CN106432411A - Method for extracting alanyl-glutamine from enzymatic conversion liquid - Google Patents
Method for extracting alanyl-glutamine from enzymatic conversion liquid Download PDFInfo
- Publication number
- CN106432411A CN106432411A CN201610844851.3A CN201610844851A CN106432411A CN 106432411 A CN106432411 A CN 106432411A CN 201610844851 A CN201610844851 A CN 201610844851A CN 106432411 A CN106432411 A CN 106432411A
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- liquid
- glutamine
- alanyl
- ceramic membrane
- alanyl glutamine
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/10—Process efficiency
Abstract
The invention relates to the technical field of preparation of alanyl-glutamine and specifically discloses a method for extracting alanyl-glutamine from enzymatic conversion liquid. The method comprises the following steps: adding sodium chloride into inactivated conversion liquid, wherein the quantity of the added sodium chloride is 3%-5% of the quantity of the conversion liquid; reducing temperature after the sodium chloride is fully dissolved; after the temperature of the conversion liquid is below 60 DEG C, successively filtering through a micro-filtration ceramic membrane with a pore diameter from 100nm to 50nm and an ultra-filtration ceramic membrane; concentrating the acquired clear liquid with a nano-filtration ceramic membrane; removing electrolyte and a large amount of water, thereby acquiring an alanyl-glutamine concentrated solution; and crystallizing, thereby acquiring an alanyl-glutamine crude product. The method provided by the invention has the advantages of simple operation, easiness in control, short process flow, low energy consumption, low cost, low impurity content of the alanyl-glutamine acquired after separation, few varieties, stable product quality and suitability for large-scale production of the alanyl-glutamine.
Description
Technical field
The present invention relates to the preparing technical field of alanyl glutamine.
Background technology
N (2)-L-Ala-L-Glu amine(Also known as alanyl glutamine)Belong to the derivant of aminoacid, referred to as third
Paddy dipeptides, is mainly used as amino acidses parenteral nutrition agent, with important medical value and economic worth.
The technology of preparing of glutamine dipeptide mainly has chemical synthesiss and microbial enzyme conversion method, wherein microbial enzyme conversion method
Have the advantages that the production time short, technology controlling and process is simple, production process is pollution-free, inexpensive, but due to obtained conversional solution
In in addition to product glutamine dipeptide also include a certain amount of reaction substrate, enzyme etc. so that enzymatic conversion liquid separation and Extraction glutamine dipeptide be stranded
Difficulty, separating obtained glutamine dipeptide impurity content is high, species is many, of poor quality.
Publication No. is carried out vacuum distillation after directly being processed using nanofiltration for conversional solution in the patent of CN104480075A and obtains
Solids crude crystalline product, its impurity is wayward, and filter efficiency is low, and NF membrane loss is big.
Content of the invention
The technical problem to be solved in the present invention is to provide a kind of method for extracting alanyl glutamine liquid from enzymatic conversion,
Simple to operate, easy to control, flow process is short, energy consumption is low, low cost, and separating obtained alanyl glutamine impurity content is few, purity height,
Product quality is stable, it is adaptable to the large-scale production of alanyl glutamine.
For solving above-mentioned technical problem, the technical solution used in the present invention is:One kind extracts the third ammonia from enzymatic conversion liquid
The method of acyl glutamine, comprises the steps:
(1)By the conversional solution after the production alanyl glutamine gained inactivation of microbial enzyme conversion method by membrane aperture be 100~
The microfiltration ceramic membrane of 50nm, 0.02~0.1Mpa of control pressure removes macromolecular substances and obtains cleaner liquid of making pottery;
Its ablation method is:Enzymatic conversion liquid is 65~90 DEG C in temperature, under the conditions of pH=5~9,30~45 min of normal pressure heat-insulating;
(2)Pottery cleaner liquid is passed through ultrafiltration ceramic membrane, is removed material of the molecular weight more than 1500 and obtain ultrafiltration clear liquid, control pressure
0.3~0.7Mpa of power;
(3)By ultrafiltration clear liquid by nanofiltration pottery membrance concentration, 0.5~1.2Mpa of control pressure, electrolyte and a large amount of water is removed, is obtained
To alanyl glutamine concentrated solution, crystallize, obtain alanyl glutamine crude product.
Further, step(1)The middle temperature to after inactivation is 65~90 DEG C, add chlorination in the conversional solution of pH=5~9
Sodium, adds Sodium Chloride quality for the 3%~5% of conversional solution quality, is lowered the temperature after Sodium Chloride dissolving completely, and liquid temp to be transformed is low
Passed through microfiltration ceramic membrane after 60 DEG C.
It is miscellaneous that ultrafiltration ceramic membrane removes other macromole that brings in the high molecular weight protein such as enzyme in conversional solution and biological enzyme liquid
Matter.
Nanofiltration ceramic membrane energy molecular cut off more than 200 Organic substance and multivalent ion, it is allowed to small organic molecule and
Monovalention is passed through, and has higher removal efficiency to bivalence or multivalent ion and molecular weight between the Organic substance between 200~500.Profit
With this feature of NF membrane, ultrafiltration clear liquid is passed through NF membrane so that electrolyte ion(Sodium Chloride), reaction substrate and a large amount of
Water passes through NF membrane, and molecular cut off is 217 alanyl glutamine, realizes separation and concentration to ultrafiltration clear liquid, obtains
To alanyl glutamine concentrated solution.
Further, step(2)Middle Sodium Chloride addition is the 3% of conversional solution quality.
Further, step(2)Middle conversional solution is cooled to 20~25 DEG C, then pass through microfiltration ceramic membrane.
Further, step(3)Middle nanofiltration pottery membrane aperture is 0.5~2 nanometer.
It is using the beneficial effect produced by technique scheme:
1)The present invention first passes through conversional solution after pottery filter is processed with ultrafiltration and carries out condensing crystallizing using nanofiltration again, and effect is more
Good, it is more convenient in product purity and operating procedure.
2)Separation method of the present invention is simple to operate, easy to control, easily realizes automatization, and flow process is short.
3)Separation method energy consumption of the present invention is low, does not use soda acid, organic reagent, separation process safety non-toxic, to environment no
Pollution.
4)The separating obtained alanyl glutamine impurity content of separation method of the present invention is few, species is few, and product purity is high, matter
Amount is stable, it is adaptable to the large-scale production of alanyl glutamine.
Description of the drawings
Fig. 1-3 is respectively the liquid chromatogram of some embodiment gained alanyl glutamine crude products of the present invention.
High performance liquid chromatography detection, the spectrum of Fig. 1-Fig. 3 are carried out according to 2015 editions alanyl glutamy standards of Chinese Pharmacopoeia
In figure, the chromatographic peak of alanyl glutamine is respectively 19.943min, 19.825min, 19.844min, alanyl in each crude product
The content of glutamine is all higher than 99%.
Specific embodiment
Separation and Extraction glutamine dipeptide difficulty in the conversional solution of glutamine dipeptide is obtained to solve microbial enzyme conversion method, is separated
Gained glutamine dipeptide impurity content is high, species is many, ropy technical problem, the invention provides one kind utilizes membrane filtration technique
The method for extracting alanyl glutamine from enzymatic conversion liquid, microbial enzyme conversion method production alanyl glutamine gained is gone out
Conversional solution after work passes sequentially through microfiltration ceramic membrane, ultrafiltration ceramic membrane and goes the removal of impurity, then is concentrated to give third by nanofiltration ceramic membrane
Aminoacyl glutamine concentrated solution, crystallized obtains the dense crude product of aminoacyl glutamine.The inventive method gained alanyl glutamine
Impurity content is few, species is few, and product quality is stable, it is adaptable to the large-scale production of alanyl glutamine.
The inventive method comprises the steps:
(1)Enzymatic conversion liquid is 65~90 DEG C in temperature, under the conditions of pH=5~9,30~45 min of normal pressure heat-insulating, it is therefore intended that make
Enzymatic conversion liquid is inactivated;
(2)Sodium Chloride is added in the conversional solution after inactivation, adds Sodium Chloride quality for the 3%~5% of conversional solution quality, Sodium Chloride
Lowered the temperature after dissolving completely, liquid temp to be transformed is passed through the microfiltration pottery that membrane aperture is 50~100nm later less than 60 DEG C
Porcelain film, removes macromolecular substances and obtains cleaner liquid of making pottery;
(3)Pottery cleaner liquid is passed through ultrafiltration ceramic membrane, is removed material of the molecular weight more than 1500 and obtain ultrafiltration clear liquid;
(4)By ultrafiltration clear liquid by nanofiltration pottery membrance concentration, electrolyte and a large amount of water is removed, obtain alanyl glutamine concentration
Liquid, recrystallization, obtain alanyl glutamine crude product.
Preferably, step(2)Middle Sodium Chloride addition is the 3% of conversional solution quality.
Step(2)Middle conversional solution is cooled to 20~25 DEG C, then pass through microfiltration ceramic membrane.
Step(3)Middle nanofiltration pottery membrane aperture is 0.5~2 nanometer.
It is illustrated to extracting alanyl glutamine from enzymatic conversion liquid using said method below.
Embodiment 1
Will be using active immobilized enzyme(Enzyme activity 4405U) conversional solution for completing is converted, it is brought rapidly up to 70 DEG C, pH to 9.0 is adjusted,
It is incubated 45min afterwards, is inactivated.
Glutamine dipeptide conversional solution 70L, the about 7.6kg of inactivation is taken, after testing the content of wherein product alanyl glutamine
For 109.5g/L, add the dissolving of Sodium Chloride 420g complete in conversional solution, conversion liquid temp is dropped in 55 DEG C, is passed through membrane aperture
For the microfiltration ceramic membrane of 50~100nm, remove the macromolecular substances cleaner liquid that obtains making pottery and amount to 63L, the containing of alanyl glutamine
Measure as 104.2 g/L, turbid phase 9.5L of pottery filter is obtained, the content of alanyl glutamine is 85.4g/L, and cleaning mixture amounts to 50L, contains
Amount 25g/L;Gained pottery cleaner liquid is passed through ultrafiltration ceramic membrane, is removed material of the molecular weight more than 1500 and obtain ultrafiltration clear liquid
59L, the content of alanyl glutamine is 102.5g/L;By gained ultrafiltration clear liquid by nanofiltration pottery membrance concentration, electrolysis is removed
Matter obtains alanyl glutamine concentrated solution 55L, and it is 71.5% that its product assay is 98.2g/L, yield.Gained concentration feed liquid is entered
Row crystallization obtains alanyl glutamine crude product.
Embodiment 2
Using metabolizing enzymes(Enzyme activity 3980U) the common 45ml of the conversional solution for completing is processed, conversional solution is brought rapidly up to 75 DEG C, when
Temperature adjusts pH to 5.0 after rising to preset temperature, is incubated 30min afterwards.Enzyme liquid 70L of inactivation is taken, content is 104.5g/L, to enter
Row pretreatment, removes the macromolecular substances cleaner liquid that waits until to make pottery by microfiltration ceramic membrane and amounts to 62L, content 102.1g/L, and pottery filter is turbid
Phase 9.7L, content 83.8g/L;Cleaning mixture amounts to 50L, content 22.3g/L;Pottery cleaner liquid is carried out ultrafiltration ceramic membrane and removes molecule
Material of the amount more than 1500 waits until that ultrafiltration clear liquid there are ultrafiltration clear liquid 58L, content 100.5g/L;Ultrafiltration clear liquid is carried out nanofiltration
Ceramic membrane, removes the electrolyte in conversional solution and obtains concentrated phase 55L, content 97.9g/L, and yield is 77.6%.Afterwards by gained concentrate feed
Liquid carries out crystallization and obtains alanyl glutamine crude product.
Embodiment 3
Take the enzyme liquid after processing(Enzyme activity 5100U) amount to 35ml, the conversional solution for completing is processed, conversional solution is brought rapidly up to 75
DEG C, pH to 6.0 is adjusted after temperature rises to preset temperature, is incubated 45min afterwards.Enzyme liquid 70L of inactivation is taken, content is
112.3g/L, crosses microfiltration ceramic membrane and removes the macromolecular substances cleaner liquid that waits until to make pottery and amount to 62.5L, content 112.2g/L, and pottery filter is turbid
Phase 11L, content 55.4g/L;Pottery cleaner liquid is carried out ultrafiltration ceramic membrane removal small-molecule substance and waits until that ultrafiltration clear liquid there are ultrafiltration
Clear liquid 60L, content 118.0g/L;Ultrafiltration clear liquid is carried out nanofiltration ceramic membrane, the electrolyte for removing in conversional solution obtains concentrated phase
55.5L, content 115.9g/L, yield is 80.3%.Afterwards gained concentration feed liquid being carried out crystallization, to obtain alanyl glutamine thick
Product.
Embodiment 4
Using active immobilized enzyme(Enzyme activity 6405U) the conversional solution 28ml for completing is processed, conversional solution is brought rapidly up to 70 DEG C, when
Temperature adjusts pH to 5.5 after rising to preset temperature, is incubated 45min afterwards.Enzyme liquid 70L of inactivation is taken, content is 130.7g/L, to enter
Row pretreatment, removes macromolecular substances by microfiltration ceramic membrane and waits until that pottery cleaner liquid amounts to 62L, and content 126.4g/L, by pottery filter
Clear liquid carries out ultrafiltration ceramic membrane removal small-molecule substance and waits until that ultrafiltration clear liquid there are ultrafiltration clear liquid 55L, content 122.8g/L;Will be super
Cleaner liquid carries out nanofiltration ceramic membrane, obtains concentrated phase 51L, content 117.3g/L, and yield is 65.4%.Afterwards gained concentration feed liquid is entered
Row crystallization obtains alanyl glutamine crude product.
Claims (5)
1. a kind of method for extracting alanyl glutamine liquid from enzymatic conversion, it is characterised in that:Comprise the steps:
(1)By the conversional solution after the production alanyl glutamine gained inactivation of microbial enzyme conversion method by membrane aperture be 100~
The microfiltration ceramic membrane of 50nm, 0.02~0.1Mpa of control pressure removes macromolecular substances and obtains cleaner liquid of making pottery;
(2)Pottery cleaner liquid is passed through ultrafiltration ceramic membrane, is removed material of the molecular weight more than 1500 and obtain ultrafiltration clear liquid, control pressure
0.3~0.7Mpa of power;
(3)By ultrafiltration clear liquid by nanofiltration pottery membrance concentration, 0.5~1.2Mpa of control pressure, electrolyte and a large amount of water is removed, is obtained
To alanyl glutamine concentrated solution, crystallize, obtain alanyl glutamine crude product.
2. the method for extracting alanyl glutamine a kind of liquid from enzymatic conversion according to claim 1, it is characterised in that
Step(1)The middle temperature to after inactivation is 65~90 DEG C, add Sodium Chloride in the conversional solution of pH=5~9, adds Sodium Chloride quality
For the 3%~5% of conversional solution quality, lowered the temperature after Sodium Chloride dissolving completely, liquid temp to be transformed less than 60 DEG C after is led to
Cross microfiltration ceramic membrane.
3. the method for extracting alanyl glutamine a kind of liquid from enzymatic conversion according to claim 1, it is characterised in that
Step(1)Middle Sodium Chloride addition is the 3% of conversional solution quality.
4. the method for extracting alanyl glutamine a kind of liquid from enzymatic conversion according to claim 1, it is characterised in that
Step(1)Middle conversional solution is cooled to 20~25 DEG C, then pass through microfiltration ceramic membrane.
5. the method for extracting alanyl glutamine a kind of liquid from enzymatic conversion according to claim 1, it is characterised in that
Step(3)Middle nanofiltration pottery membrane aperture is 0.5~2 nanometer.
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Cited By (1)
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CN110885357A (en) * | 2019-12-20 | 2020-03-17 | 大连医诺生物股份有限公司 | Method for separating and purifying glutamine dipeptide by nanofiltration membrane |
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Application publication date: 20170222 |