CN100345820C - Method for separating and parifying 15N-L-alanine from 15N-amino-acid mixing liquid - Google Patents

Abstract

The present invention provides a method for separating and purifying 15N-L-alanine from 15N-amino acid mixing liquid. The method comprises the following main procedures: a, thallus and metal ions are removed from 15N-amino acid mixing liquid; b, 15N-L-alanine is separated according to the different amino acid types and content in the 15N-amino acid mixing liquid, and the mixing liquid obtained in procedure a is absorbed and eluted by a single step or multiple steps respectively via different kinds of ion exchange resin; c, the 15N-L-alanine solution obtained in procedure b is purified so as to obtain pure white powdery 15N-L-alanine. The present invention has the advantages of simple technical line, convenient operation and little investment. In addition, the method increases the yield of 15N-L-alanine, avoids the losses of 15N-L-alanine and other 15N-amino acid in research and preparation processes, and saves 15N raw materials to the greatest extent. The quality of the product obtained by using the method reaches medicine standards, and the method is suitable for industrial application.

Description

From 15Separate in the N-amino acid mixing liquid and purify 15The method of N-L-L-Ala

Technical field

The present invention relates to a kind of separation method of chemical substance, particularly a kind of from ¹⁵Separate in the N-amino acid mixing liquid and purify ¹⁵The method of N-L-L-Ala.

Background technology

Isotopic labeling ¹⁵The N-L-L-Ala is a kind of amino acid with special purpose, is applied to various fields such as medical science pharmacology, biochemical metabolism research and instrumental analysis, has higher value more than common L-Ala.In recent years, along with ¹⁵The developing of N-amino acid world market, ¹⁵The market requirement of N-L-L-Ala also increases day by day.In its research and development and production process (comprising enzyme transforming process production and fermentative Production), on the one hand, ¹⁵After once extracting, the N-L-L-Ala leaves over down a large amount of several ¹⁵N-amino acid (heteroacid) with ¹⁵The separation mother liquor of N-L-L-Ala (purpose amino acid) coexistence; On the other hand, because the change of study condition and the fluctuation of working condition, can produce a large amount of several inevitably ¹⁵N-amino acid with ¹⁵The mixed liquid of the reaction of N-L-L-Ala coexistence.In these above two kinds of mixed liquid, it is all little that the content between purpose amino acid and heteroacid differs, and adopts existing method to be difficult to further separation.Because ¹⁵The N raw materials for production reach ¹⁵N-amino acid all is worth high, for improving ¹⁵The N utilization ratio reduces ¹⁵Research and development of N-amino acid and production cost must be in the amino acid mixing liquid ¹⁵The N-L-L-Ala separates purification.

At present, general both at home and abroad employing storng-acid cation exchange resin directly extracts from reaction solution ¹⁵The N-L-L-Ala is as the research (Analytical Biochemistry 126,389-393,1982) of Zvi E.Kahana and the technical scheme of Chinese patent application CN03141991.7.But this method is only in enzyme catalysis ¹⁵Under the situation that transformation efficiency is high in the N-L-aspartic acid reaction process or ¹⁵Be suitable in the once extraction of N-L-L-Ala.For following situation: 1. transformation efficiency is low, and more substrate is arranged ¹⁵The situation that the N-L-aspartic acid is residual; 2. fermentative Production ¹⁵In the N-L-L-Ala process because the complicacy of bacterial classification metabolic process, have part other ¹⁵The situation that N-amino acid accumulates simultaneously; 3. the organic nitrogen source in the fermention medium is utilized not exclusively and the situation of decomposing other common amino acid that induces one; 4. ¹⁵The N-L-L-Ala once extracts the situation that the back remainder contains the second extraction of heteroacid mother liquor.The existing method of above-mentioned situation utilization all can not obtain pure ¹⁵N-L-L-Ala and reclaim other ¹⁵N-amino acid.And do not have relevant at present both at home and abroad yet ¹⁵The report of N-L-L-Ala second extraction.

Summary of the invention

Purpose of the present invention, be to provide a kind of from ¹⁵Separate in the N-amino acid mixing liquid and purify ¹⁵The method of N-L-L-Ala.This method can be from contain the amino acid whose mixed liquid of different non-purposes separation and Extraction ¹⁵The N-L-L-Ala obtains other simultaneously ¹⁵N-amino acid.

To achieve these goals, the present invention has adopted following technical scheme: a kind of from ¹⁵Separate in the N-amino acid mixing liquid and purify ¹⁵The method of N-L-L-Ala, described ¹⁵N-is amino-acid mixed to comprise Production by Enzymes with liquid ¹⁵Enzyme reaction solution and fermentative Production in the N-L-L-Ala process ¹⁵Fermented liquid in the N-L-L-Ala process comprises following key step:

The removal of a, thalline and metal ion

Will ¹⁵Amino-acid mixed and the liquid oxalic acid treatment of N-is removed wherein thalline and precipitation by metallic ion separation;

B, ¹⁵The separation of N-L-L-Ala

According to ¹⁵N-amino-acid mixed with liquid in amino acid kind and content different, adopt different ion exchange resin respectively, to step a gained ¹⁵N-is amino-acid mixed to carry out single step or multistep absorb-elute with liquid, isolates ¹⁵The N-L-L-Ala;

C, ¹⁵Making with extra care of N-L-L-Ala

With step b gained ¹⁵N-L-L-Ala solution removes NH ₄ ⁺, decolouring, concentrate, make with extra care out after the crystallization, separation, drying ¹⁵The N-L-L-Ala.

Among the described step b, for transformation efficiency lower enzyme reaction solution or the higher mother liquor of enzyme reaction solution after conventional method is once extracted of transformation efficiency, in its solution ¹⁵N-L-aspartate content＞1%, adjusting pH is 9-12, adopts the basic anion resin to adsorb, with deionized water or weakly acidic solution wash-out, fraction collection ¹⁵The N-L-L-Ala and ¹⁵The N-L-aspartic acid is isolated ¹⁵The N-L-L-Ala.

Among the described step b, during in containing Xie Ansuan, Methionin in the enzyme reaction solution one or both, perhaps contain in the fermented liquid ¹⁵The N-L-Xie Ansuan, ¹⁵The N-L-phenylalanine, ¹⁵During in the N-L-Methionin one or more, adopt strongly-acid or weakly acidic cation-exchange resin,, isolate by multistep absorption and wash-out ¹⁵The N-L-L-Ala.

Described multistep absorption and wash-out may further comprise the steps:

The first step, elder generation are 1.0-5.0 with enzyme reaction solution or fermented liquid adjusting pH, adopt NH ₄ ⁺-storng-acid cation exchange resin absorption, Methionin wherein or ¹⁵N-L-Methionin is adsorbed, in effluent liquid, at first obtain Xie Ansuan and ¹⁵The N-L-L-Ala or ¹⁵The N-L-L-Ala and ¹⁵The mixed liquid of N-L-Xie Ansuan then obtains ¹⁵The N-L-phenylalanine, in the weakly alkaline elutriant, obtain at last ( ¹⁵N-) L-Methionin, fraction collection;

Second the step, with the first step gained ¹⁵N-L-L-Ala and Xie Ansuan or ¹⁵The N-L-L-Ala and ¹⁵It is 1.0-5.0 that the mixed liquid of N-L-Xie Ansuan is regulated pH, adopts the absorption of strongly-acid or weakly acidic cation-exchange resin, with strong acid weak base salts solution wash-out, at first obtains in elutriant ¹⁵The N-L-L-Ala, obtain thereafter ( ¹⁵N-) L-Xie Ansuan, fraction collection is isolated ¹⁵The N-L-L-Ala.

Described isolated ¹⁵The N-L-L-Ala adopts acidic cation-exchange resin absorption again, washes post with deionized water, can not check acid ion to effluent liquid, uses the weakly alkaline solution wash-out again, is not contained the pure of acid ion ¹⁵The N-L-L-Ala.

Activated carbon decolorizing is adopted in decolouring described in the step c; Concentrate and adopt the heating reduction vaporization to concentrate, Heating temperature is 40-70 ℃; Crystallization is crystallization in anhydrous ethanol medium; The dry vacuum and heating drying that adopts, Heating temperature is 40-80 ℃.

Described weakly acidic solution is hydrochloric acid or the acetate of 0.025-0.5mol/L.

Described weakly alkaline solution is the ammoniacal liquor of 0.025-0.5mol/L, and described strong acid weak base salts solution is the ammonium chloride solution of 0.025-0.5mol/L.

Of the present invention from ¹⁵Separate in the N-amino acid mixing liquid and purify ¹⁵The method of N-L-L-Ala has solved enzyme process and fermentative Production effectively ¹⁵Cause because of heteroacid in the N-L-L-Ala process more ¹⁵The N-L-L-Ala is difficult to isolating problem, has improved ¹⁵The output of N-L-L-Ala has also increased ¹⁵The N utilization ratio of raw materials.Realized from enzyme reaction solution and fermented liquid, isolating respectively ¹⁵The N-L-aspartic acid, ¹⁵The N-L-Xie Ansuan, ¹⁵The N-L-phenylalanine and ¹⁵N-L-Methionin, and obtain ¹⁵The N-L-L-Ala.

Operational path of the present invention is simple, and is easy to operate, and less investment has improved ¹⁵The production yield of N-L-L-Ala has been avoided in research and development and the production process ¹⁵N-L-L-Ala and other ¹⁵The amino acid whose loss of N has been saved to greatest extent ¹⁵The N raw material.Quality product reaches the drug standard.Be fit to industrial application.

Embodiment

Embodiment 1.

Raw material is a Production by Enzymes ¹⁵Enzyme reaction solution in the N-L-L-Ala process, its main component comprises: ¹⁵N-L-L-Ala 52g/L, ¹⁵N-L-aspartic acid 12g/L and a small amount of KH ₂PO ₄, MgSO ₄7H ₂O, the pH=7-9 of solution, cumulative volume are 50mL.

At first in enzyme reaction solution, when constantly stirring, add oxalic acid, add, reach 3.0, place refrigerated centrifuge centrifugal 30min under 5000rpm then, remove thalline and metal ion until the pH value that mixes liquid while dissolve.Collect supernatant liquor, precipitation is with the deionized water wash that is equivalent to its volume 3 times, and it is standby that washings and supernatant liquor are merged the back.

Then above-mentioned mixed solution is regulated pH=9.0 with the NaOH solution of 10mol/L, flow velocity with 80mL/h feeds strongly basic anion exchange resin 201 * 4 posts (717 resins, 3.4 * 64cm) absorption is washed post to the limpid colourless and pH=7 of effluent liquid with deionized water with the flow velocity of 50mL/h.Use the flow velocity wash-out of the HCl solution of 0.025mol/L again, adsorb effluent liquid and deionized water elutriant and HCl wash-out effluent volume at last sample and collect for the 1-1800mL section with 50mL/h ¹⁵The N-L-L-Ala, the 2700-4200mL section is collected ¹⁵The N-L-aspartic acid, thus the two is separated fully.Obtain ¹⁵The N-L-L-Ala and ¹⁵The N-L-aspartic acid is regulated pH=2.5 with the HCl of 6mol/L respectively, again through one time 001 * 7 resin (732 resins, 2.5 * 55cm) flow velocity with 50mL/h adsorbs, washing post to effluent liquid with deionized water with the flow velocity of 30mL/h adds AgI and white precipitate do not occur, during limpid colourless the and pH=7 of effluent liquid, change the ammoniacal liquor wash-out of 2mol/L, obtain not anion-containing pure ¹⁵The N-L-L-Ala or ¹⁵The N-L-aspartic acid.

Will ¹⁵N-L-L-Ala pure component merges, being evaporated to solid and water earlier dissolves repeatedly and is evaporated to phlegma pH=7, add deionized water again and be diluted to 1.5% concentration (weight ratio), the gac that adds 0.2g when being heated to 80 ℃, and after under agitation keeping 80-90 ℃ of heating 30min, repeatedly suction filtration mixes liquid repeatedly, and is limpid bright until solution.Again solution is concentrated in 60 ℃ of following reduction vaporizations, be about in batches, on a small quantity, repeatedly to add again when saturated temperature to evaporated liquor and be-10 ℃ dehydrated alcohol, and stir while adding, until white ¹⁵The N-L-L-Ala separate out in a large number and when no longer increasing till.The placement under-20 ℃ of this crystalline mother solution is spent the night, and suction filtration is isolated then ¹⁵The crystallization of N-L-L-Ala is placed 3h. with it in 60 ℃ of vacuum drying ovens, obtain purified white powder ¹⁵N-L-L-Ala 2.18g, extract yield are 83.6%, ¹⁵The N abundance is 9.88%, and purity is 98.24%. ¹⁵The N-L-aspartic acid is also refining with similar approach, gets white powder product 0.48g, and extract yield is 80.0%, ¹⁵The N abundance is 10.17%, and purity is 98.34%.

Embodiment 2

Raw material is with embodiment 1.

After at first enzyme reaction solution being removed thalline and metal ion by the step of embodiment 1, NaOH solution with 10mol/L is regulated pH=12.0, (3.4 * 64cm) go up samples absorption, wash post to the limpid colourless and pH=7 of effluent liquid with deionized water with the flow velocity of 50mL/h to feed weak base anion-exchange resin 331 posts with the flow velocity of 80mL/h.Use the flow velocity wash-out of the HCl solution of 0.05mol/L again, in the effluent liquid of last sample absorption and deionized water elutriant, promptly obtain with 50mL/h ¹⁵The N-L-L-Ala is collected for the 2500-6100mL section at the HCl effluent volume ¹⁵The N-L-aspartic acid, thus the two is separated fully.Thereafter, it is refining to use the method identical with embodiment 1 to remove behind the negatively charged ion.Obtain ¹⁵N-L-L-Ala product 2.25g, yield is 86.4%, ¹⁵The N abundance is 98.31%, and purity is 98.26%; ¹⁵N-L-aspartic acid product 0.52g, yield is 86.7%, ¹⁵The N abundance is 98.8%, and purity is 98.33%.

Embodiment 3.

Raw material is a Production by Enzymes ¹⁵Enzyme reaction solution in the N-L-L-Ala process, its main component comprises: ¹⁵N-L-L-Ala 55g/L, Xie Ansuan 3.4g/L, Methionin 2.8g/L, ¹⁵N-L-aspartic acid 4.3g/L and a small amount of KH ₂PO ₄, MgSO ₄7H ₂O, cumulative volume are 50mL.

001 * 7 (732) resin is filled in the glass column of 2.5 * 55cm, feed the ammoniacal liquor of 0.5mol/L, finish washing when treating effluent liquid pH=10, behind the static 4h, finish transition when being washed to pH=7, obtain NH with the flow velocity of 50mL per hour ₄ ⁺Type 001 * 7 resin column is standby.

At first in enzyme reaction solution, when constantly stirring, add oxalic acid, add, reach 1.0, be placed in the refrigerated centrifuge centrifugal 30min under 5000rpm then, remove thalline and metal ion until the pH of solution while dissolve.Collect supernatant liquor, precipitation is with the deionized water wash that is equivalent to its volume 3 times, and it is standby that washings and supernatant liquor are merged the back.

Then above-mentioned solution is adjusted to pH=2.5 with the HCl of 6mol/L, passes through NH with the flow velocity of 22mL/h ₄ ⁺Type 001 * 7 resin column is collected effluent liquid, the ply of paper of effluent liquid is analysed test show there is not the Methionin spot, has ¹⁵The N-L-L-Ala, ¹⁵N-L-aspartic acid and Xie Ansuan spot.Illustrate that Methionin is adsorbed, and contains in the effluent liquid ¹⁵The N-L-L-Ala, ¹⁵N-L-aspartic acid and Xie Ansuan.With the HCl adjusting pH=2.5 of this three seed amino acids effluent liquid with 6mol/L, through one time 001 * 7 resin (2.5 * 55cm) flow velocity absorption with 35mL/h, when washing post to the limpid colourless and pH=7 of effluent liquid with the flow velocity of 30mL/h, adopt 0.05mol/L ammonium chloride solution wash-out with deionized water.Collect for the 1500-2200mL section at the elutant volume ¹⁵The N-L-aspartic acid, the 8000-8600mL section is collected ¹⁵The N-L-L-Ala, the 7500-7900mL section is collected Xie Ansuan, thereby the three is separated fully.To obtain thereafter, ¹⁵The N-L-L-Ala is transferred pH=2.5 with the HCl of 6mol/L, again through one time 001 * 7 resin (2.5 * 55cm) flow velocity absorption with 50mL/h, it is limpid colourless and add AgI when white precipitate not occurring to effluent liquid to wash post with deionized water with the flow velocity of 35mL/h, changes 2mol/L ammoniacal liquor wash-out, obtains ¹⁵N-L-L-Ala pure component.Obtain after refining with the method identical with embodiment 1 ¹⁵N-L-L-Ala product 2.12g, extract yield is 77.2%, and abundance is 98.31%, and purity is 98.29%.Right ¹⁵N-L-aspartic acid pure component is with handling together ¹⁵The method dechlorination ion that the N-L-L-Ala is identical, refining obtains ¹⁵N-L-aspartic acid product 0.68g, extract yield is 78.8%, abundance is 98.8%.

Embodiment 4

Raw material is a fermentative Production ¹⁵Fermented liquid in the N-L-L-Ala process, main component comprises: ¹⁵N-L-L-Ala 16.6g/L, ¹⁵N-L-Xie Ansuan 2.2g/L, ¹⁵N-L-phenylalanine 0.91g/L, ¹⁵N-L-Methionin 0.46g/L, glucose and small amounts of inorganic salt, cumulative volume are 400mL.

001 * 7 (732) resin is filled in the glass column of 4.4 * 84cm, feed the ammoniacal liquor of 3mol/L with the flow velocity of 100mL per hour, finish washing when treating effluent liquid pH=10, behind the static 4h, be NH transition when being washed to pH=7 ₄ ⁺Type 001 * 7 resin column is standby.

Above-mentioned fermented liquid when stirring, glass rod is added oxalic acid, add while dissolving, reach 4.0 until mixed liquid pH, place refrigerated centrifuge centrifugal 30min under 5000rpm then, collect supernatant liquor, precipitation merges washings and supernatant liquor afterwards standby with the deionized water wash of 1 times of volume 3 times.

Above-mentioned mixed solution is regulated pH=2.5 in the mode of similar embodiment 3 with the HCl of 6mol/L, pass through NH with the flow velocity of 120mL/h ₄ ⁺Type 001 * 7 resin column, ¹⁵N-L-Methionin is adsorbed, and in the effluent liquid in the 220-2500mL section is ¹⁵The N-L-L-Ala and ¹⁵The mixed solution of N-L-Xie Ansuan is pure in the 2700-3300mL section ¹⁵N-L-phenylalanine solution.With NH ₄ ⁺Type 001 * 7 resin column is used 1.0mol/L ammoniacal liquor wash-out again, and elutriant is pure ¹⁵The N-L-lysine solution.In the mode of similar embodiment 3, separate thereafter, ¹⁵The N-L-L-Ala with ¹⁵The mixed liquid of N-L-Xie Ansuan, with the HCl accent pH=2.5 of 6mol/L, (3.4 * 64cm) the flow velocitys absorption with 70mL/h obtain for the 4000-7200mL section at effluent volume through one time 001 * 7 resin ¹⁵The N-L-L-Ala, the 7200-8100mL section obtains ¹⁵The N-L-Xie Ansuan, thus both are separated fully. ¹⁵The N-L-L-Ala obtains product 4.88g after using the method identical with embodiment 1 refining, and extract yield is 73.5%, ¹⁵The N abundance is 98.22%, and purity is 98.27%. ¹⁵The N-L-Xie Ansuan, ¹⁵The N-L-phenylalanine, ¹⁵The N-L-lysine solution is used the method dechlorination ion identical with embodiment 3, refining respectively, as a result shown in the table 1.

Table 1

Title	Crystal mass (g)	Extract yield (%)	15N abundance (atom%)	Purity (%)
					15The N-L-Xie Ansuan	3.86	88.1	98.16	98.36
15The N-L-phenylalanine	1.65	90.5	98.19	98.40
					15N-L-Methionin	0.85	92.4	98.14	98.34

Embodiment 5

Raw material is a Production by Enzymes ¹⁵Mother liquor after enzyme reaction solution in the N-L-L-Ala process (transformation efficiency 99.60%) once extracts, main component comprises: ¹⁵N-L-L-Ala 5.1g/L, ¹⁵N-L-aspartic acid 6.5g/L, cumulative volume are 200mL (for successively once extracting the amalgamation liquid of back mother liquor for four times).Above-mentioned mother liquor is regulated pH=10.0 with 10mol/L NaOH solution, and (2.5 * 55cm) go up sample absorption, wash post to the limpid colourless and pH=7 of effluent liquid with deionized water with the flow velocity of 30mL/h to feed weak base anion-exchange resin 331 posts with the flow velocity of 30mL/h.Use the flow velocity wash-out of the HCl solution of 0.05mol/L again, in the effluent liquid of last sample absorption and deionized water elutriant, promptly obtain with 30mL/h ¹⁵The N-L-L-Ala is collected for the 1700-4500mL section at the HCl effluent volume ¹⁵The N-L-aspartic acid, thus the two is separated fully.Thereafter, it is refining to use the method identical with embodiment 1 to remove behind the negatively charged ion.Obtain ¹⁵N-L-L-Ala 0.84g, extract yield are 82.7%, ¹⁵The N abundance is 9.88%, and purity is 98.21%; Obtain ¹⁵N-L-aspartic acid 1.10g, extract yield are 84.5%, ¹⁵The N abundance is 10.17%, and purity is 98.36%.

Claims (6)

1, a kind of from ¹⁵Separate in the N-amino acid mixing liquid and purify ¹⁵The method of N-L-L-Ala, described ¹⁵N-is amino-acid mixed to comprise Production by Enzymes with liquid ¹⁵Enzyme reaction solution and fermentative Production in the N-L-L-Ala process ¹⁵Fermented liquid in the N-L-L-Ala process is characterized in that, may further comprise the steps:

A, general ¹⁵Amino-acid mixed and the liquid oxalic acid treatment of N-is removed wherein thalline and precipitation by metallic ion separation;

B, basis ¹⁵N-amino-acid mixed with liquid in amino acid kind and content different, adopt different ion exchange resin respectively, to step a gained ¹⁵N-is amino-acid mixed to carry out single step or multistep absorb-elute with liquid, isolates ¹⁵The N-L-L-Ala;

C, with step b gained ¹⁵N-L-L-Ala solution removes NH ₄ ⁺, decolouring, concentrate, make with extra care out after the crystallization, separation, drying ¹⁵The N-L-L-Ala.

2, according to claim 1 from ¹⁵Separate in the N-amino acid mixing liquid and purify ¹⁵The method of N-L-L-Ala is characterized in that: among the described step b, and for transformation efficiency lower enzyme reaction solution or the higher mother liquor of enzyme reaction solution after conventional method is once extracted of transformation efficiency, in its solution ¹⁵N-L-aspartate content＞1%, adjusting pH is 9-12, adopts the basic anion resin to adsorb, with deionized water or weakly acidic solution wash-out, fraction collection ¹⁵The N-L-L-Ala and ¹⁵The N-L-aspartic acid is isolated ¹⁵N-L-L-Ala, described weakly acidic solution are hydrochloric acid or the acetate of 0.025-0.5mol/L.

3, according to claim 1 from ¹⁵Separate in the N-amino acid mixing liquid and purify ¹⁵The method of N-L-L-Ala is characterized in that: among the described step b, during in containing Xie Ansuan, Methionin in the enzyme reaction solution one or both, perhaps contain in the fermented liquid ¹⁵The N-L-Xie Ansuan, ¹⁵The N-L-phenylalanine, ¹⁵During in the N-L-Methionin one or more, adopt strongly-acid or weakly acidic cation-exchange resin,, isolate by multistep absorption and wash-out ¹⁵The N-L-L-Ala.

4, according to claim 3 from ¹⁵Separate in the N-amino acid mixing liquid and purify ¹⁵The method of N-L-L-Ala is characterized in that: described multistep absorption and wash-out may further comprise the steps:

The first step, elder generation are 1.0-5.0 with enzyme reaction solution or fermented liquid adjusting pH, adopt NH ₄ ⁺-storng-acid cation exchange resin absorption, Methionin wherein or ¹⁵N-L-Methionin is adsorbed, in effluent liquid, at first obtain Xie Ansuan and ¹⁵The N-L-L-Ala or ¹⁵The N-L-L-Ala and ¹⁵The mixed liquid of N-L-Xie Ansuan then obtains ¹⁵The N-L-phenylalanine, in the weakly alkaline elutriant, obtain at last ( ¹⁵N-) L-Methionin, fraction collection;

Second the step, with the first step gained ¹⁵N-L-L-Ala and Xie Ansuan or ¹⁵The N-L-L-Ala and ¹⁵It is 1.0-5.0 that the mixed liquid of N-L-Xie Ansuan is regulated pH, adopts the absorption of strongly-acid or weakly acidic cation-exchange resin, with strong acid weak base salts solution wash-out, at first obtains in elutriant ¹⁵The N-L-L-Ala, obtain thereafter ( ¹⁵N-) L-Xie Ansuan, fraction collection is isolated ¹⁵The N-L-L-Ala;

Described weakly alkaline elutriant is 0.025-0.5mol/L or the ammoniacal liquor of 1.0mol/L, and described strong acid weak base salts solution is the ammonium chloride solution of 0.025-0.5mol/L.

5, according to claim 2 or 3 or 4 described from ¹⁵Separate in the N-amino acid mixing liquid and purify ¹⁵The method of N-L-L-Ala is characterized in that: described isolated ¹⁵The N-L-L-Ala adopts acidic cation-exchange resin absorption again, washes post with deionized water, can not check acid ion to effluent liquid, uses the weakly alkaline solution wash-out again, is not contained the pure of acid ion ¹⁵The N-L-L-Ala; Described weakly alkaline solution is the ammoniacal liquor of 0.025-0.5mol/L.

6, according to claim 1 from ¹⁵Separate in the N-amino acid mixing liquid and purify ¹⁵The method of N-L-L-Ala is characterized in that: activated carbon decolorizing is adopted in the decolouring described in the step c; Concentrate and adopt the heating reduction vaporization to concentrate, Heating temperature is 40-70 ℃; Crystallization is crystallization in anhydrous ethanol medium; The dry vacuum and heating drying that adopts, Heating temperature is 40-80 ℃.