CN1166634C - 15N-L-proline separating and purifying method - Google Patents

15N-L-proline separating and purifying method Download PDF

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Publication number
CN1166634C
CN1166634C CNB011268069A CN01126806A CN1166634C CN 1166634 C CN1166634 C CN 1166634C CN B011268069 A CNB011268069 A CN B011268069A CN 01126806 A CN01126806 A CN 01126806A CN 1166634 C CN1166634 C CN 1166634C
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proline
pro
separating
crude product
purifying method
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CN1405153A (en
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侯静华
孙建春
杜晓宁
李良君
宋明鸣
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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

The present invention relates to a method for separating and purifying 15N-L-proline. Strong acidic resin is adopted to remove pigment and impurities in fermentation liquor, strong alkaline resin is adopted to separate proline from other amino acid heteroacid, and then, anhydrous alcohol is adopted for refining. The obtained 15N-L-proline has the advantages of high purity, whiteness and no yellowing in long term storage. The method has the advantages of simple technology and convenient operation, and the total extracting yield reaches more than 80%.

Description

15The separating and purifying method of N-L-proline(Pro)
Technical field
The present invention relates to a kind of amino acid whose separating and purifying method, relate to a kind of or rather 15The separating and purifying method of N-L-proline(Pro).
Background technology
The L-proline(Pro) is one of important composition of human body protein, belongs to heterocycle family amino acid, in industries such as medicine, agricultural, chemical industry and food important purposes is arranged. 15The N-L-proline(Pro) is because the singularity of its structure, in research fields such as medical science, pharmacology, agricultural sciences, life sciences as tracer agent and widespread use. 15Organic synthesis method and microbe fermentation method are generally adopted in the preparation of N-L-proline(Pro), but no matter which kind of method of employing all can run into the product separation issues of purification.Because proline(Pro) is unique amino acid that is dissolved in alcohol, and solubleness is very big in water, is 162.30g/100mL water (25 ℃) that therefore, purification difficult is bigger.
The purification process of existing L-proline(Pro), comprise nitrosification method (" Food science ", 1998 (10), 26-29), the alumina column chromatography method, Pentachlorophenol becomes salt precipitation method (" medicine industry ", 1981 (4), 8-9), the alcohol-ether method, purifying mixture method of water and low-carbon alcohol (January 17 1991 applying date, application number 911003703, publication number CN1063280A) and ion-exchange-resin process etc.Problems such as the nitrosification method exists operation steps many, and pre-processing period is long; The alumina column chromatography method exist time-consuming many, should not the expansion scale etc. problem; Though Pentachlorophenol becomes salt precipitation method to have the good characteristics of specificity, the Pentachlorophenol severe toxicity, labour protection is a problem; The separation and purification of alcohol-ether method is not thorough, and easily makes proline(Pro) crystallization oxidation jaundice, and the boiling point of ether is low in addition, and is inflammable and explosive, not easy to be recycled; And the purifying mixture method of described water of patent and low-carbon alcohol is to unite as the purification solvent with methyl alcohol, ethanol, Virahol, propyl carbinol, still 15The tracer agent that the N-L-proline(Pro) is used as industries such as medicine, food should not adopt methyl alcohol, Virahol, propyl carbinol as the purification solvent; And general ion-exchange-resin process, before upper prop, all to add flocculation agent fermented liquid is carried out pre-treatment (" Huadong Chemical College journal ", 1989 (4), 456-460), perhaps use bleaching agent bleaching (" microorganism journal ", 1993 (2), 87-90), before refining, also to decolour to elutriant.
Summary of the invention
At the deficiency of above-mentioned separation purification method, the invention provides a kind of good separating effect, few the purifying of operation steps from the bio-fermented liquid separation 15The method of N-L-proline(Pro).
The present invention is that a kind of spent ion exchange resin separates purification 15The improved method of N-L-proline(Pro) adopts highly acidic resin and basic resin extraction separation from fermented liquid respectively 15The N-L-proline(Pro), and then will obtain 15N-L-proline(Pro) list spot crude product is refining with dehydrated alcohol, finally obtains purity greater than 98% 15N-L-proline(Pro) product.
Of the present invention 15The separating and purifying method of N-L-proline(Pro) comprises following operation steps:
(1) the centrifugal mycelium of removing in the fermented liquid, centrifugal clear liquid is regulated pH=3～5 with oxalic acid, and with highly acidic resin absorption, with the desorb of 0.1～0.2M ammoniacal liquor, stripping liquid vacuum concentration drying obtains 15N-L-proline(Pro) crude product,
(2) step (1) obtains 15N-L-proline(Pro) crude product water dissolution is regulated pH=9～11 with ammoniacal liquor, with basic resin absorption, with the desorb of 0.1M hydrochloric acid, will contain 15N-L-proline(Pro) list spot stripping liquid vacuum concentration drying obtains 15N-L-proline(Pro) list spot crude product,
(3) step (2) obtains 15N-L-proline(Pro) list spot crude product dissolves down at 60～80 ℃ with dehydrated alcohol, filtered while hot, and filtrate is through cooling, and crystallization is filtered, and promptly obtains after the vacuum-drying purifying 15N-L-proline(Pro) product.
Of the present invention 15The separating and purifying method of N-L-proline(Pro), described highly acidic resin are that the gel superacicd styrene is a resin, void column flow velocity SV=0.1～1.0%, and described basic resin is a styrene series anion exchange resin, void column flow velocity SV=0.1～1.0%.
Of the present invention 15The separating and purifying method of N-L-proline(Pro), described drying temperature are 50～60 ℃.
Of the present invention 15The separating and purifying method of N-L-proline(Pro), the described anhydrous alcohol solution of using 15During N-L-proline(Pro) list spot crude product, the dosage of dehydrated alcohol is 15N-L-proline(Pro) list spot crude product: dehydrated alcohol=1: (4～15) (weight).
Of the present invention 15The separating and purifying method of N-L-proline(Pro), step (2) obtains 15N-L-proline(Pro) list spot crude product also can be earlier with dehydrated alcohol band water before using anhydrous alcohol solution, band water final vacuum drying.
Of the present invention 15The separating and purifying method of N-L-proline(Pro), the temperature of crystallisation by cooling are controlled at room temperature to-18 ℃.
Of the present invention 15The separating and purifying method of N-L-proline(Pro) compared with prior art has the following advantages:
1. method of the present invention adopts highly acidic resin can remove a large amount of pigments and impurity, and fermented liquid needn't flocculate or pre-treatment such as decolouring, also need not to decolour before refining, adopts basic resin to make 15The N-L-proline(Pro) separates substantially fully with heteroacid such as L-glutamic acid, methionine(Met), Methionins, 15N-L-proline(Pro) list spot yield＞95%, so it is technology is simple, easy to operate.
2. with the easy oxidation jaundice of ether-Ethanol Method purified L-proline(Pro) product, and method of the present invention with dehydrated alcohol as refining solvent, pigment all can be flowed in refinement mother liquor, the product purity height that makes, color and luster is snow-white, long storage can not turned to be yellow, and purity＞98% always extracts yield and reaches more than 80%.
Embodiment
Further set forth the present invention below by detailed description, but embodiment not a limitation of the present invention to the specific embodiment of separating and purifying method of the present invention.
Embodiment 1
Obtain after the fermentation 15N-L-proline(Pro) high abundance fermented liquid is regulated pH=3.2 with oxalic acid, with the centrifugal supernatant liquor that gets of whizzer, gets supernatant liquor 350mL, and this supernatant liquor contains 15N-L-proline(Pro) 2.11% (weight), last gel superacicd styrene are resin (Amberjet 1500H highly acidic resin) post, and void column flow velocity SV=0.2% cleans resin column to neutral with distilled water afterwards, is adsorbed on the resin column with the desorb of 0.1M ammoniacal liquor then 15N-L-proline(Pro), stripping liquid be 50 ℃ of following vacuum concentration dryings, obtains containing heteroacid 15N-L-proline(Pro) crude product 9.24g.
Add distilled water 350mL dissolving 15N-L-proline(Pro) crude product, regulate its pH=9.9 with ammoniacal liquor, last styrene series anion exchange resin (201 * 4OH type basic resin) post, void column flow velocity SV=0.15%, clean resin column to neutral with distilled water afterwards, be adsorbed on the resin column with the 0.1MHCl desorb then 15The N-L-proline(Pro) is collected 15N-L-proline(Pro) list spot stripping liquid, 50 ℃ of following vacuum concentration dryings, with dehydrated alcohol band water once, complete drying in vacuum drying oven obtains again 15N-L-proline(Pro) list spot crude product 7.53g.
Dissolve down in 70 ℃ of water-baths with the 140mL dehydrated alcohol 15N-L-proline(Pro) list spot crude product, filtered while hot, filtrate is cooled to room temperature, leaves standstill to make 15The crystallization of N-L-proline(Pro) is separated out, and filters, and with absolute ethanol washing once, refilters, and 50 ℃ of following vacuum-dryings, obtains 5.97g 15N-L-proline(Pro) product, product color is snow-white, and purity is 98.41%, 15The N abundance is 96.14%, and always extracting yield is 80.8%.
Embodiment 2
Obtain after the fermentation 15The low abundance fermented liquid of N-L-proline(Pro) is regulated pH=5.0 with oxalic acid, with the centrifugal supernatant liquor that gets of whizzer, gets supernatant liquor 150mL, and this supernatant liquor contains 15N-L-proline(Pro) 2.50% (weight), last gel superacicd styrene are resin (001 * 7H type highly acidic resin) post, and void column flow velocity SV=1.0% cleans resin column to neutral with distilled water afterwards, uses the desorb of 0.1M ammoniacal liquor then, is contained 15The stripping liquid of N-L-proline(Pro), stripping liquid be 55 ℃ of following vacuum concentration dryings, obtains containing heteroacid 15N-L-proline(Pro) crude product 4.65g.
Add distilled water 150mL dissolving 15N-L-proline(Pro) crude product is regulated its pH=10.2 with ammoniacal liquor, last styrene series anion exchange resin (201 * 7OH type basic resin) post, and void column flow velocity SV=1.0% cleans resin column to neutral with distilled water afterwards, uses the 0.1MHCl desorb then, collects 15N-L-proline(Pro) list spot stripping liquid, 55 ℃ of following vacuum concentration dryings, with dehydrated alcohol band water once, complete drying in vacuum drying oven obtains again 15N-L-proline(Pro) list spot crude product 3.58g.
Dissolve down in 85 ℃ of water-baths with the 20mL dehydrated alcohol 15N-L-proline(Pro) list spot crude product, filtered while hot, filtrate is cooled to room temperature, and standing over night is filtered, and drains, and with absolute ethanol washing once, drains again, 55 ℃ of following vacuum-dryings, obtains 3.01g 15N-L-proline(Pro) product, product color is snow-white, and purity is 98.36%, 15The N abundance is 10.14%, and always extracting yield is 80.2%.
Embodiment 3
Obtain after the fermentation 15N-L-proline(Pro) high abundance fermented liquid is regulated pH=4.1 with oxalic acid, with the centrifugal supernatant liquor that gets of whizzer, gets supernatant liquor 400mL, and this supernatant liquor contains 15N-L-proline(Pro) 2.11% (weight), last gel superacicd styrene are resin (Amberjet 1500H highly acidic resin) post, and void column flow velocity SV=0.6% cleans resin column to neutral with distilled water afterwards, uses the desorb of 0.2M ammoniacal liquor then, is contained 15N-L-proline(Pro) stripping liquid in 60 ℃ of following vacuum concentration dryings, obtains containing heteroacid 15N-L-proline(Pro) crude product 10.59g.
Add distilled water 400mL dissolving 15N-L-proline(Pro) crude product is regulated its pH=10.8 with ammoniacal liquor, last styrene series anion exchange resin (201 * 4OH type basic resin) post, and void column flow velocity SV=0.6% cleans resin column to neutral with distilled water afterwards, uses the 0.1MHCl desorb then, collects 15N-L-proline(Pro) list spot stripping liquid, 60 ℃ of following vacuum concentration dryings, with dehydrated alcohol band water once, complete drying in vacuum drying oven obtains again 15N-L-proline(Pro) list spot crude product 8.32g.
Dissolve down in 80 ℃ of water-baths with the 85mL dehydrated alcohol 15N-L-proline(Pro) list spot crude product, filtered while hot, filtrate is cooled to room temperature, freezing spending the night, temperature is-18 ℃, filters, and drains, and with absolute ethanol washing once, drains again, in 60 ℃ of following vacuum-dryings, obtains 7.02g 15N-L-proline(Pro) product, product color is snow-white, and purity is 98.36%, 15The N abundance is 96.11%, and always extracting yield is 83.2%.

Claims (9)

1, a kind of 15The separating and purifying method of N-L-proline(Pro) is characterized in that this method comprises following operation steps:
(1) the centrifugal mycelium of removing in the fermented liquid, centrifugal clear liquid is regulated pH=3～5 with oxalic acid, and with highly acidic resin absorption, with the desorb of 0.1～0.2M ammoniacal liquor, stripping liquid vacuum concentration drying obtains 15N-L-proline(Pro) crude product,
(2) step (1) obtains 15N-L-proline(Pro) crude product water dissolution is regulated pH=9～11 with ammoniacal liquor, with basic resin absorption, with the desorb of 0.1M hydrochloric acid, will contain 15N-L-proline(Pro) list spot stripping liquid vacuum concentration drying obtains 15N-L-proline(Pro) list spot crude product,
(3) step (2) obtains 15N-L-proline(Pro) list spot crude product dissolves down at 60～80 ℃ with dehydrated alcohol, filtered while hot, and filtrate is through cooling, and crystallization is filtered, and promptly obtains after the vacuum-drying purifying 15N-L-proline(Pro) product.
2, as claimed in claim 1 15The separating and purifying method of N-L-proline(Pro) is characterized in that described highly acidic resin is that the gel superacicd styrene is a resin.
3, as claimed in claim 1 15The separating and purifying method of N-L-proline(Pro) is characterized in that described basic resin is a styrene series anion exchange resin.
4, as claimed in claim 1 or 2 15The separating and purifying method of N-L-proline(Pro), when it is characterized in that using resin absorption, void column flow velocity SV=0.1～1.0% of centrifugal clear liquid.
5, described as claim 1 or 3 15The separating and purifying method of N-L-proline(Pro), when it is characterized in that using resin absorption, 15Void column flow velocity SV=0.1～1.0% of N-L-proline(Pro) dissolving crude product liquid.
6, as claimed in claim 1 15The separating and purifying method of N-L-proline(Pro) is characterized in that the described anhydrous alcohol solution of using 15During N-L-proline(Pro) list spot crude product, 15N-L-proline(Pro) list spot crude product and dehydrated alcohol weight ratio are 1: (4～15).
7, as claimed in claim 1 15The separating and purifying method of N-L-proline(Pro) is characterized in that described 15N-L-proline(Pro) list spot crude product earlier with dehydrated alcohol band water, is with water final vacuum drying before using anhydrous alcohol solution.
8, as claimed in claim 1 15The separating and purifying method of N-L-proline(Pro), the temperature that it is characterized in that crystallisation by cooling are that room temperature is to-18 ℃.
9, described as claim 1 or 7 15The separating and purifying method of N-L-proline(Pro) is characterized in that described drying temperature is 50～60 ℃.
CNB011268069A 2001-09-19 2001-09-19 15N-L-proline separating and purifying method Expired - Fee Related CN1166634C (en)

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Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101348453B (en) * 2008-08-29 2010-12-29 广东肇庆星湖生物科技股份有限公司 Method for purifying proline
CN102675180A (en) * 2012-05-31 2012-09-19 精晶药业有限公司 Method for extracting proline
CN103265467B (en) * 2013-05-06 2015-07-29 华南理工大学 A kind of crystallisation by cooling refines the method for L-PROLINE
CN103333094B (en) * 2013-06-19 2015-05-13 广东肇庆星湖生物科技股份有限公司 Process method for crystallization purification of proline
CN104922932B (en) * 2015-05-26 2016-10-26 安徽皖东树脂科技有限公司 A kind of aminoacid extracts resin dedicated using method
CN106543063A (en) * 2016-09-23 2017-03-29 合肥信达膜科技有限公司 A kind of proline extraction element and method
CN108640865A (en) * 2018-07-02 2018-10-12 无锡晶海氨基酸股份有限公司 A kind of preparation method of medicinal proline

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