CN110372773A - The production method of high-purity glutamine dipeptide - Google Patents
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Abstract
The production method of high-purity glutamine dipeptide, include the steps that the reaction solution to enzymatic glutamine dipeptide synthetic reaction post-processes, the post-processing include the steps that for Candida tropicalis mutant strain thallus being inoculated with cultivated in enzymatic glutamine dipeptide synthesis reaction solution except heteroacid；The deposit number of the Candida tropicalis mutant strain is: CGMCC No.17928.Whole process of the present invention does not need re-crystallization step more without adding crystal seed, wash brilliant agent, effectively simplifies and isolates and purifies and process for refining, saves production cost；Yeast can reuse three batches or more, and forage protein can be made in waste yeast, generate without a large amount of solid waste and waste water, and technique is environmentally protective.Entire production process is easy to control, and production process is pollution-free, and product purity height (99% or more), high income (95% or more) are suitble to heavy industrialization application.
Description
Background technique
Glutamine dipeptide is a kind of important amino acid parenteral nutrition agent, is mainly used in injection, powder needle, water needle, piece at present
In agent.Microbial enzyme method synthesis glutamine dipeptide has many advantages, such as that reaction condition is mild, low in the pollution of the environment, yield is high, at low cost, because
Many enterprises use α-aminoacidesters acyltransferase to prepare glutamine dipeptide raw material one after another in recent years for this.
The substrate that α-aminoacidesters acyltransferase synthetic method uses for the ester derivative of l-Alanine and glutamine,
Wherein the ester derivative of l-Alanine is easily degraded in the reaction system as l-Alanine (L-Ala), because its physicochemical property with
Product is close, to influence product yield and purity.In order to obtain high-purity glutamine dipeptide, usually adopted during separation and Extraction
Glutamine dipeptide crude product is refined with ion-exchange or recrystallization method.CN103623777A is adsorbed using hypersober
To decoloration, removal of impurities, the endotoxic effect of removal, hypersober includes white carbon black 30-45%, the black 50-65% of black wood charcoal, ion friendship
Resin 0.5-3% is changed, solid waste and waste water are more, and subsequent treatment cost is high.CN104163849A is set using continuous chromatography separation
Standby to carry out continuous chromatography separation impurity and inorganic salts, higher cost, the high cost of high-purity glutamine dipeptide is also to cause
Limited one of the reason of products application range.CN104480075A refines glutamine dipeptide using methanol, and product yield exists
85% or so, technique is relatively environment-friendly, but the addition of methanol also brings dissolvent residual risk.
From the point of view of the state of the art, in the preparation process of high-purity glutamine dipeptide, product purification technique also need more into
One step.
Summary of the invention
The purpose of the present invention is to provide one kind can the specific free l-Alanine (L- eliminated in production system
Ala), L-Glutamine (L-Gln), thus the method for improving glutamine dipeptide product purity.Based on Candida tropicalis
(Candida tropicalis) (CICC number 1253) is starting strain, obtains a plant mutant strain IBEC- by mutagenesis screening
16, which can specifically remove free amino acid L-Ala and L-Gln in reaction system, and in L-Ala and L-
Dipeptides is not utilized in the presence of Gln.Candida tropicalis mutant strain IBEC-16 is now preserved in the micro- life of China of BeiJing, China
Object culture presevation administration committee common micro-organisms center (CGMCC), deposit number are CGMCC No.17928, the deposit date is
On 06 16th, 2019.
Based on above-mentioned Candida tropicalis mutant strain, the present invention is intended to provide a kind of producer of high-purity glutamine dipeptide
Method, this method include the steps that the reaction solution to enzymatic glutamine dipeptide synthetic reaction post-processes, and the post-processing includes
Candida tropicalis mutant strain thallus is inoculated with cultivated in enzymatic glutamine dipeptide synthesis reaction solution except heteroacid step
Suddenly；The deposit number of the Candida tropicalis mutant strain is: CGMCC No.17928.
The production method of high-purity glutamine dipeptide provided by the present invention, using Candida tropicalis mutant strain specificity
Yeast is accessed in glutamine dipeptide reaction solution and is reacted by the characteristic for eliminating free amino acid L-Ala, L-Gln, filtrate after filtration sterilization
Desalination is filtered by film, condensing crystallizing, drying obtain glutamine dipeptide product.Whole process is without adding crystal seed, washing brilliant agent, less
Re-crystallization step is needed, effectively simplifies and isolates and purifies and process for refining, saves production cost；Yeast can reuse three batches
More than secondary, and forage protein can be made in waste yeast, generate without a large amount of solid waste and waste water, and technique is environmentally protective.It is whole
A production process is easy to control, and production process is pollution-free, and product purity height (99% or more), high income (95% or more) are fitted
Close heavy industrialization application.
Detailed description of the invention
3 width of attached drawing of the present invention:
Fig. 1 is consumption result figure of the candida tropicalis mutant strain IBEC-16 to L-Ala, L-Gln and glutamine dipeptide.
Fig. 2 is utilization power figure of the candida tropicalis mutant strain IBEC-16 to L-Ala, L-Gln and glutamine dipeptide.
Fig. 3 is candida tropicalis CICC NO.1253 and its mutant strain IBEC-16 to l-Alanine and glutamine benefit
With situation difference test result.
Specific embodiment
A kind of production method of the present invention about high-purity glutamine dipeptide is suitable for enzymatic (especially microbial enzyme) and synthesizes
The processing of reaction system after glutamine dipeptide, the step post-processed including the reaction solution to enzymatic glutamine dipeptide synthetic reaction
Suddenly, the post-processing include Candida tropicalis mutant strain thallus is inoculated in enzymatic glutamine dipeptide synthesis reaction solution into
The step of removing heteroacid of row culture；The deposit number of the Candida tropicalis mutant strain is: CGMCC No.17928.
(it is also referred to as reaction solution herein) in the reaction solution of the enzymatic glutamine dipeptide synthetic reaction, has the residual of heteroacid
It stays, the heteroacid includes l-Alanine (L-Ala) and/or L-Glutamine (L-Gln).The specific implementation of this case is to remove this
Two kinds of heteroacid.
More specifically in embodiment, described is the step of removing heteroacid: by enzymatic glutamine dipeptide synthesis reaction solution
PH is adjusted to 6.0, accesses Candida tropicalis mutant strain thallus, makes initial OD=8~12；Then in 30 ± 2 DEG C, 300~
It is cultivated under the conditions of 500rpm, incubation controls dissolved oxygen amount (DO) 30~50%, pH value 5.5~6.0；After culture, centrifugation
And filtrate and thallus are collected respectively.In the reaction process, the content of amino acid and glutamine dipeptide is surveyed in sampling per hour, until reaction
Completely, it is centrifuged, collection filtrate and thallus, thallus recycle and reuse respectively.Wherein, the fully reacting refers to L- in system
Alanine content is consumed to 0.01g/L (concentration) below.The content detection of amino acid and glutamine dipeptide is referring to Chinese Pharmacopoeia
The two annex V D high performance liquid chromatographies of version in 2010.
Candida tropicalis mutant strain thallus described in above-mentioned any technical solution is prepared by following methods: taking one
Candida tropicalis mutant strain (IBEC-16) access of ring activation is placed in 30 equipped in the conical flask of 50ml YPD culture medium
DEG C, cultivate about 16h in 220rpm shaking table；Then 1ml culture solution is taken to access in another conical flask for filling 50ml YPD culture medium,
It is placed in 30 DEG C, cultivates 20h or so in 220rpm shaking table, thalline were collected by centrifugation.
The activation culture for the Candida tropicalis mutant strain (IBEC-16) that inclined-plane saves uses YPD culture medium, specific side
Formula is: taking in 100 μ L glycerol pipes in bacterium solution access YPD culture medium, is placed in 30 DEG C, cultivates about 16h culture in 220rpm shaking table, draw
Picking monoclonal access inclined-plane saves after line culture.
Unless otherwise specified, heretofore described and YPD culture medium it is equal are as follows: glucose 20g/L, yeast extract 10g/L, egg
White peptone 20g/L, 121 DEG C of sterilizing 20min.
In the application of heretofore described Candida tropicalis mutant strain, thallus may be reused.Described
The method that thallus is reused is identical as using for the first time, with a batch thallus reusable 3 times.
It further include to except filter obtained after heteroacid step after the reaction of heteroacid in further specific embodiment
Liquid carries out the step of nanofiltration membrane desalination and evaporative crystallization.Specifically, it can depressurize and steam through molecular cut off 400Da nanofiltration membrane desalination
It evaporates to a large amount of solids and is precipitated, cooling filtering, solid obtains glutamine dipeptide sterling in 50 DEG C of decompression dryings.
More specifically, method of the present invention includes following processing steps to glutamine dipeptide reaction solution:
(1) heteroacid is removed
By the reaction solution tune pH to 6.0 of enzymatic glutamine dipeptide synthetic reaction, Candida tropicalis mutant strain thallus is accessed,
Make initial OD=10；Then it is cultivated under the conditions of 30 ± 2 DEG C, incubation control dissolved oxygen amount (DO) 30~50%, pH value 5.5~
6.0；After culture, it is centrifuged and collects filtrate and thallus respectively；
(2) desalination, crystallization
Filtrate obtained by step (1) is precipitated through molecular cut off 400Da nanofiltration membrane desalination, vacuum distillation to a large amount of solids, drop
Temperature filtering, solid obtain glutamine dipeptide sterling in 50 DEG C of decompression dryings.
On the other hand, for glutamine dipeptide reaction solution, the present invention is not particularly limited, this is in those skilled in the art
It can choose and determine.The reaction solution of glutamine dipeptide can be it is obtained according to the correlation technique in existing record,
Reaction step before being coupled to processing step of the invention, non-limiting citing are as follows:
Enzymatic reaction synthesizes glutamine dipeptide: l-Alanine methyl ester hydrochloride and L-Glutamine are dissolved in phosphate buffer
In, glutamine dipeptide synzyme is added, carries out enzymatic reaction under the conditions of 25-30 DEG C, pH value 8.0-9.0, terminates after twenty minutes anti-
It answers, collects reaction solution；Wherein: the concentration of reaction substrate l-Alanine methyl ester hydrochloride is 200-300mM；Reaction substrate L- paddy ammonia
The concentration of amide is 200-300mM；The concentration of glutamine dipeptide synzyme is 0.1-1.0% (v/v)；Phosphate buffer 0.2M,
pH8.7；The control of enzymatic reaction process pH value uses 6M sodium hydroxide solution.
The reaction solution obtained after above-mentioned reaction can be directly used for of the present invention except heteroacid post-processing step.
The contents of the present invention are further described with reference to the accompanying drawings and embodiments.These non-limiting embodiments are not
It should be understood as to any form of restriction of the content of present invention.
1. saccharomycete of embodiment obtains and culture
With candida tropicalis (CICC number 1253) for starting strain, a plant mutant strain is obtained by Uv-induced screening
IBEC-32, which can specifically remove L-Ala and L-Gln, and not utilize in the presence of L-Ala and L-Gln
Glutamine dipeptide.
Used culture medium composition is as follows:
Slant medium: glucose 20g/L, yeast extract 10g/L, peptone 20g/L, 15-20g/L agar, 121 DEG C of sterilizings
20min。
YPD culture medium: glucose 20g/L, yeast extract 10g/L, peptone 20g/L, 121 DEG C of sterilizing 20min.
Shake flask medium: glutamine dipeptide 20g/L, l-Alanine 20g/L, glutamine 10g/L, KH2PO4·3H2O2.5g/
L, MgSO4·7H2O 1.0g/L, KCl 0.5g/L, pH6.0.
1, mutant strain IBEC-16 Breeding Process, includes the following steps:
(1) preparation of mutant strain IBEC-16 bacteria suspension
Candida tropicalis (the CICC number 1253) access for taking a ring to activate is equipped with the conical flask of 50ml YPD culture medium
In, be placed in 30 DEG C, in 220rpm shaking table culture to logarithmic growth phase.Take culture solution in sterile centrifugation tube, 8000rpm centrifugation
10min discards supernatant liquid, washs centrifugation again using physiological saline, vibrates 10min after cell is resuspended using physiological saline, adjusts
Whole cell concentration is 1 × 108A/ml is mutant strain IBEC-16 bacteria suspension.
(2) ultraviolet mutagenesis and domestication
It takes bacteria suspension 5mL obtained by step (1) to be added in sterilized petri dishes, is placed on magnetic stirring apparatus and stirs, in 20W ultraviolet light
30~180s is irradiated at lower 30cm, and samples dilution spread plate, is selected and is growed single colonie fast, that bacterium colony is big and be inoculated in inclined-plane training
Base is supported, inclined-plane preservation is carried out.
Bacterial strain access on a ring inclined-plane is taken to be placed in 30 DEG C, 220rpm shaking table equipped in the conical flask of 50ml YPD culture medium
Then middle culture is accessed in domestication culture medium, the bacterial strain for selecting tolerance l-Alanine maximum concentration is used to logarithmic growth phase
In shaking flask secondary screening.
(3) shaking flask secondary screening
It takes the bacterial strain access that inclined-plane saves in a ring step (2) equipped in the conical flask of 50ml YPD culture medium, is placed in 30
DEG C, cultivate 20h in 220rpm shaking table, thalline were collected by centrifugation, accessed in Shake flask medium, 30 DEG C, 220rpm culture 24 it is small
When, it samples per hour and controls pH6.0 in incubation or so, contained using various amino acid in liquid-phase chromatographic analysis reaction solution
Amount.In all detection bacterial strains, the mutant strain IBEC-16 that the most bacterial strain of l-Alanine is target is consumed for 24 hours.
(4) detection of mutant strain IBEC-16 genetic stability
The candida tropicalis IBEC-16 that step (3) are obtained is passed on 10 times on continuous inclined-plane, and every generation is inoculated with respectively
One ring is placed in 30 DEG C, then culture is transferred into shaking to logarithmic growth phase in 220rpm shaking table in the conical flask of YPD culture medium
In bottle culture medium, 30 DEG C, 220rpm shaker fermentation for 24 hours, sample per hour in incubation and control pH6.0 or so, measurement is anti-
Answer amino acid content in liquid.It is compared simultaneously with the Candida IBEC-16 in 1 generation.
Embodiment 2. obtains high-purity glutamine dipeptide using mutant strain IBEC-16
Glutamine dipeptide reaction solution used in the present embodiment is obtained by following methods: according to " Production of
L-alanyl-L-glutamine by recycling E.coli expressing a-amino acid ester
Method documented by an acyltransferase " text prepares glutamine dipeptide reaction solution, " Bioresource technology ",
2017。
The mutant strain IBEC-16 access for taking a ring to activate is placed in 30 DEG C, 220rpm equipped in the conical flask of YPD culture medium
20h is cultivated in shaking table, thalline were collected by centrifugation.By reaction solution (41.9g/L containing glutamine dipeptide, alanine 4.3g/L, glutamine
PH to 6.0 7.8g/L) is adjusted, access thallus (reaction initial OD=10 or so) is cultivated, DO under the conditions of 30 DEG C, 300-500rpm
Control is in 30-50%, and in 5.5-6.0, the content of amino acid and glutamine dipeptide, about 8-10h knot are surveyed in sampling per hour for pH control
Filtrate is collected by centrifugation in beam.Filtrate is through nanofiltration desalination, and to there is a large amount of solids to be precipitated, cooling filtering, solid subtracts at 50 DEG C for vacuum distillation
Pressure dries to obtain glutamine dipeptide sterling 40g, yield 95%, purity 99%.
Degrading experiment of the 3. mutant strain IBEC-16 of embodiment to different aminoacids
Take a ring activate mutant strain IBEC-16 access equipped with 50ml YPD culture medium conical flask in, be placed in 30 DEG C,
20h is cultivated in 220rpm shaking table.Thalline were collected by centrifugation, access Shake flask medium (glutamine dipeptide 20g/L, l-Alanine 20g/L,
Glutamine 10g/L, KH2PO4·3H2O 2.5g/L, MgSO4·7H2O 1.0g/L, KCl 0.5g/L) in, 30 DEG C, 220rpm
Culture 24 hours, samples per hour and controls pH5.5-6.0, using the third ammonia of L- in liquid-phase chromatographic analysis reaction solution in incubation
The content of acid, glutamine and glutamine dipeptide.As a result as shown in Figure 1.As seen from Figure 1, glutamine is in 6h or so by complete benefit
With l-Alanine is then degradable in 20h or so, and the content of glutamine dipeptide is basically unchanged.
Mutant strain IBEC-16 application test in 3. fermentor of embodiment
Glutamine dipeptide reaction solution used in the present embodiment is obtained by following methods: according to " Production of
L-alanyl-L-glutamine by immobilized Pichia pastoris GS115expressing a-amino
Method documented by an acid ester acyltransferase " text prepares glutamine dipeptide reaction solution, " Microbial Cell
Factories ", 2019.
The mutant strain IBEC-16 access for taking a ring to activate is placed in 30 DEG C, 220rpm equipped in the conical flask of YPD culture medium
20h is cultivated in shaking table；It is initial ventilatory capacity 0.7VVM in 10% 50L fermentor of the access containing YPD culture medium by inoculum concentration,
It is cultivated under the conditions of 30 DEG C, 150-400rpm, DO control, 5.0, cultivates 6-8h or so in 20%, pH control.300L is reacted
Liquid (60g/L containing glutamine dipeptide, alanine 12.5g/L, glutamine 4.5g/L) adjusts pH to 6.0, access thallus (reaction initial OD
=10 or so), initial ventilatory capacity 0.7VVM is cultivated under the conditions of 30 DEG C, 150-350rpm, and DO control is controlled in 30-50%, pH
In 5.5-6.0, the content of sample detection amino acid and glutamine dipeptide per hour.From the point of view of Fig. 2, glutamine and l-Alanine point
Not in 4h, 12h or so using completely.Filtrate is collected by centrifugation after reaction, for filtrate through nanofiltration desalination, vacuum distillation is a large amount of to having
Solid is precipitated, and cooling filtering, solid obtains glutamine dipeptide sterling 58.2g, yield 97%, purity 99% in 50 DEG C of decompression dryings.
The recycling test of 4. mutant strain IBEC-16 thallus of embodiment
Glutamine dipeptide reaction solution used in the present embodiment is obtained by following methods: according to " Production of
L-alanyl-L-glutamine by recycling E.coli expressing a-amino acid ester
Method documented by an acyltransferase " text prepares glutamine dipeptide reaction solution, " Bioresource technology ",
2017。
The mutant strain IBEC-32 access for taking a ring to activate is placed in 30 DEG C, 220rpm equipped in the conical flask of YPD culture medium
20h is cultivated in shaking table, thalline were collected by centrifugation.By reaction solution tune pH to 6.0, thallus (reaction initial OD=10 or so) are accessed,
30 DEG C, cultivate under the conditions of 300-500rpm, DO control is in 30-50%, and pH control is in 5.5-6.0, and amino acid is surveyed in sampling per hour
With the content of glutamine dipeptide, filtrate is collected by centrifugation.Filtrate cooled down through nanofiltration desalination, vacuum distillation to there is a large amount of solids to be precipitated
Filter, solid obtain glutamine dipeptide sterling in 50 DEG C of decompression dryings.Recycling yeast thallus is reused with reference to above-mentioned steps, is recycled
It see the table below 1 using yeast removal heteroacid result.
Table 1
5. candida tropicalis CICC NO.1253 of embodiment and mutant strain IBEC-16 are to l-Alanine and glutamine
Utilize difference test
The candida tropicalis CICC NO.1253 and mutant strain IBEC-16 for taking a ring to activate are respectively connected to equipped with 100ml
In the conical flask of YPD culture medium, it is placed in 30 DEG C, cultivates 20h in 220rpm shaking table.Thalline were collected by centrifugation, is then respectively connected to anti-
Answer liquid (glutamine dipeptide 20g/L, l-Alanine 20g/L, glutamine 10g/L, KH2PO4·3H2O2.5g/L, MgSO4·7H2O
1.0g/L, KCl 0.5g/L) in, react initial OD620=10 or so, 30 DEG C, 220rpm culture 24 hours, pH controls 5.5-6.0
Left and right samples for every eight hours and using the content of amino acid in liquid-phase chromatographic analysis reaction solution.As a result as shown in Figure 3.
From the point of view of Fig. 3 result, two plants of bacterium will not utilize glutamine dipeptide (Ala-Gln) substantially, and to l-Alanine (L-Ala)
It is different with the producing level of glutamine (Gln).Two plants of bacterium all can be first with glutamine, the utilization of mutant strain IBEC-16
Speed ratio candida tropicalis CICC NO.1253 is slightly fast；L- can not be fully utilized in starting strain CICC NO.1253 culture for 24 hours
Alanine, and 20g/L or so can be then fully utilized in mutant strain IBEC-16, improve to the utilization efficiency of l-Alanine
100%.
Claims (7)
1. the production method of high-purity glutamine dipeptide is post-processed including the reaction solution to enzymatic glutamine dipeptide synthetic reaction
Step, which is characterized in that the post-processing includes that Candida tropicalis mutant strain thallus is inoculated with into enzymatic glutamine dipeptide
Cultivated in synthesis reaction solution except heteroacid the step of；
The deposit number of the Candida tropicalis mutant strain is: CGMCC No.17928.
2. the method according to claim 1, wherein the heteroacid includes l-Alanine and/or L- glutamy
Amine.
3. according to the method described in claim 2, it is characterized in that, described is the step of removing heteroacid: by enzymatic glutamine dipeptide
The pH of synthesis reaction solution is adjusted to 6.0, accesses Candida tropicalis mutant strain thallus, makes initial OD=8~12；Then in 30 ±
2 DEG C, cultivate under the conditions of 300~500rpm, incubation controls dissolved oxygen amount 30~50%, pH value 5.5~6.0；After culture,
It is centrifuged and collects filtrate and thallus respectively.
4. according to the method described in claim 3, it is characterized in that, the thallus is reused.
5. according to the method described in claim 3, it is characterized in that, the post-processing further includes to except being obtained after heteroacid step
The filtrate obtained carries out the step of nanofiltration membrane desalination and evaporative crystallization.
6. according to the method described in claim 2, it is characterized by comprising the following steps:
(1) heteroacid is removed
By the reaction solution tune pH to 6.0 of enzymatic glutamine dipeptide synthetic reaction, Candida tropicalis mutant strain thallus is accessed, is made just
Beginning OD=10；Then it is cultivated under the conditions of 30 ± 2 DEG C, incubation controls dissolved oxygen amount 30~50%, pH value 5.5~6.0；Culture
After, it is centrifuged and collects filtrate and thallus respectively；
(2) desalination, crystallization
Filtrate obtained by step (1) is precipitated, cooled down through 400 Da nanofiltration membrane desalination of molecular cut off, vacuum distillation to a large amount of solids
Filter, solid obtain glutamine dipeptide sterling in 50 DEG C of decompression dryings.
7. the method according to claim 1, wherein under the Candida tropicalis mutant strain thallus passes through
State method preparation: the Candida tropicalis mutant strain thallus access for taking a ring to activate is equipped with the conical flask of 50ml YPD culture medium
In, it is placed in 30 DEG C, cultivates about 16h in 220rpm shaking table；Then 1ml culture solution is taken to access another 50ml YPD culture medium of filling
In conical flask, it is placed in 30 DEG C, cultivates 20h or so in 220rpm shaking table, thalline were collected by centrifugation.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113817790A (en) * | 2021-10-18 | 2021-12-21 | 精晶药业股份有限公司 | Method for preparing alanyl glutamine by immobilized enzyme |
| CN113817790B (en) * | 2021-10-18 | 2023-11-03 | 精晶药业股份有限公司 | Method for preparing alanyl glutamine by immobilized enzyme |
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| CN110372773B (en) | 2022-06-10 |
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