CN1487087A - Prepn of isotopically labeled 15 N-L-aspartic acid and 15 N-L-alanine - Google Patents

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

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CN1487087A
CN1487087A CNA031419917A CN03141991A CN1487087A CN 1487087 A CN1487087 A CN 1487087A CN A031419917 A CNA031419917 A CN A031419917A CN 03141991 A CN03141991 A CN 03141991A CN 1487087 A CN1487087 A CN 1487087A
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aspartic acid
labeled
isotope
ala
nitrogenous source
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毛展伟
劳含章
钱志良
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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-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

Isotopic labeling 15The N-L-aspartic acid and 15The preparation method of N-L-L-Ala
Technical field
The present invention relates to a kind of 15The N-L-aspartic acid and 15The preparation method of N-L-L-Ala is specifically related to utilize the microbial enzyme reaction method to prepare isotopic labeling 15The N-L-aspartic acid and 15The method of N-L-L-Ala.
Technical background
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.
Conventional L-aspartic acid and L-L-Ala, because the enzyme process reactions steps is simple, by-product amino acid is few, is production method commonly used at present.But do not see as yet by the Production by Enzymes isotopic labeling at present 15The N-L-aspartic acid and 15The method of N-L-L-Ala.
Summary of the invention
One of technical issues that need to address of the present invention be disclose a kind of utilization contain L-Aspartase travel cell will contain isotopic labeling nitrogen 15 ( 15N) inorganic raw material and carbon source are synthetic through enzyme process 15The method of N-L-aspartic acid;
Two of the technical issues that need to address of the present invention are to disclose a kind of utilization to contain travelling cell and will containing of aspartic acid β-decarboxylase 15The N-L-aspartic acid through enzymatic conversion method is 15The method of N-L-L-Ala.
The present invention prepares isotopic labeling 15The method of N-L-aspartic acid comprises the steps:
Carbon source, nitrogenous source (ammoniacal liquor, ammonium sulfate, ammonium chloride etc.), trace element are configured to fermention medium, through high-temperature sterilization, insert intestinal bacteria bacterial classifications (E.coli) A.S.1881, shake the cell of travelling that bottle (or fermentor tank) fermentative production contains L-Aspartase, the cell of travelling that obtains is joined in the reaction solution that contains substrate, isotope-labeled nitrogenous source and trace element, transfer pH to 7-10, preferably at 8.2-8.8, in 30-50 ℃, preferably in 40-43 ℃ constant incubator the reaction 24-48 hour;
Said carbon source is one or more in FUMARIC ACID TECH GRADE, glucose, fructose or the sucrose;
Said nitrogenous source is one or more in ammoniacal liquor, ammonium sulfate or the ammonium chloride etc.;
Said substrate is a FUMARIC ACID TECH GRADE;
Said isotope-labeled nitrogenous source comprises 15N-ammonium sulfate, 15N-ammonium chloride, 15N-urea, 15In the N-ammoniacal liquor etc. one or more;
Said trace element comprises NaCl, MgSO 4, KH 2In PO4, corn steep liquor or the peptone one or more;
Enzyme reaction is heated to 85-95 ℃ with reaction solution after finishing, and adds the 0.5-2.0% gac, through stirring, being incubated vacuum filtration, washing leaching cake, merging filtrate and with the filtrate vacuum concentration to 20-30%, be heated to 70-90 ℃, slowly add sulfuric acid while stirring and be adjusted to pH=2.5～3.0, in the 20-200r/m constant speed stirrer, stir crystallisation by cooling, filter, washing, 60-80 ℃ of drying promptly obtains isotope-labeled 15The N-L-aspartic acid.
The present invention prepares isotopic labeling 15The method of N-L-L-Ala comprises the steps:
A certain proportion of carbon source, nitrogenous source, nutritive substance are configured to fermention medium, through high-temperature sterilization, insert false monospore bacillus (Pseudomonas) B186, shake the cell of travelling that bottle or ferment tank production contain aspartic acid β-decarboxylase, in enzyme reaction solution, add gradually 15N-L-aspartic acid, pH are 2-3, react in 30～40 ℃ of constant-temperature shaking culture casees, and pH is reached more than 6.0～7.0, filter, and get isotope-labeled 15N crude product L-Ala.
Said carbon source is one or more in FUMARIC ACID TECH GRADE, glucose, fructose or the sucrose;
Said nitrogenous source is one or more in ammoniacal liquor, ammonium sulfate or the ammonium chloride etc.;
Said nutritive substance comprises one or more in sal epsom, manganous sulfate, zinc sulfate, phosphoric acid salt, peptone or the corn steep liquor;
With isotope-labeled 15N crude product L-Ala is dissolved in the distilled water by the concentration of 10-15%, is heated to 85-90 ℃, adds 0.5～2% gac, and insulation, suction filtration stir evaporative crystallization, naturally cool to room temperature, and ice bath is cooled to 0～5 ℃, and vacuum filtration gets isotope-labeled 15N makes with extra care L-Ala;
Merge all mother liquors, transfer pH to 3.0-3.5, separate from handing over H type #732 highly acidic cation ion-exchange resins with HCl, upper prop speed is 50-1000ml/l, washes post through deionized water, with 2-10% ammoniacal liquor wash-out, elution speed is 50-1000ml/l, collects the pH6-8 elutriant.More than elutriant vacuum concentration to 20%, add dehydrated alcohol alcohol and analyse, crystallization, suction filtration, drying promptly obtains isotope-labeled 15N makes with extra care L-Ala.
The microorganism of using:
The microorganism that the present invention is used to produce L-Aspartase is intestinal bacteria bacterial classification (E.coli) A.S.1881, by the preservation of DSMZ of institute of microbiology of the Chinese Academy of Sciences and provide and public offering (microbiology circular: the 10th volume the 1st phase p8-10,1983).The microorganism that the present invention is used to produce aspartic acid β-decarboxylase is that moral A Kun breathes out false monospore bacillus (Pseudomonas) B186, by preservation center preservation in the bacterium of Shanghai City Industry Wei Biological Research Institute and provide and public offering (industrial microorganism: the 6th phase of nineteen eighty-three p1-4).
Above-mentioned bacterial classification has following character:
1, attitude physiological and biochemical property:
Morphological specificity: (1) intestinal bacteria (E.coli) A.S.1881: direct rod shape, 1.1-1.5 μ m * 2.0-6.0
μ, Gram-negative, single existence in the fermented liquid.
(2) moral A Kun breathes out false monospore bacillus (Pseudomonas) B186: direct rod shape, 0.5-1.0
μ m * 1.5-5.0 μ, Gram-negative, single existence in the fermented liquid.
2, the substratum of Cai Yonging:
Slant medium (g/l): peptone 10 extractum carniss 5.0 NaCl 5.0 agar 20 distilled water
Dissolving transfers pH to 7.2-7.5, adds agar and constant volume 1000ml, packing test tube slant, 121
℃ the sterilization 30 minutes.
L-Aspartase enzyme liquid culture medium (g/l)
Fumaric acid 10 NaCl 1.45 MgSO 4.7H2O 0.2 KH 2PO4 1.0 dry powder corn steep liquors
PH7.0-7.5 is transferred with ammoniacal liquor in 11 dissolving backs, constant volume 1000ml, and packing 50ml/250ml triangular flask,
Sterilized 30 minutes for 121 ℃.
Aspartic acid β-cocarboxylase liquid culture medium (g/l)
Fumaric acid 15 peptones 5.0 MgSO 4.7H 2O 0.2 KH 2PO4 0.1 dry powder corn steep liquor
4.0 pH7.0-7.5 is transferred with 4NNaOH, constant volume in NaOH 7.0 ammoniacal liquor 6.0 dissolving backs
1000ml, packing 50ml/250ml triangular flask was sterilized 30 minutes for 121 ℃.
Aspartic acid substrate reactions liquid
Carbon source: FUMARIC ACID TECH GRADE;
Nitrogenous source: 15N-ammonium sulfate, 15N-ammonium chloride, 15N-urea, 15N-ammoniacal liquor etc. one or more;
Trace element: 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.
3, culture condition:
L-Aspartase enzyme liquid cell culture condition:
Culture temperature: 30-40 ℃, temperature preferably: 36-38 ℃.
Shake bottled liquid measure: 250ml and shake bottled substratum 30-100ml, liquid amount 45-55ml preferably.
Shaking table frequency: rotary shaker (or reciprocal shaking table) 100-250r/m, frequency 200-220r/m preferably.
Aspartic acid β-cocarboxylase liquid cell culture condition:
Culture temperature: 25-40 ℃, temperature preferably: 29-33 ℃.
Shake bottled liquid measure: 250ml and shake bottled substratum 30-100ml, liquid amount 45-55ml preferably.
Shaking table frequency: rotary shaker (or reciprocal shaking table) 100-250r/m, frequency 200-220r/m preferably.
15N-L-L-Aspartase reaction conditions:
Add enzyme liquid thalline 0.1-1% in enzyme reaction solution, reaction is 24-48 hour in 35-50 ℃ of (temperature 40-45% preferably) constant incubator.
15N-L-L-Ala condition:
Every 50ml enzyme reaction solution can add 15N-L-aspartic acid 30-50g, reaction is 48-72 hour in 30-40 ℃ of (temperature 36-38 preferably ℃) constant incubator.
Analytical procedure of the present invention:
Adopt the potassium permanganate method to detect fumaric amount;
Adopt the HPLC method to measure aspartic acid and L-Ala content;
Adopt micro-example conversion processes sample, detect by the gas isotope mass spectrograph 15N-Threonine abundance.
By above-mentioned disclosed technical scheme as seen, method of the present invention can prepare isotopic labeling easily 15N-L-aspartic acid and isotope-labeled 15N makes with extra care L-Ala, product purity more than 99%, abundance is more than 98.5%, this shows that the present invention has bigger industrializing implementation prospect.
The present invention will specify the embodiment of technology by following non-limiting instance.
Embodiment
Embodiment 1
Bacterial classification intestinal bacteria Asp-4 is 37 ℃ of cultivation 24 maturations in slant medium, insert L-Aspartase enzyme liquid culture medium, shake bottled substratum 50ml in 250ml, and 220rpm cultivated 24 hours on 37 ℃ of rotary shakers.After cultivation is finished nutrient solution was placed aseptic centrifuge tube 4000rpm centrifugal 15 minutes, the wet thallus cell 0.5g after centrifugal is added contain 5% FUMARIC ACID TECH GRADE, 8% 15Among the enzyme reaction solution 200ml of N-ammonium sulfate, 0.0125% sal epsom, transfer pH8.2-8.8 with NaOH, reaction is 24 hours in 42 ℃ of constant incubators.It is as shown in table 1 that reaction finishes the back result:
Table 1
Project No.1 No.2 No.3 is average
15N-(NH 4) 2SO 4Addition (g) 40 40 40
Fumaric acid substrate addition (g) 10 10 10
Reaction solution volume (decolouring back) (ml) 625 630 618
Aspartic acid concentration (%) 7.93 8.14 8.25 8.11
Aspartic acid growing amount (g) 49.56 51.28 50.99
Fumaric acid residual concentration (%) 0.28 0.36 0.35
Fumaric acid transformation efficiency (%) 96.55 95.45 95.68
Nitrogenous source molar yield (%) 61.45 63.59 63.23 62.76
Embodiment 2
Contain 5% FUMARIC ACID TECH GRADE, 0.0125% sal epsom and 3% with cultivating sophisticated wet thallus cell 0.5g adding in example 1 method 15N-ammonium sulfate, 3% 15Among the mixed nitrogen reaction solution 200ml of N-ammonium chloride, use 15N-ammoniacal liquor is transferred pH8.2-8.8, and reaction is 24 hours in 42 ℃ of constant incubators.The aspartic acid growing amount is 52.0g as a result, and the fumaric acid transformation efficiency is 96.8%, and the nitrogenous source molar yield is 62.4%.
Embodiment 3
The reaction solution that example 1 reaction is finished is heated to 85-90 ℃, adds 0.5% gac, stirs, is incubated 45 minutes, vacuum filtration, distilled water wash filter cake.Merging filtrate and with filtrate vacuum concentration to 20% under 65 ℃, 680mmHg is heated to 80 ℃, slowly adds the vitriol oil while stirring, has thin partial crystallization to go out during pH3.0, stirs crystallisation by cooling in the 100rpm constant speed stirrer.When being cooled to room temperature, 5 ℃ of refrigerator overnight.Crystal solution through vacuum filtration, to wash brilliant finished product wet brilliant, 60 ℃ of dried overnight.The result is as shown in table 2:
Table 2
Project No.1 No.2 No.3 is average
Reaction solution volume (ml) 625 630 618
Aspartate content (%) 7.93 8.14 8.25 8.11
Obtain product (g) 42.5 43.1 43.9
Total recovery (%) 85.8 84.0 86.1 85.3
Raw material abundance (atom%) 98.90 98.90 98.90
Product abundance (atom%) 98.23 98.44 98.32 98.33
Product purity (%) 98.89 98.99 99.11 99.00
Embodiment 4
The false monospore bacillus of bacterial classification is 37 ℃ of cultivation 24 maturations in slant medium, insert aspartic acid β-cocarboxylase liquid culture medium, shake bottled substratum 50ml in 250ml, and 220rpm cultivated 24 hours on 37 ℃ of rotary shakers.Cultivation adds after finishing 15N-L-aspartic acid 40g, reaction is 72 hours in 37 ℃ of constant incubators.The result is as shown in table 3:
Table 3
Project No.1 No.2 No.3 is average
Enzyme liquid amasss (ml) 50 50 50
Aspartic acid addition (g) 40 40 40
Reaction finishes back substrate content (%) 0.2 0.3 0.11
Transformation efficiency (%) 99.75 99.63 99.86 99.75
Embodiment 5
With example 3 conversion fluid vacuum filtrations, get crude product 15The N-L-L-Ala.After suction filtration finishes, with crude product 15The N-L-L-Ala is dissolved in the distilled water by the concentration of 15wt%, is heated to 85-90 ℃, adds 2% gac, be incubated 60 minutes, remove gac behind the suction filtration, stir evaporative crystallization, naturally cool to room temperature, ice bath is cooled to 5 ℃, and vacuum filtration must be made with extra care L-Ala.Merge all mother liquors, transfer pH to 3.5 with 1NHCl, stand-by.
Get φ 4 * 50cm from handing over post, interior dress 500ml#732 highly acidic cation ion-exchange resins, change the H type, will merge the mother liquor upper prop after being washed to neutrality, upper prop speed is 500ml/h, upper prop finishes the back and washes post with 2 times of column volume deionized waters, with 2.2% ammoniacal liquor wash-out, elution speed is 500ml/h, collects pH7 wash-out section.More than elutriant vacuum concentration to 20%, add 5 times of volume dehydrated alcohol alcohol and analyse, crystallization behind vacuum filtration in 65 ℃ of loft drier dryings.The result is as shown in table 4:
Table 4
Project No.1 No.2 No.3 is average
Aspartic acid addition (g) 40 40 40
Obtain product (g) 23.10 22.70 23.05
Total recovery (%) 86.3 84.8 86.1 85.3
Raw material abundance (atom%) 98.32 98.32 98.32
Product abundance (atom%) 98.23 98.14 98.22 98.20
Product purity (%) 99.11 99.20 99.18 99.16

Claims (8)

1. isotopic labeling 15The preparation method of N-L-aspartic acid is characterized in that, comprises the steps:
Earlier carbon source, nitrogenous source and trace element are mixed with fermention medium and cultivate enterobacteria bacterial classification (E.coli) A.S.1881, fermentative production contains the cell of travelling of L-Aspartase, then it is joined in the reaction solution that contains substrate, isotope-labeled nitrogenous source and trace element, transfer pH to 7-10, in 30-50 ℃, constant temperature culture reaction 24-48 hour.
2. method according to claim 1 is characterized in that, said carbon source is one or more in FUMARIC ACID TECH GRADE, glucose or the sucrose; Said nitrogenous source is one or more in ammoniacal liquor, ammonium sulfate or the ammonium chloride etc.; Said substrate is a FUMARIC ACID TECH GRADE; Said isotope-labeled nitrogenous source comprises 15N-ammonium sulfate, 15N-ammonium chloride, 15N-urea, 15In the N-ammoniacal liquor one or more; Said trace element comprises NaCl, MgSO 4, KH 2In PO4, corn steep liquor or the peptone one or more.
3. method according to claim 1 is characterized in that, after enzyme reaction finishes, reaction solution is heated to 85-95 ℃, adds gac, stir, be incubated, suction filtration, washing is concentrated into 20-30%, be heated to 70-90 ℃, add sulfuric acid and be adjusted to pH=2.5～3.0, crystallisation by cooling, filter, washing, drying promptly obtains isotope-labeled 15The N-L-aspartic acid.
4. one kind prepares isotopic labeling 15The method of N-L-L-Ala is characterized in that comprising the steps:
Earlier carbon source, nitrogenous source, nutritive substance are mixed with fermention medium, insert false monospore bacillus (Pseudomonas) B186, fermentative production contains the cell of travelling of aspartic acid β-decarboxylase, adds gradually in enzyme reaction solution then 15N-L-aspartic acid, pH are 2-3, react in 30～37 ℃ of constant-temperature shaking culture casees, and pH is reached more than 6.6～8.0, filter, and get isotope-labeled 15N crude product L-Ala.
5. method according to claim 4 is characterized in that, said carbon source is a FUMARIC ACID TECH GRADE; Said nitrogenous source is a kind of in ammoniacal liquor, ammonium sulfate or the ammonium chloride etc.; Said nutritive substance comprises one or more in sal epsom, manganous sulfate, zinc sulfate, phosphoric acid salt, peptone or the corn steep liquor;
6. method according to claim 4 is characterized in that, with isotope-labeled 15N crude product L-Ala is dissolved in the distilled water, is heated to 85-90 ℃, adds 0.5～2% gac, insulation, suction filtration, and evaporative crystallization naturally cools to room temperature, is cooled to 0～5 ℃, and suction filtration gets isotope-labeled 15The N L-Ala.
7. method according to claim 6 is characterized in that, merges all mother liquors, transfers pH to 3.0-3.5, separate from handing over the highly acidic cation ion-exchange resins, collect the pH6-8 elutriant, concentrate, add dehydrated alcohol alcohol and analyse, crystallization, suction filtration, drying promptly obtains isotope-labeled 15N makes with extra care L-Ala.
8. according to claim 6 or 7 described methods, it is characterized in that, isotope-labeled 15N crude product L-Ala is dissolved in the distilled water by the concentration of 10-15wt%.
CNA031419917A 2003-07-31 2003-07-31 Prepn of isotopically labeled 15 N-L-aspartic acid and 15 N-L-alanine Pending CN1487087A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100396782C (en) * 2005-05-27 2008-06-25 上海化工研究院 Preparation method of stable isotop labelled 15N-L-asparagic acid
CN102805052A (en) * 2012-07-31 2012-12-05 中国人民解放军军事医学科学院放射与辐射医学研究所 Method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and special culture medium
CN101824445B (en) * 2009-03-06 2013-01-23 中国科学院生态环境研究中心 Method for preparing 13C stable isotope labeled cellulose

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100396782C (en) * 2005-05-27 2008-06-25 上海化工研究院 Preparation method of stable isotop labelled 15N-L-asparagic acid
CN101824445B (en) * 2009-03-06 2013-01-23 中国科学院生态环境研究中心 Method for preparing 13C stable isotope labeled cellulose
CN102805052A (en) * 2012-07-31 2012-12-05 中国人民解放军军事医学科学院放射与辐射医学研究所 Method for integrally marking drosophila proteomes by using stable isotope labeling with amino acids in cell culture (SILAC) and special culture medium

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