CN100396782C - Preparation method of stable isotop labelled 15N-L-asparagic acid - Google Patents

Preparation method of stable isotop labelled 15N-L-asparagic acid Download PDF

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CN100396782C
CN100396782C CNB2005100262467A CN200510026246A CN100396782C CN 100396782 C CN100396782 C CN 100396782C CN B2005100262467 A CNB2005100262467 A CN B2005100262467A CN 200510026246 A CN200510026246 A CN 200510026246A CN 100396782 C CN100396782 C CN 100396782C
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preparation
aspartic acid
cold labeling
labeling according
phosphate
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CN1869245A (en
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侯静华
周震
李良君
杜晓宁
宋明鸣
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Shanghai Research Institute of Chemical Industry SRICI
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Abstract

The present invention relates to a preparation method of stable isotope labeling <15>N-L-aspartic acid. The preparation method comprises the following steps: (1), fermentation of microorganisms, namely the fermentation of Escherichia coli ATCC11303 or CGMCC1.881, which are used for producing enzymes; (2), slant cultivation; (3), fermentation cultivation; (4), enzyme reaction; (5), extraction, refining and recovery of <15>N. Compared with the prior art, the present invention has the advantages that a set of laboratory scale of production processes is established, the superiority of an enzyme method for preparing <15>N-L-amino acid is embodied, the present invention is favorable for improving the utilization rate of <15>N and simplifying the post-extraction processes, which reduces the production cost and enhances the quality of the products; the products with small drop-out values of abundance are obtained at the high <15>N utilization rate, and the extracting yield is high, so the market competitiveness of the products is greatly improved.

Description

A kind of cold labeling 15The preparation method of N-L-aspartic acid
Technical field
The present invention relates to a kind of 15The preparation method of N-L-aspartic acid specifically relates to a kind ofly do the enzyme source with intestinal bacteria free whole cell and prepare cold labeling 15The method of N-L-aspartic acid; Belong to stable isotope tagged compound production field, relate to microbial enzyme and biological separation technique.
Background technology
Because 15N is than common 14The many mass units of N, and its nuclear spin I=1/2 have NMR signal, therefore can utilize its mass effect and isotopic effect, by measuring technologies such as mass spectrums, obtain valuable structural information.So the amino acid of cold cold labeling is widely used because of unique spike effect is arranged. 15The amino acid of N mark is researching human body protein metabolism and indivedual amino acid metabolism, and the indispensable tracer of transamination between the amino acid.Along with the mensuration of Human Genome Sequencing, the development of protein engineering and the fast development of other related sciences, 15Labeled amino acids such as N-L-aspartic acid will obtain using more widely in fields such as medical science, pharmacology, biology, life science, agricultural sciences, organic chemistry.
Traditional production 15The amino acid whose method of N-L-can adopt organic synthesis method, microbe fermentation method or enzyme process and labelled protein decomposition and separation preparation method etc.The employing synthesis method is fairly simple, but needs optical resolution to make 15The N raw material availability greatly reduces, and cost rises.Labelled protein decomposition and separation preparation method is usually used in preparing aminoacids complex, separate to obtain single amino acid and be difficult to.Adopt Production by Enzymes 15The N-L-aspartic acid, product is more single, and heteroacid seldom separates fairly simple.But, often make owing to have organic nitrogen source in the fermentating formula of cultivation enzyme liquid 15The more quality product requirement that do not reach that descends of the abundance of N-L-aspartic acid.Reach so need descend abundance 15The technical control in addition of the utilization ratio of N.At present, exist 15Rarely seen one piece of domestic patent report in the production field of N stable isotope label L-aspartic acid, application number is 03141991.7.The abundance drop-out value of this patent application is bigger, 15The utilization ratio of N is lower, does not address reaction remnants' 15The recovery of N raw material.
Summary of the invention
Purpose of the present invention provides a kind of cold labeling for the defective that overcomes above-mentioned prior art existence 15The preparation method of N-L-aspartic acid, this preparation method is by adopting free whole cell Production by Enzymes 15The technological line of N-L-aspartic acid is intended to relax with certain technical qualification 15The problem that the N abundance descends, and improve as far as possible 15The N utilization ratio.
Purpose of the present invention can be achieved through the following technical solutions: a kind of cold labeling 15The preparation method of N-L-aspartic acid is characterized in that, this preparation method comprises following processing step:
(1). the microorganism that enzymatic production adopted:
Intestinal bacteria (Escherichia coli) ATCC 11303 or CGMCC 1.881;
(2). slant culture:
Substratum is a nutrition nutrient agar substratum (g/L): peptone 10.0, extractum carnis 3.0, sodium-chlor 5.0, agar 15.0, distilled water 1.0L, pH value 6.8～7.2 is settled to 1.0L, packing, 0.1MPa (121 ℃) sterilization 20 minutes, bacterial classification inserts back 28 ℃～32 ℃ and cultivated 16～24 hours;
(3). fermentation culture:
Become fermention medium with carbon source, nitrogenous source, micro-ion configuration, regulate its pH value 5～9, intestinal bacteria inclined-plane seed is inserted in the sterilization back, in 150～260rpm, 22 ℃～40 ℃, cultivates 16～24 hours; Liquid amount is 50ml/500ml, and sterilising conditions is 0.1MPa (121 ℃), 20 minutes;
The growth concentration of thalline is analyzed by spectrophotometry;
(4). enzyme reaction:
Substrate reactions liquid (g/L): the fumaric concentration of substrate is 50g/L～200g/L, is preferable with 100g/L～150g/L, adds micro-ion and reaches 15Regulating the pH value with NaOH behind the N nitrogenous source is 7.5～9.5;
The free whole cell of the gained that will ferment adds in the substrate solution as the enzyme source, thalline add-on 0.5 weight %～1.2 weight %, under 30 ℃～50 ℃, 35 ℃～40 ℃ reactions preferably 20～28 hours, the stopped reaction when centre is tested fumaric concentration less than 0.25 weight % through sampling with spectrophotometry;
Fumaric concentration is measured by ultraviolet spectrophotometry when reaching the end reaction end in the reaction process, thereby can calculate transformation efficiency;
(5). extract refining reaching 15The recovery of N:
After reaction finishes, enzyme reaction solution is carried out centrifugal, supernatant liquor catches up with ammonia to concentrate on Rotary Evaporators, and institute overflows ammonia and absorbed recovery, is 6.5～7.5 until phlegma pH value; This solution is heated to 75 ℃～85 ℃ then, added 0.2～0.8 weight % activated carbon decolorizing 30～45 minutes, filter colourless filtrate; Filtrate placed be concentrated into 1/5～1/3 of original volume on constant temperature blender with magnetic force or the Rotary Evaporators, treat temperature maintenance, add acid for adjusting pH value to 2.80～3.00 such as hydrochloric acid, have crystal to separate out gradually at 60 ℃～70 ℃; After being chilled to room temperature, putting refrigerator cold-storage and spend the night, suction filtration got white crystal in second day, washed, drained, and crystal was scraped in the container, in 50 ℃～60 ℃ vacuum dryings; Product is carried out purity, abundance detect, purity＜98.5% can be gone recrystallization again, finally obtains 15N-L-aspartic acid product; 15The molar yield of N is more than 70%, and is final 15The utilization ratio of N reaches 65.99%;
Can transfer again after the iso-electric point mother liquor concentrates pH or again upper prop purify and can obtain product again; To reacting remnants' 15The recovery of N raw material reaches more than 80%.
Described step (3) fermentation culture: become fermention medium with carbon source, nitrogenous source, micro-ion configuration, regulate its pH value 6～8, intestinal bacteria inclined-plane seed is inserted in the sterilization back, in 180～200rpm, 25 ℃～37 ℃, cultivates 16～24 hours.
Described carbon source is selected from glucose, FUMARIC ACID TECH GRADE, fructose, sucrose, or contains these sugared molasses, starch.
Described nitrogenous source is selected from ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, ammoniacal liquor, or in the yeast extract, corn steep liquor, W-Gum one or more.
In the described step (3), micro-ion is selected from one or more in potassium phosphate,monobasic, potassium primary phosphate, trimagnesium phosphate, sal epsom, sodium-chlor, ferrous sulfate, manganous sulfate, copper sulfate, the lime carbonate.
In the described step (3), the interpolation of organic nitrogen source corn steep liquor can be controlled by measuring cell concentration, is 0.680 as basis for estimation with the absorbance that records in the 650nm place after 10 times of the fermented liquid dilutions.
Described fermention medium (g/L): corn steep liquor 60, FUMARIC ACID TECH GRADE 20, MgSO 4.7H 2O 0.2, KH 2PO 40.5 ammoniacal liquor adjust pH 5～9 boils after-filtration.
In the described step (4), substrate is a FUMARIC ACID TECH GRADE, and is used in the substrate solution 15The N nitrogenous source is 15In the ammonium chloride of N mark, ammonium sulfate, ammonium acetate, ammonium phosphate, ammoniacal liquor, the urea one or more.
In the described step (4), micro-ion is selected from one or more in potassium phosphate,monobasic, potassium primary phosphate, disodium-hydrogen, SODIUM PHOSPHATE, MONOBASIC, trimagnesium phosphate, sal epsom, sodium-chlor, ferric sulfate, manganous sulfate, copper sulfate, the lime carbonate.
Technological method of the present invention has been set up the laboratory scale production technique of a cover, has embodied the enzyme process preparation 15The amino acid whose superiority of N-L-helps 15The simplification of the raising of N utilization ratio and back extraction process has reduced production cost, has improved product quality, with higher 15The N utilization ratio obtains the very little product of abundance drop-out value, and it is very high to extract yield, has improved the competitiveness of product in market greatly.
Embodiment
The present invention will illustrate the implementation process of present technique by following specific embodiment.
Embodiment 1
18 hours intestinal bacteria of slant culture are inserted fermention medium, and dress 50ml substratum was cultivated 20 hours rotating speed 180rpm in the 500ml triangular flask in 30 ℃ of constant temperature shaking tables.Cultivation gained fermented liquid centrifugal 30 minutes in 4800rpm, the supernatant liquor that inclines, it is 10% that wet thallus 0.5 gram of obtaining joins concentration of substrate, contain abundance is 10.39at%'s 15Among the substrate solution 100ml of the sal epsom of N-ammonium sulfate 7.7 weight %, 0.01 weight %, regulating the pH value with NaOH was 7.9, in 35 ℃ of isothermal reactions 23 hours.The reaction solution that obtains, adds activated carbon 0.2 gram decolouring and gets colourless solution after 30 minutes after supernatant liquor catches up with ammonia to be concentrated into pH value 7.5 through centrifugal, be concentrated into 25ml, add sulfuric acid and transfer to pH value 2.85, separate out than polycrystal, cooling, second day suction filtration, washing, snow-white crystal, oven dry, get product 9.92 grams, abundance is 10.38at%, only than the absolute decline 0.01at% of raw material, relative decline 0.09at%, extracting yield is 89.3 weight %.
Embodiment 2
Get wet thallus 2.0 gram with embodiment 1 the same cultivation, joining concentration of substrate is 11.6 weight %, and containing abundance is 99.53at%'s 15Among the substrate solution 200ml of the sal epsom of N-ammonium chloride 7.4 weight %, 0.01 weight %, regulating the pH value with 10MNaOH was 9.0, in 39 ℃ of isothermal reactions 27 hours.The reaction solution that obtains, adds activated carbon 0.6 gram decolouring and gets colourless solution after 35 minutes after supernatant liquor catches up with ammonia to be concentrated into pH value 6.5 through centrifugal, be concentrated into 55ml, add hydrochloric acid and transfer to pH value 3.00, separate out a large amount of white crystals, cooling, second day suction filtration, washing, snow-white crystal, oven dry, get product 23.65 grams, abundance is 99.18at%, only than the absolute decline 0.35at% of raw material, decline 0.35at% extracts yield and reaches 90.3 weight % relatively.
Embodiment 3
20 hours intestinal bacteria of slant culture are inserted fermention medium, and dress 50ml substratum was cultivated 24 hours rotating speed 200rpm in the 500ml triangular flask in 32 ℃ of constant temperature shaking tables.Cultivation gained fermented liquid centrifugal 30 minutes in 4800rpm, the supernatant liquor that inclines, it is 10% that wet thallus 0.5 gram of obtaining joins concentration of substrate, contain abundance is 98.50at%'s 15Among the substrate solution 100ml of the sal epsom of N-ammonium sulfate 8 weight %, 0.01 weight %, regulating the pH value with NaOH was 8.3, in 37 ℃ of isothermal reactions 25 hours.The reaction solution that obtains, adds activated carbon 0.25 gram decolouring and gets colourless solution after 40 minutes after supernatant liquor catches up with ammonia to be concentrated into pH value 6.8 through centrifugal, be concentrated into 20ml, add sulfuric acid and transfer to pH value 2.80, separate out than polycrystal, cooling, second day suction filtration, washing, snow-white crystal, oven dry, get product 10.05 grams, abundance is 98.19at%, only than the absolute decline 0.31at% of raw material, relative decline 0.31at%, extracting yield is 90.5 weight %.
Embodiment 4
Get wet thallus 2.2 gram with embodiment 3 the same cultivations, joining concentration of substrate is 15.0 weight %, and containing abundance is 99.62at%'s 15Among the substrate solution 200ml of the sal epsom of N-ammonium chloride 9 weight %, 0.01 weight %, regulating the pH value with 10MNaOH was 9.2, in 40 ℃ of isothermal reactions 26 hours.The reaction solution that obtains, adds activated carbon 1.0 gram decolourings and gets colourless solution after 45 minutes after supernatant liquor catches up with ammonia to be concentrated into pH value 6.5 through centrifugal, be concentrated into 60ml, add hydrochloric acid and transfer to pH value 2.90, separate out a large amount of white crystals, cooling, second day suction filtration, washing, snow-white crystal, oven dry, get product 31.08 grams, abundance is 99.28at%, only than the absolute decline 0.34at% of raw material, decline 0.34at% extracts yield and reaches 91.5 weight % relatively.

Claims (12)

1. a cold labeling 15The preparation method of N-L-aspartic acid is characterized in that, this preparation method comprises following processing step:
(1). the microorganism that enzymatic production adopted:
Intestinal bacteria (Escherichia coli) ATCC 11303 or CGMCC 1.881;
(2). slant culture:
Substratum is a nutrition nutrient agar substratum, in g/L: peptone 10.0, extractum carnis 3.0, sodium-chlor 5.0, agar 15.0, distilled water 1.0L, pH value 6.8～7.2 is settled to 1.0L, packing, 0.1MPa sterilized 20 minutes for 121 ℃, and bacterial classification inserts back 28 ℃～32 ℃ and cultivated 16～24 hours;
(3). fermentation culture:
Become fermention medium with carbon source, nitrogenous source, micro-ion configuration, regulate its pH value 5～9, intestinal bacteria inclined-plane seed is inserted in the sterilization back, in 150～260rpm, 22 ℃～40 ℃, cultivates 16～24 hours; Liquid amount is 50ml/500ml, and sterilising conditions is 0.1MPa, 121 ℃, and 20 minutes;
The growth concentration of thalline is analyzed by spectrophotometry;
(4). enzyme reaction:
Substrate reactions liquid is in g/L: the fumaric concentration of substrate is 50g/L～200g/L, add micro-ion and 15Regulating the pH value with NaOH behind the N nitrogenous source is 7.5～9.5;
The free whole cell of the gained that will ferment adds in the substrate solution as the enzyme source, thalline add-on 0.5 weight %～1.2 weight %, reacted 20～28 hours stopped reaction when the centre is tested fumaric concentration less than 0.25 weight % through sampling with spectrophotometry down at 30 ℃～50 ℃;
Fumaric concentration is measured by ultraviolet spectrophotometry when reaching the end reaction end in the reaction process, thereby calculates transformation efficiency;
(5). extract refining reaching 15The recovery of N:
After reaction finishes, enzyme reaction solution is carried out centrifugal, supernatant liquor catches up with ammonia to concentrate on Rotary Evaporators, and institute overflows ammonia and absorbed recovery, is 6.5～7.5 until phlegma pH value; This solution is heated to 75 ℃～85 ℃ then, added 0.2～0.8 weight % activated carbon decolorizing 30～45 minutes, filter colourless filtrate; Filtrate placed be concentrated into 1/5～1/3 of original volume on constant temperature blender with magnetic force or the Rotary Evaporators, treat temperature maintenance, add acid for adjusting pH value to 2.80～3.00, have crystal to separate out gradually at 60 ℃～70 ℃; After being chilled to room temperature, putting refrigerator cold-storage and spend the night, suction filtration got white crystal in second day, washed, drained, and crystal was scraped in the container, in 50 ℃～60 ℃ vacuum dryings; Product is carried out purity, abundance detection, and the capable again recrystallization of purity＜98.5% finally obtains 15N-L-aspartic acid product; 15The molar yield of N is more than 70%, and is final 15The utilization ratio of N reaches 65.99%, after the iso-electric point mother liquor that above-mentioned recrystallization obtains concentrates again adjust pH or again upper prop purify and can obtain product again; To reacting remnants' 15The recovery of N raw material reaches more than 80%.
2. a kind of cold labeling according to claim 1 15The preparation method of N-L-aspartic acid, it is characterized in that, described step (3) fermentation culture: become fermention medium with carbon source, nitrogenous source, micro-ion configuration, regulate its pH value 6～8, intestinal bacteria inclined-plane seed is inserted in the sterilization back, in 180～200rpm, 25 ℃～37 ℃, cultivated 16～24 hours.
3. a kind of cold labeling according to claim 1 and 2 15The preparation method of N-L-aspartic acid is characterized in that, described carbon source is selected from glucose, FUMARIC ACID TECH GRADE, fructose, sucrose, or contains these sugared molasses, starch.
4. a kind of cold labeling according to claim 1 and 2 15The preparation method of N-L-aspartic acid is characterized in that, described nitrogenous source is selected from ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, ammoniacal liquor, or in the yeast extract, corn steep liquor, W-Gum one or more.
5. a kind of cold labeling according to claim 1 and 2 15The preparation method of N-L-aspartic acid, it is characterized in that, in the described step (3), micro-ion is selected from one or more in potassium phosphate,monobasic, potassium primary phosphate, trimagnesium phosphate, sal epsom, sodium-chlor, ferrous sulfate, manganous sulfate, copper sulfate, the lime carbonate.
6. a kind of cold labeling according to claim 1 and 2 15The preparation method of N-L-aspartic acid, it is characterized in that, in the described step (3), the interpolation of organic nitrogen source corn steep liquor can be controlled by measuring cell concentration, is 0.680 as basis for estimation with the absorbance that records in the 650nm place after 10 times of the fermented liquid dilutions.
7. a kind of cold labeling according to claim 1 and 2 15The preparation method of N-L-aspartic acid is characterized in that, described fermention medium is in g/L: corn steep liquor 60, FUMARIC ACID TECH GRADE 20, MgSO 4.7H 2O 0.2, KH 2PO 40.5 ammoniacal liquor adjust pH 5～9 boils after-filtration.
8. a kind of cold labeling according to claim 1 15The preparation method of N-L-aspartic acid is characterized in that, in the described step (4), substrate is a FUMARIC ACID TECH GRADE, and is used in the substrate solution 15The N nitrogenous source is 15In the ammonium chloride of N mark, ammonium sulfate, ammonium acetate, ammonium phosphate, ammoniacal liquor, the urea one or more.
9. a kind of cold labeling according to claim 1 15The preparation method of N-L-aspartic acid, it is characterized in that, in the described step (4), micro-ion is selected from one or more in potassium phosphate,monobasic, potassium primary phosphate, disodium-hydrogen, SODIUM PHOSPHATE, MONOBASIC, trimagnesium phosphate, sal epsom, sodium-chlor, ferric sulfate, manganous sulfate, copper sulfate, the lime carbonate.
10. a kind of cold labeling according to claim 1 15The preparation method of N-L-aspartic acid is characterized in that, in described step (4) enzyme reaction, the fumaric concentration of substrate is 100g/L～150g/L.
11. a kind of cold labeling according to claim 1 15The preparation method of N-L-aspartic acid is characterized in that, in described step (4) enzyme reaction, the free whole cell of the gained that will ferment adds in the substrate solution as the enzyme source, 35 ℃～40 ℃ reactions down.
12. a kind of cold labeling according to claim 1 15The preparation method of N-L-aspartic acid is characterized in that, described step (5) is extracted refining reaching 15In the recovery of N, add salt acid for adjusting pH value to 2.80～3.00.
CNB2005100262467A 2005-05-27 2005-05-27 Preparation method of stable isotop labelled 15N-L-asparagic acid Expired - Fee Related CN100396782C (en)

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CN1487087A (en) * 2003-07-31 2004-04-07 上海新立工业微生物科技有限公司 Prepn of isotopically labeled 15 N-L-aspartic acid and 15 N-L-alanine

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1487087A (en) * 2003-07-31 2004-04-07 上海新立工业微生物科技有限公司 Prepn of isotopically labeled 15 N-L-aspartic acid and 15 N-L-alanine

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