CN104120101B - A kind of E. coli mutant strain and the method producing chiral aspartic acid using this bacterium - Google Patents
A kind of E. coli mutant strain and the method producing chiral aspartic acid using this bacterium Download PDFInfo
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Abstract
The invention discloses one plant is used for the coli strain HT ME that waste water recycling process produces aspartic acid, deposit number is CGMCC No.9288.Due to all not salt tolerants of the escherichia coli in existing aspartic acid production technology, Facultative Halophiles HT ME provides a preferable microorganism resource for the improvement of aspartic acid production technology.Using HT ME bacterial strain, waste water recycling transformation is carried out to existing aspartic acid production technology, fermentation waste water discharge capacity can be made to decline 2/3rds, pollution can be reduced and reduce production cost.
Description
Technical field
The invention belongs to microbial technique and field of fermentation engineering, specifically, be related to a kind of E. coli mutant strain and
Carry out the method that waste water recycling process produces chiral aspartic acid using this bacterial strain.
Background technology
Aspartic acid (Aspartic acid) also known as aminosuccinic acid, be constitute body protein primary amino acid it
One.Structural formula is:HOOC-CH2-CH (NH2)-COOH, molecular weight 133.10.It has two kinds of optical isomers:D type and L-type.Logical
Often described aspartic acid refers to the D type of equivalent and the mixture of L-type, is the prismatic crystallization of colourless monocline, is dissolved in water, is slightly soluble in
75% (mass fraction) ethanol, insoluble in ether.
Aspartic acid is one of acidic amino acid, belongs to non essential amino acid, in vivo by glutamic acid rotating ammonia to oxaloacetic acid
And obtain, decompose and then oxaloacetic acid or the generation butene dioic acid entrance tricarboxylic of the effect deamination through Aspartase are produced by deaminizating
Acid circulation.It is the conjunction of the biological internal aminoacid such as lysine, threonine, isoleucine, Methionine and purine, pyrimidine bases
Become precursor.It can convey electrolyte as the carrier Cardiomyocytes of potassium ion, magnesium ion, thus improving myocardium shrinkage function, simultaneously
Reduce oxygen consumption, in coronary artery circulation obstacle anoxia, have protective effect to cardiac muscle.It participates in ornithine cycle, promotes ammonia
Generate carbamide with carbon dioxide.Reduce the content of ammonia and carbon dioxide in blood, strengthen liver function, allaying tiredness.
The synthesis of aspartic acid mainly has chemical method and two kinds of enzymatic clarification at present.Chemical method is mainly for the production of Radix Asparagi ammonia
The racemic modification DL- aspartic acid of acid.In recent years, Poland, Japan, Germany are with through maleic anhydride (maleic anhydride) and ammonification
Agent directly carries out additive reaction and synthesizes aspartic acid.This method have that raw material is easy to get, process is simple, reaction condition is gentle and
The features such as product is easily separated, is suitable for large-scale production.
Machowski S of Poland et al. is reacted with NH4Cl with maleic anhydride and excessive ammonia, and reaction is in an interval
In reactor, react 2.5h under 140 DEG C, 0.4MPa.The mixture obtaining is cooled to after 70 DEG C and is acidified to hydrochloric acid
PH2.5, is cooled to room temperature, now aspartic acid Precipitation, and precipitation is filtered, and obtains product, yield is at 4 DEG C after washing
55%.
2002, the Xing second place of Zhejiang University waited in a titanium autoclave, to maleic anhydride) carry out aminating reaction
Synthesize DL- aspartic acid.Improvement has been made to original synthesis technique by experimentation, make product yield bring up to 70% with
On, and replace hydrochloric acid to carry out acidizing crystal using maleic acid (maleic acid) thus improve anti-product separation purification
Answer the utilization of thing and the pollution reducing reaction residual.
Wang Yaquan of University Of Tianjin in 2003 etc. proposes a kind of simplicity, chemosynthesis that are not using any catalyst
Method.Experiment is carried out in the 250ml autoclave of polytetrafluoro lining.Experimental procedure is as follows：By (the analysis of 32.5g maleic anhydride
Pure) it is dissolved in 70ml water, add ammonia (analysis is pure) to pH8, to fill 1MPa nitrogen, then under electromagnetic agitation, be heated to 453K,
Reaction 6h, is cooled to room temperature after reaction, obtain clear crystal, yield 63.1%, 338 DEG C of fusing point after filtration.The infrared spectrum of product
It coincide with the standard spectrogram of DL- aspartic acid, volumetric analysiss show aspartate content >=98%.
And enzymatic clarification is the main method of the industrial synthesis left-handed monomer of aspartic acid at present, this method not only yield
High and optical purity is high.Enzyme process is to occur additive reaction to obtain with aspartic acid enzyme catalysiss ammonia and fumaric acid, and its reaction equation is such as
Shown in lower：
Biological enzyme can be divided into IC method and FWC method again.IC method is immobilized cell reaction or immobilized enzyme method technique, Ji Jiangtian
Winter propylhomoserin enzyme is prepared as immobilized enzyme or the escherichia coli with aspartic acid enzyme activity is prepared as immobilized cell, by enzyme
Promote reaction to promote to be converted into the industrialized production of L-Aspartic acid by fumaric acid.And produce L-Aspartic acid with immobilized cell reaction
Compare, free whole cell method has higher work vigor and production capacity, technological process is more succinct, the equipment of needs is thrown
Money is less, thus production cost is lower, has the stronger market competitiveness.Free whole cell method produces the master of L-Aspartic acid
The flow process is wanted to be：With maleic acid as raw material, generate fumaric acid, ammonification under the catalytic action with ammonium persulfate for the sodium bromide
Afterwards with E. coli broth for enzyme source in escherichia coli bioreactor, generate L-Aspartic acid through enzymatic reaction,
Adjust isoelectric point, IP by persalt to reclaim, waste liquid is then discharged and carried out harmless treatment.If technique is improved, with anti-butylene two
Acid instead of HCl carries out isoelectric point, IP regulation, reclaims thin stillage recycling after aspartic acid, adds after fumaric acid and catalyst again
Carry out new round production, then wastewater discharge will greatly reduce.The key of technologic improvement is selection-breeding tolerance reuse waste liquid accumulation
The escherichia coli of the salinities such as sodium bromide.
In current aspartic acid production technology, existing engineered strain is not good to the tolerance degree of salinity, in reaction system
Sodium bromide can have inhibitory action to enzymatic reaction with the accumulation of ammonium persulfate, therefore thin stillage recycling cannot be caused discharging of waste liquid
Amount is big, with serious pollution problem.
The present invention is positioned the process modification of aspartic acid production, using microbial technique pointedly selection-breeding salt tolerant large intestine
Bacillus, and the bacterial strain obtaining is analyzed and has applied.
Content of the invention
It is an object of the present invention to provide a kind of salt tolerant E. coli mutant strain, can be used for waste water recycling process and produce sky
Winter propylhomoserin.
Inventor separates from the cesspool of Anhui aspartic acid manufacturing enterprise, identification obtains one plant of salt tolerant escherichia coli
Bacterial strain.Multi irradiation mutagenesis screening is carried out to this bacterial strain, finally obtains one plant of mutant HT- with high aspartase activity
ME.This bacterial strain has been preserved in China General Microbiological culture presevation administrative center, and Classification And Nomenclature is Escherichia coli, protects
Hiding numbering is CGMCC No.9288, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Chinese Academy of Sciences microbe research
Institute (100101), preservation date is on June 9th, 2014.
Through identification of morphology and biological characteristic research, preserving number is that the bacterial strain of CGMCC No.9288 has following characteristics：
(1), bacterial strain HT-ME required growth conditionss in PYG culture medium：Temperature 10-40 DEG C (the most suitable 35 DEG C), pH3.5-
8.5 (the most suitable 6.5), salinity 0-8% (w/v, the most suitable 0%).
(2), bacterial strain HT-ME is gram negative bacilli, size 0.5-1.0 × 2-10(μm).
(3), in PYG culture medium bacterial strain HT-ME for when be about 0.2h.
(4), bacterial strain HT-ME is to penicillin, vancomycin, streptomycin, erythromycin, chloromycetin, kanamycin, neomycin etc.
Multiple Classes of Antibiotics is sensitive.
Wherein, be preferably used in pass on, the PYG medium standard formula of preservation strain as follows：10g peptone, 10g yeast
Cream, 5g glucose, 40ml saline solution (0.25g calcium chloride, 0.5g magnesium chloride, 1g dipotassium hydrogen phosphate, 1g potassium dihydrogen phosphate, 10g carbon
Sour hydrogen sodium, 2g sodium chloride, ultrapure water dissolution, it is settled to 1L), ultrapure water dissolution is settled to 1L, pH=7.
For the 16S rRNA gene of CGMCC No.9288 bacterial strain, performing PCR amplification and sequencing are entered to preserving number, records
16S rRNA Gene Partial fragment length is 1465bp (Seq ID No.1), the 16S rRNA sequence of CGMCC No.9288 bacterial strain
It is specifically shown in sequence table.Bacterial strain CGMCC No.9288 is similar to the 16S rRNA gene order of known escherichia coli reference culture
Property be 96.9%, DNA hybridization rate (Tm method) be 80.3%, there is the salt-tolerant trait that reference culture does not possess, therefore, bacterial strain
CGMCC No.9288 is the mutant of escherichia coli reference culture.
It is a further object of the present invention to provide a kind of method producing chiral aspartic acid using this bacterial strain.
The method that utilization E. coli mutant bacterial strain of the present invention produces chiral aspartic acid, including following step
Suddenly：
(1), with maleic acid as raw material, generate fumaric acid under the catalytic action with Ammonium persulfate. for the sodium bromide and sink
Form sediment, after ammonification, make resolution of precipitate；
(2), there is aspartase activity fermentation liquid as enzyme using obtain after the culture of CGMCC No.9288 strain fermentation
Source is mixed with the solution of step (1), generates L-Aspartic acid through enzymatic reaction；
(3), adjust pH to acid, reclaim target product L-Aspartic acid.
In methods described step (1), for temperature control at 50-80 DEG C, sodium bromide dosage is suitable to preferred reaction condition
The 0.5-5% of butene dioic acid quality, the dosage of Ammonium persulfate. is the 0.5-5% of maleic acid.Preferred reaction condition
For temperature control at 60-75 DEG C, sodium bromide dosage is the 1-2.5% of maleic acid quality, and the dosage of Ammonium persulfate. is
The 1.5-3% of maleic acid, centrifugation after having reacted obtains fumaric acid solid.Most preferably reaction condition is in 75 DEG C of water
Under the conditions of bath, first add the sodium bromide of maleic acid quality 1.5%, then add the over cure of maleic acid quality 2.6%
Sour ammonium, centrifugation after having reacted obtains fumaric acid solid.
In methods described step (2), preferred enzymatic reaction condition is to stir 1-6 hour, preferred enzyme at 20-40 DEG C
Promoting reaction condition is to stir 2-4 hour at 30-35 DEG C.
In methods described step (2), CGMCC No.9288 inoculation is the fermentation culture of 68 (preferably 7.2) in pH value
In base, culture 12-36 hour (preferably 24 hours) at a temperature of 15 35 DEG C (preferably 30-32 DEG C), obtain and there is Aspartase
The fermentation liquid of activity.Fermentation medium may be selected Escherichia coli fermentation culture medium generally in the art, such as LB, Carnis Bovis seu Bubali cream albumen
Peptone culture medium etc., a preferred fermentation medium composition is as follows：10g peptone, 10g yeast extract, 5g glucose, 40ml salt is molten
Liquid (0.25g calcium chloride, 0.5g magnesium chloride, 1g dipotassium hydrogen phosphate, 1g potassium dihydrogen phosphate, 10g sodium bicarbonate, 2g sodium chloride, ultrapure
Water dissolution, is settled to 1L), ultrapure water dissolution is settled to 1L, pH=7.
The fermentation liquid with aspartase activity is added in the reaction solution of step (1), the amount of being preferably added is
0.1 5 times of step (1) reaction solution volume, preferably 0.5 3 times, more preferably 1 1.5 times.
In methods described step (3), preferred reaction condition, by solution pH regulator 1.0-6.0, preferably adjusts pH to 2.5-
3.0 between.Standing, to be precipitated completely after be filtrated to get L-Aspartic acid crude product.Preferably carry out at lower than room temperature when adjusting pH.
The reagent of regulation pH used is common mineral acid or organic acid, example hydrochloric acid, sulphuric acid, phosphoric acid, acetic acid, citric acid, maleic
Diacid, hydroxymethane sulfonic acid, hydrobromic acid, methanesulfonic acid, acetic acid, trifluoracetic acid, maleic acid, benzenesulfonic acid, toluenesulfonic acid, sulfamic acid,
Glycolic, stearic acid, lactic acid, malic acid, flutter acid, p-aminobenzene sulfonic acid, 2 one acetoxy-benzoic acids, fumaric acid, toluenesulfonic acid,
Methanesulfonic acid, ethane disulfonic acid, oxalic acid, hydroxyethylsulfonic acid. etc. or its mixture, preferably hydrochloric acid or maleic acid.
It is a further object of the present invention to provide one kind utilizes this bacterial strain to produce chiral aspartic acid for waste water recycling process
Method.
Aforesaid utilization enzymatic clarification produces L-Aspartic acid technique, and reaction inevitably produces waste liquid.Existing
Engineered strain is not good to the tolerance degree of salinity, and sodium bromide and the accumulation of ammonium persulfate in reaction system can have suppression to enzymatic reaction
Make and use, therefore discharging of waste liquid amount cannot be caused big, with serious pollution problem thin stillage recycling.And the ability of selection-breeding of the present invention
The escherichia coli CGMCC No.9288 of the salinities such as the sodium bromide being accumulated by reuse waste liquid can preferably solve this difficult problem, can be right
Prior art processes are improved, and by thin stillage recycling, carry out new round production again, then after adding maleic acid and catalyst
Wastewater discharge will greatly reduce.
Therefore, present invention also offers a kind of method producing chiral aspartic acid for waste water recycling process, including such as
Lower step：
(1), recycle the waste liquid that enzymatic clarification aspartic acid produces, add maleic acid, and catalyst bromination
Sodium and Ammonium persulfate. generate fumaric acid precipitation, make resolution of precipitate after ammonification；
(2), using obtain after the culture of CGMCC No.9288 strain fermentation have the fermentation liquid of aspartase activity as
Enzyme source is mixed with the solution of step (1), generates L-Aspartic acid through enzymatic reaction；
(3), adjust pH to acid with maleic acid, reclaim target product L-Aspartic acid.
The waste liquid that this technique produces can continue to be circulated waste water recycling using said method step, until waste water accumulation
Till total salt concentration is no longer appropriate for CGMCC No.9288 growth.
In methods described step (1), the maleic acid final concentration added to more than 100g/L, preferably to 500-700g/L
More than.Preferably for temperature control at 50-80 DEG C, sodium bromide dosage is the 0.1-2% of maleic acid quality to reaction condition,
The dosage of Ammonium persulfate. is the 0.1-2% of maleic acid.Preferred reaction condition be temperature control at 60-75 DEG C, bromine
Change the 0.1-0.5% that sodium dosage is maleic acid quality, the dosage of Ammonium persulfate. is the 0.2-1% of maleic acid,
After having reacted, centrifugation obtains fumaric acid solid.Most preferably reaction condition is under 75 DEG C of water bath condition, first adds along fourth
The sodium bromide of enedioic acid quality 0.15%, then adds the Ammonium persulfate. of maleic acid quality 0.26%, centrifugation after having reacted
Obtain fumaric acid solid.
In methods described step (2), preferred enzymatic reaction condition is to stir 1-6 hour, preferred enzyme at 20-40 DEG C
Promoting reaction condition is to stir 2-4 hour at 30-35 DEG C.The condition of CGMCC No.9288 strain fermentation culture is as previously mentioned.
In methods described step (3), preferred reaction condition is to adjust pH to 1.0-6.0 with maleic acid, preferably adjusts
Between section pH to 2.5-3.0.Solution left standstill, to be precipitated completely after be filtrated to get L-Aspartic acid crude product.Preferably exist when adjusting pH
Low carry out at room temperature.
The present invention cultivates and obtains one plant of mutant HT-ME (CGMCC No.9288) with high aspartase activity.Greatly
Enterobacteria HT-ME requires simple to growth conditionss, and growth is rapid, 15-45 DEG C, pH3.0-8.0, salinity 0-8% (w/v) scope
Interior equal can growth, the best growing condition about 35 DEG C, pH6.5, salinity 0% about.HT-ME bacterial strain produces work for aspartic acid
Skill improvement provides a preferable microorganism resource, overcomes in current aspartic acid production technology conventional project bacterial strain to salinity
Tolerance degree is good, in reaction system the accumulation of sodium bromide and Ammonium persulfate. inhibitory action can be had therefore cannot enzymatic reaction
By thin stillage recycling, cause that discharging of waste liquid amount is big, with serious pollution shortcoming.HT-ME bacterial strain salt tolerant scope is relatively broad, also protects simultaneously
Hold preferable fumaric acid-aspartic acid conversion ratio.Carry out waste water using HT-ME bacterial strain to existing aspartic acid production technology to return
With transformation so that fermentation waste water discharge capacity declines 2/3rds, decrease pollution, reduce production cost, thus can produce
Good economic results in society.
Specific embodiment
Embodiment 1 carries out small-scale fermenting and producing aspartic acid using HT-ME bacterial strain
(1), with 680g maleic acid for material dissolution in 1 liter of water, in 6.8g sodium bromide and 13.6g ammonium persulfate
Fumaric acid precipitation is generated, centrifugation obtains precipitation, plus makes resolution of precipitate obtain anti-fourth after 1L22% strong aqua ammonia under catalytic action
Enedioic acid ammonia solution；
(2), by CGMCC No.9288 bacterial strain with PYG culture medium (pH7.0) at 35 DEG C, dissolved oxygen 4ppm, fermentation culture 24
Obtain after hour has aspartase activity fermentation liquid as enzyme source, mixes with the solution even of step (1), 35 DEG C of stirrings
Incubation 3 hours, generates L-Aspartic acid through enzymatic reaction；
(3), it is adjusted with the regulations speed of 0.5pH per minute with 8% hydrochloric acid under 10 DEG C of cryogenic conditions, until pH=
2.5, subsequently stand and filter within 6 hours to obtain aspartic acid crude product.The conversion ratio of reaction is 98.6%
Embodiment 2 carries out waste water recycling using HT-ME bacterial strain and produces chiral aspartic acid
(1), filter the solution after aspartic acid crude product with the waste liquid of embodiment 1 step (1) catalytic reaction and step (3)
Mixing carries out waste water recycling process, adds maleic acid to final concentration of 700g/L, then add 1.3g sodium bromide with
2.8g Ammonium persulfate., generates fumaric acid precipitation under its catalytic action, and centrifugation obtains precipitation, makes to sink after the amount of filling up strong aqua ammonia
Shallow lake dissolving obtains fumaric acid ammonia solution；
(2), by CGMCC No.9288 bacterial strain with LB culture medium (pH7.0) 35 DEG C, dissolved oxygen 4ppm condition bottom fermentation train
Obtain after supporting 24 hours has aspartase activity fermentation liquid as enzyme source, mixes with the solution even of step (1), 35 DEG C
Stirring incubation 3 hours, generates L-Aspartic acid through enzymatic reaction；
(3), it is adjusted with the regulations speed of 0.5pH per minute with maleic acid pressed powder at ambient temperature,
Until pH=2.5, subsequently stand and filter within 6 hours to obtain aspartic acid crude product.Reclaim the waste water after aspartic acid can add again
Maleic acid is repeated production for after 700g/L as raw material to concentration, until the conversion ratio of HT-ME bacterial strain fermentation liquor (is surveyed
Determine method as described below) do not reach 80% till.
In production process, using titration of potassium permanganate fumaric acid conversion ratio：2ml reactant liquor+30ml10%
Sulfuric acid solution is titrated with 0.1mol/L potassium permanganate solution after boiling.Conversion ratio=(the permanganic acid that before reaction, solution titration consumes
After potassium solution volume-reaction solution titration consume potassium permanganate solution volume)/reaction before solution titration consume potassium permanganate
Liquor capacity.
Producing average conversion for the first time is 98.3%, and second circulation average conversion is 92.1%, and third time circulates
Average conversion is 85.5%, and the 4th time circulation average conversion is 60.2%, thus can obtain, HT-ME bacterial strain can be used for waste liquid
The continuous aspartic acid production technology circulating three times, makes conversion ratio be maintained at more than 85%, the three cyclic production discharges of this technique
Waste water, wastewater discharge is about 1/3rd of common process.
Claims (38)
1. a kind of salt tolerant E. coli mutant strain, deposit number is CGMCC No.9288.
2. salt tolerant E. coli mutant strain according to claim 1 is it is characterised in that described bacterial strain 16S rRNA gene portion
Divide fragment sequence consistent with described in Seq ID No.1.
3. salt tolerant E. coli mutant strain according to claim 1 is it is characterised in that described bacterial strain has following characteristics：
(1), bacterial strain required growth conditionss in PYG culture medium：Temperature 10-40 DEG C, pH3.5-8.5, salinity 0-8% (w/v)；
(2), bacterial strain is gram negative bacilli, size 0.5-1.0 × 2-10μm；
(3), in PYG culture medium bacterial strain Dai Shiwei 0.2h；
(4), bacterial strain is to penicillin, vancomycin, streptomycin, erythromycin, chloromycetin, kanamycin or neomycin sensitive.
4. the method that salt tolerant E. coli mutant strain described in a kind of utilization claim 1 produces chiral aspartic acid, including as follows
Step：
(1), with maleic acid as raw material, generate fumaric acid precipitation under the catalytic action with Ammonium persulfate. for the sodium bromide,
Resolution of precipitate is made after ammonification；
(2), there is the fermentation liquid of aspartase activity as enzyme source using obtain after the culture of CGMCC No.9288 strain fermentation
Mix with the solution of step (1), generate L-Aspartic acid through enzymatic reaction；
(3), adjust pH to acid, reclaim target product L-Aspartic acid.
5. method according to claim 4 it is characterised in that in methods described step (1) reaction condition be temperature control
At 50-80 DEG C, sodium bromide dosage is the 0.5-5% of maleic acid quality, and the dosage of Ammonium persulfate. is maleic acid
The 0.5-5% of quality.
6. method according to claim 5 it is characterised in that in methods described step (1) reaction condition be temperature control
At 60-75 DEG C, sodium bromide dosage is the 1-2.5% of maleic acid quality, and the dosage of Ammonium persulfate. is maleic acid
The 1.5-3% of quality, centrifugation after having reacted obtains fumaric acid solid.
7. method according to claim 6 it is characterised in that in methods described step (1) reaction condition be in 75 DEG C of water
Under the conditions of bath, first add the sodium bromide of maleic acid quality 1.5%, then add the over cure of maleic acid quality 2.6%
Sour ammonium, centrifugation after having reacted obtains fumaric acid solid.
8. method according to claim 4 it is characterised in that in methods described step (2) enzymatic reaction condition be in 20-
40 DEG C of stirring 1-6 hours.
9. method according to claim 8 is it is characterised in that in methods described step (2), enzymatic reaction condition be
30-35 DEG C of stirring 2-4 hour.
10. method according to claim 4 is it is characterised in that in methods described step (2), CGMCC No.9288 bacterial strain
It is inoculated in the fermentation medium that pH value is 6-8, at a temperature of 15-35 DEG C, cultivates 12-36 hour, obtain and there is Aspartase
The fermentation liquid of activity.
11. methods according to claim 10 it is characterised in that in methods described step (2) fermentation medium be ability
The general Escherichia coli fermentation culture medium in domain.
12. methods according to claim 11 it is characterised in that in methods described step (2), fermentation medium be LB or
Beef-protein medium.
13. methods according to claim 12 are it is characterised in that in methods described step (2), fermentation medium forms
As follows：10g peptone, 10g yeast extract, 5g glucose, 40ml saline solution, ultrapure water dissolution is settled to 1L, pH=7；Wherein, institute
Every liter of the saline solution stated consists of 0.25g calcium chloride, 0.5g magnesium chloride, 1g dipotassium hydrogen phosphate, 1g potassium dihydrogen phosphate, 10g carbonic acid
Hydrogen sodium, 2g sodium chloride, ultrapure water dissolution, it is settled to 1L.
14. methods according to claim 4 will be it is characterised in that in methods described step (2), will have Aspartase
The fermentation liquid of activity is added in the reaction solution of step (1), and the addition of fermentation liquid is step (1) reaction solution volume
0.1-5 times.
15. methods according to claim 14 will be it is characterised in that in methods described step (2), will have Aspartase
The fermentation liquid of activity is added in the reaction solution of step (1), and the addition of fermentation liquid is step (1) reaction solution volume
0.5-3 times.
16. methods according to claim 15 will be it is characterised in that in methods described step (2), will have Aspartase
The fermentation liquid of activity is added in the reaction solution of step (1), and the addition of fermentation liquid is the 1- of step (1) reaction solution volume
1.5 again.
17. methods according to claim 4 are it is characterised in that in methods described step (3), reaction condition is by solution
PH regulator to 1.0-6.0, to be precipitated completely after be filtrated to get L-Aspartic acid crude product.
18. methods according to claim 17 are it is characterised in that in methods described step (3), reaction condition is by solution
PH regulator to 2.5-3.0, standing, to be precipitated completely after be filtrated to get L-Aspartic acid crude product.
19. methods according to claim 4 it is characterised in that in methods described step (3), the examination of regulation pH used
Agent is common mineral acid or organic acid, selected from hydrochloric acid, sulphuric acid, phosphoric acid, acetic acid, citric acid, maleic acid, hydroxyl first sulphur
Acid, hydrobromic acid, methanesulfonic acid, trifluoracetic acid, benzenesulfonic acid, sulfamic acid, glycolic, stearic acid, lactic acid, malic acid, flutter sour, right
Aminobenzenesulfonic acid, fumaric acid, ethane disulfonic acid, oxalic acid, hydroxyethylsulfonic acid. or its mixture.
20. methods according to claim 19 it is characterised in that in methods described step (3), the examination of regulation pH used
Agent is hydrochloric acid or maleic acid.
21. one kind carry out waste water recycling process using salt tolerant E. coli mutant strain described in claim 1 and produce chiral Radix Asparagi ammonia
The method of acid, comprises the steps：
(1), recycle enzymatic clarification aspartic acid produce waste liquid, add maleic acid, and catalyst sodium bromide with
Ammonium persulfate. generates fumaric acid precipitation, makes resolution of precipitate after ammonification；
(2), there is the fermentation liquid of aspartase activity as enzyme source using obtain after the culture of CGMCC No.9288 strain fermentation
Mix with the solution of step (1), generate L-Aspartic acid through enzymatic reaction；
(3), adjust pH to acid with maleic acid, reclaim target product L-Aspartic acid.
22. methods according to claim 21 are it is characterised in that the waste liquid that this technique produces can continue to adopt said method
Step is circulated waste water recycling, till the salinity of waste water accumulation is no longer appropriate for CGMCC No.9288 growth.
23. methods according to claim 21 it is characterised in that in methods described step (1), the maleic acid added
Final concentration is to more than 100g/L.
24. methods according to claim 23 it is characterised in that in methods described step (1), the maleic acid added
Final concentration is to 500-700g/L.
25. methods according to claim 21 are it is characterised in that in methods described step (1), reaction condition is temperature
Control at 50-80 DEG C, sodium bromide dosage is the 0.1-2% of maleic acid quality, the dosage of Ammonium persulfate. is maleic
The 0.1-2% of diacid quality.
26. methods according to claim 25 are it is characterised in that in methods described step (1), reaction condition is temperature control
At 60-75 DEG C, sodium bromide dosage is the 0.1-0.5% of maleic acid quality to system, and the dosage of Ammonium persulfate. is maleic
The 0.2-1% of diacid quality, centrifugation after having reacted obtains fumaric acid solid.
27. methods according to claim 26 are it is characterised in that in methods described step (1), reaction condition is at 75 DEG C
Under water bath condition, first add the sodium bromide of maleic acid quality 0.15%, then add maleic acid quality 0.26%
Ammonium persulfate., centrifugation after having reacted obtains fumaric acid solid.
28. methods according to claim 21 it is characterised in that in methods described step (2), enzymatic reaction condition be
20-40 DEG C of stirring 1-6 hour.
29. methods according to claim 28 it is characterised in that in methods described step (2), enzymatic reaction condition be
30-35 DEG C of stirring 2-4 hour.
30. methods according to claim 21 it is characterised in that in methods described step (2), CGMCC No.9288 bacterium
Strain is inoculated in the fermentation medium that pH value is 6-8, cultivates 12-36 hour at a temperature of 15-35 DEG C, obtains and has aspartic acid
The fermentation liquid of enzymatic activity.
31. methods according to claim 30 it is characterised in that in methods described step (2) fermentation medium be ability
The general Escherichia coli fermentation culture medium in domain.
32. methods according to claim 31 it is characterised in that in methods described step (2), fermentation medium be LB or
Beef-protein medium.
33. methods according to claim 32 are it is characterised in that in methods described step (2), fermentation medium forms such as
Under：10g peptone, 10g yeast extract, 5g glucose, 40ml saline solution, ultrapure water dissolution is settled to 1L, pH=7；Wherein, described
Every liter of saline solution consist of 0.25g calcium chloride, 0.5g magnesium chloride, 1g dipotassium hydrogen phosphate, 1g potassium dihydrogen phosphate, 10g bicarbonate
Sodium, 2g sodium chloride, ultrapure water dissolution, it is settled to 1L.
34. methods according to claim 21 will be it is characterised in that in methods described step (2), will have Aspartase
The fermentation liquid of activity is added in the reaction solution of step (1), and the addition of fermentation liquid is step (1) reaction solution volume
0.1-5 times.
35. methods according to claim 34 will be it is characterised in that in methods described step (2), will have Aspartase
The fermentation liquid of activity is added in the reaction solution of step (1), and the addition of fermentation liquid is step (1) reaction solution volume
0.5-3 times.
36. methods according to claim 35 will be it is characterised in that in methods described step (2), will have Aspartase
The fermentation liquid of activity is added in the reaction solution of step (1), and the addition of fermentation liquid is the 1- of step (1) reaction solution volume
1.5 again.
37. methods according to claim 21 are it is characterised in that in methods described step (3), reaction condition is with along fourth
Enedioic acid adjusts pH to 1.0-6.0.
38. methods according to claim 37 are it is characterised in that in methods described step (3), reaction condition is with along fourth
Enedioic acid adjusts between pH to 2.5-3.0, solution left standstill, to be precipitated completely after be filtrated to get L-Aspartic acid crude product.
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