ion resin (as #732) absorption with 100-1000ml/h speed, after finishing, absorption washs from handing over post to effluent liquid pH5-7 with the 500-5000ml pure water, basic solution wash-out with 2-10wt%, collect elutriant, use the 1-5% activated carbon decolorizing, reduced vacuum is concentrated into 15-30% concentration, adds 3-5 times of raw spirit precipitation, vacuum filtration, 60-80 ℃ of drying, can obtain purity more than 99%, abundance is more than 98.5% 15N-L-Threonine finished product.
Said acidic substance comprise sulfuric acid, hydrochloric acid or nitric acid;
Said basic solution comprises a kind of and composition thereof in ammoniacal liquor or the sodium hydroxide.
By above-mentioned disclosed technical scheme as seen, method of the present invention can prepare isotopic labeling easily 15The N-L-Threonine, product purity more than 99%, abundance is more than 98.5%, this shows that the present invention has bigger industrializing implementation prospect.
Embodiment
The present invention will specify the embodiment of technology by following non-limiting instance.
Embodiment 1
Fermention medium, glucose 8%, 15N-ammonium sulfate 2.5%, potassium primary phosphate 0.2%, sal epsom 0.1%, lime carbonate 2%, the 500ml triangular flask 50ml that packs into sterilized 20 minutes for 121 ℃, inserted 1ml bacterial classifications of 24 hours of 37 ℃ of amplifications in the liquid proliferated culture medium, 37 ℃ of cultivations, detected in the time of 48 hours 15The N-L-threonine content is 2.2%.
Embodiment 2
Fermention medium, sucrose 8%, 15N-ammonium sulfate 2.5%, potassium primary phosphate 0.2%, sal epsom 0.1%, the 3L substratum of in the 5L fermentor tank, packing into, 121 ℃ of sterilizations 20 minutes, insert 5% in the liquid proliferated culture medium 24 hours bacterial classification of amplification, ventilate at 37 ℃, 210L/h, 600r/m cultivated 48 hours under stirring, with 2NaOH control pH value 6.4, the results are shown in following table in the process.
The jar batch product acid residual ammonia of (%) C-transformation efficiency (%) N-transformation efficiency (%) (%)
01 2.13 30.4 50.48 0.082
02 2.26 31.2 53.56 0.068
03 2.20 32.1 52.14 0.072
Average 2.20 31.2 52.06 0.074
Embodiment 3～5
Fermented liquid sulphur acid for adjusting pH value 2 with embodiment 1 fermenting-ripening, being heated to 90 ℃ kept 20 minutes, the centrifugal thalline of removing, supernatant liquor is entered strong acidic ion resin #732 absorption with 500ml/h speed, after finishing, absorption washs from handing over post to effluent liquid pH5-7 with 2500 pure water, ammoniacal liquor wash-out with 6%, collect elutriant, use 2.5% activated carbon decolorizing, reduced vacuum is concentrated into 20wt%, adds 4 times of raw spirit precipitations, vacuum filtration, 70 ℃ of dryings, can obtain purity more than 99%, abundance is more than 98.5% 15N-L-Threonine finished product.Result such as following table.
Table 2
Project implementation example 3 embodiment 4 embodiment 5 are average
Fermentating liquid volume (ml) 3,240 3,300 3280
Threonine content (%) 2.13 2.26 2.20 2.20
Obtain product (g) 55.7 60.6 59.0
Total recovery (%) 80.7 81.2 81.7 82.2
Raw material abundance (atom%) 98.90 98.90 98.90
Product abundance (atom%) 98.66 98.56 98.70 98.64
Product purity (%) 99.70 99.58 99.52 99.60
Embodiment 6
Adopt the identical method of embodiment 1, with 15N-ammonium chloride 2% replaces 15N-ammonium sulfate, its result is as follows:
15The N-L-threonine content is 2.0%.
Embodiment 7
Adopt the identical method of embodiment 1, with 15N-urea 1.5%, 15N-ammonium nitrate 1% replaces 15N-ammonium sulfate, its result is as follows:
15The N-L-threonine content is 1.9%.

Claims (7)

  1. Intestinal bacteria (E.coli) (pTH08+prh-T04)/VT418 CGMCC No.0923.
  2. 2. isotopic labeling 15The preparation method of N-L-Threonine is characterized in that, comprises that earlier the bacterial classification to claim 1 increases in substratum, insert then contain carbon source, 15Fermentation culture in the substratum of N-nitrogenous source, fermented liquid pH value is 5.5-7.0.
  3. 3. method according to claim 2 is characterized in that culture temperature is 30-40 ℃, and the control dissolved oxygen concentration is 10-60% in the fermenting process.
  4. 4. method according to claim 2 is characterized in that, and is said 15The N-nitrogenous source comprises 15N-ammonium sulfate, 15N-ammonium chloride, 15N-urea, 15In the N-ammonium nitrate one or more.
  5. 5. according to claim 2,3 or 4 described methods, it is characterized in that, also comprise the steps: the fermented liquid of fermenting-ripening is regulated the pH value to 1-3 with acidic substance, be heated to 80-100 ℃, remove thalline, supernatant liquor is entered strong acidic ion resin absorption, wash from handing over post with pure water after absorption is finished to effluent liquid pH5-7, with the basic solution wash-out of 2-10wt%, collect finished product from elutriant then 15The N-L-Threonine.
  6. 6. method according to claim 5 is characterized in that said acidic substance comprise sulfuric acid, hydrochloric acid or nitric acid.
  7. 7. method according to claim 5 is characterized in that, said basic solution comprises a kind of and composition thereof in ammoniacal liquor, the sodium hydroxide.
CN 03141994 2003-07-31 2003-07-31 Prepn of isotopically labeled 15 N-L-threonine Expired - Fee Related CN1272439C (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102181502A (en) * 2011-05-17 2011-09-14 内蒙古阜丰生物科技有限公司 Method for improving yield of L-threonine produced by fermentation

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102181502A (en) * 2011-05-17 2011-09-14 内蒙古阜丰生物科技有限公司 Method for improving yield of L-threonine produced by fermentation
CN102181502B (en) * 2011-05-17 2014-03-19 内蒙古阜丰生物科技有限公司 Method for improving yield of L-threonine produced by fermentation

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