CN105603015B - A kind of production method of L-glufosinate-ammonium - Google Patents
A kind of production method of L-glufosinate-ammonium Download PDFInfo
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- CN105603015B CN105603015B CN201610045121.7A CN201610045121A CN105603015B CN 105603015 B CN105603015 B CN 105603015B CN 201610045121 A CN201610045121 A CN 201610045121A CN 105603015 B CN105603015 B CN 105603015B
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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- C12Y206/00—Transferases transferring nitrogenous groups (2.6)
- C12Y206/01—Transaminases (2.6.1)
- C12Y206/01002—Alanine transaminase (2.6.1.2), i.e. alanine-aminotransferase
Abstract
The invention discloses a kind of production methods of L-glufosinate-ammonium; this method comprises: using 2- carbonyl -4- (hydroxymethyl phosphono) butyric acid and its salt as substrate; under the conditions of existing for the amino group donor; transamination reaction occurs using the cell catalysis substrate of in vitro transaminase or vivoexpression transaminase and amino group donor, obtains L-glufosinate-ammonium；The amino group donor is alanine, and the amino acid sequence of the transaminase is as shown in NO.1~3 SEQ ID.The present invention is using 2- carbonyl -4- (hydroxymethyl phosphono) butyric acid and its salt as substrate; alanine is amino group donor, and transamination reaction occurs by specific transaminase-catalyzed substrate, can be fully converted into substrate as L-glufosinate-ammonium; feed stock conversion is high, and conversion ratio is up to 100%.
Description
Technical field
The present invention relates to technical field of biochemical industry more particularly to a kind of production methods of L-glufosinate-ammonium；Specifically
A kind of method of biological enzyme production optical voidness L-glufosinate-ammonium.
Background technique
Glufosinate-ammonium, English name are as follows: Phosphinothricin (abbreviation PPT) generally refers to compound 2- amino -4- [hydroxyl
Base (methyl) phosphono] salt for being formed of-butyric acid or itself and alkali compounds.Glufosinate-ammonium is to be developed by Hirst company the eighties
(belonging to Beyer Co., Ltd afterwards) broad-spectrum touch-out type herbicide, belong to phosphonic acid herbicide, be glutamine synthetase inhibitor,
Transfer in leaf, but other places cannot be transferred to, after glutamine synthesis is suppressed, ammonium ion is caused to be accumulated, chloroplaset disintegrates, from
And keep photosynthesis suppressed, eventually lead to Plant death.
There are two types of optical isomers, respectively L-glufosinate-ammonium (formula 2) and D- glufosinate-ammonium (formula 3) for glufosinate-ammonium.But only L-type
With phytotoxicity, activity of weeding is 2 times of racemic mixture, and is easily decomposed in the soil, to the toxicity of human and animal
Smaller, herbicidal spectrum is wide, small to the destructive power of environment.
Currently, glufosinate-ammonium available on the market is typically all racemic mixture.If glufosinate-ammonium product can be with L- configuration
Pure enantiomeric form use, can make glufosinate-ammonium usage amount reduce by 50%, this for improve Atom economy, reduce make
It is all of great significance with cost, mitigation environmental pressure.
There are mainly three types of the methods for preparing optical voidness L-glufosinate-ammonium: chemical synthesis, chiral separation and biocatalysis
Method.
Chemical synthesis is more common in laboratory research, such as from the pure L-glufosinate-ammonium of chiral raw material synthesizing optical
Minowa N etc. is that starting material synthesizes L-glufosinate-ammonium (Hirayama M.Asymmetric Synthesis of using glycine
(+)-Phosphinothricin and Related Compounds by the Michael Addition of Glycine
Schiff Bases to Vinyl Compounds[J].Bulletin of the Chemical Society of Japan,
1987,60:1761–1766.).Zeiss H J etc. has done many trials to the asymmetric syntheses of L-glufosinate-ammonium
(Enantioselective Synthesis of Both Enantiomers of Phosphinothricin via
Asymmetric Hydrogenation ofα-acylamidoAcrylates[J].Journal of Organic
Chemistry, 1991,56:1783-1788.), (Enantioselective Synthesis of L-
Phosphinothricin from L-methionine and L-glutamic Acid via L-vinylglycine[J]
.Tetrahedron,1992,48(38):8263–8270.).Dissymmetric synthesis processing step is more, yield is low, asymmetry used
Synthetic agent is mostly more expensive, causes production cost higher, is unfavorable for large scale preparation L-glufosinate-ammonium.
Chiral separation is to recycle chiral selectors by chemical synthesis racemic DL- glufosinate-ammonium or derivatives thereof,
The separation for carrying out D type and L-type isomers, so that optically pure L-glufosinate-ammonium be made.This technique has the following disadvantages, first is that
It needs using chiral selectors, second is that D- glufosinate-ammonium needs racemization again to recycle, third is that single resolution yield is low, fourth is that work
Skill is more complicated.
In contrast, biological catalysis is stringent with stereoselectivity, reaction condition is mild, high income and product are easily separated
The advantages that purifying is the potential advantages method for producing L-glufosinate-ammonium.
Just have been found that L-glufosinate-ammonium in transaminase when studying glufosinate-ammonium in edaphon intracorporal metabolic pathway
Under the action of, transamination occurs and is broken down into a kind of 2-ketoacid --- 2- carbonyl -4- (hydroxymethyl phosphono) butyric acid is (referred to as
PPO).Transamination is a reversible reaction, and 2- carbonyl -4- (hydroxymethyl phosphono) butyric acid can lead under the catalysis of transaminase
It crosses back reaction and generates L-glufosinate-ammonium.
In the world, Schulz A et al. (Stereospecific Production of the Herbicide
Phosphinothricin(glufosinate)by Transamination:Isolation and Characterization
of a Phosphinothricin-specific Transaminase from Escherichia coli[J].Applied
And Environmental Microbiology, 1990,56:1-6.) (United States Patent (USP) US5221737) early in last century 90 years
In generation, just utilizes the transaminase being separated to from E.coli K-12, using 2- carbonyl -4- (hydroxymethyl phosphono) butyric acid as substrate,
Pidolidone is catalyzed transamination reaction production L-glufosinate-ammonium (Fig. 1) as amino group donor.Transaminase is installed after immobilization to biology
Reactor, production concentration are 50g/ (Lh) up to 76.1g/L, maximum output, and the ee value of L-glufosinate-ammonium is more than 99.9%.But this
A technique has two big defects, one is raw material PPO cannot be fully converted to L-PPT, conversion ratio highest only has 90%；The second is wanting
It carries out reversible reaction to the direction for generating L-PPT, needs the Pidolidone of 4 times of equivalents or more as amino group donor, it is excessive
Glutamic acid brings very big trouble to separation, and it is anti-to turn ammonia for enzymatic when the key of problem is using Pidolidone as amino group donor
It should be a reversible reaction.
One of the approach for solving the problems, such as this is exactly to replace Pidolidone using L-Aspartic acid, and L-Aspartic acid is through turning ammonia
Oxaloacetic acid is generated after effect, oxaloacetic acid is unstable in aqueous solution, is easily decomposed into pyruvic acid, to break transamination reaction
Balance.In consideration of it, Bartsch K (United States Patent (USP) US6335186 B1) increases a grass under above-mentioned single enzyme reaction system
Ethyl acetoacetic acid transaminase organizes transaminase coupling reaction system (Fig. 2) in pairs.However, there is still a need for use paddy ammonia in coupling reaction
The formation in the reaction of acid, glutamic acid and ketoglutaric acid balances, and structure is again extremely similar with product L-PPT, it is difficult to separate
It is removed in purifying；And also add two kinds of need impurity oxaloacetic acid to be separated and L-Aspartic acid.Further, since dynamic
The difference of mechanics parameter, using the process of two kinds of enzymes than using the more difficult to optimize of a kind of enzyme.Therefore, PPO in this process
Conversion ratio also only have 85%.
Bartsch K etc. then has also been proposed a new work in the patent (Chinese patent CN1349561A) of China's application
Skill (such as Fig. 3), they screen the oxalyl second for having obtained specifically converting 2- carbonyl -4- (hydroxymethyl phosphono) butyric acid
Sour transaminase is directly amino group donor using L-Aspartic acid.But this low process efficiency, when the concentration of substrate PPO is
552mmol/L, in the case where almost consuming the raw material L-Aspartic acid of about 700mmol/L, only generate
The product L-PPT of 251.9mmol/L generates the impurity alanine of about 234.5mmol/L at the same time.Raw material PPO reaction
Conversion ratio only has 52%.
Summary of the invention
The present invention provides a kind of life of L-glufosinate-ammonium for defect existing for existing transaminase method production L-PPT technique
Production method, this method feed stock conversion is high, production cost is low, high income.
A kind of production method of L-glufosinate-ammonium, comprising: using 2- carbonyl -4- (hydroxymethyl phosphono) butyric acid and its salt the bottom of as
Object utilizes the cell catalysis substrate and ammonia of in vitro transaminase or vivoexpression transaminase under the conditions of existing for the amino group donor
Transamination reaction occurs for base donor, obtains L-glufosinate-ammonium；The amino group donor is alanine, and the amino acid sequence of the transaminase is such as
Shown in NO.1~3 SEQ ID.
Wherein, amino acid sequence is that transaminase shown in SEQ ID NO.1 derives from e. coli k12 W3110 (E.coli
K12W3110).Amino acid sequence is that transaminase shown in SEQ ID NO.2 derives from 168 (Bacillus of bacillus subtilis
subtis 168).Amino acid sequence is that transaminase shown in SEQ ID NO.3 derives from bacillus megaterium YYBM1
(Bacillus magaterium YYBM1)。
Preferably, the cell is the engineering bacteria for expressing transaminase, the host cell of the engineering bacteria is E.coli
BL21 (DE3), the amino acid sequence of the transaminase is as shown in NO.1~3 SEQ ID.
Specifically, the engineering bacteria contains expression vector pET-28a (+), and the aminotransferase gene is connected to expression vector
On pET-28a (+).
The biocatalytic reaction of above-mentioned production L-glufosinate-ammonium can also not only be adopted using the engineering bacteria of expression transaminase
With the in vitro transaminase after clasmatosis, or the transaminase after immobilization.
Preferably, 10000rpm is centrifuged the weight in wet base meter after 10min in transamination reaction, the additive amount of the engineering bacteria is
The 0.5~25% of reaction solution weight.It is further preferred that the additive amount of the cell is the 5~10% of reaction solution weight.
Preferably, the molar ratio of the alanine and substrate is 1~6:1；It is further preferred that the alanine and substrate rub
You are than being 2~4:1.
It further include coenzyme in reaction system, which is vitamin B6 type coenzyme, for example, pyridoxol, pyridoxal and pyrrole
It trembles amine or their derivative.Specifically, the coenzyme is phosphopyridoxal pyridoxal phosphate or phosphopyridoxamine；Based on mass fraction, described
The additive amount of coenzyme is 0.01~2 ‰；It is further preferred that 0.1~1 ‰.
Preferably, the temperature of the transamination reaction is 20~70 DEG C, the time is 6~72 hours；It is further preferred that temperature is 30
~60 DEG C, the time is 12~36 hours.
Preferably, the pH value of control transamination reaction is 6~9.The adjusting of pH is carried out using alkali, such as: sodium hydroxide, hydrogen-oxygen
Change potassium, ammonium hydroxide, isopropylamine, triethylamine etc..
Compared with prior art, the invention has the following advantages:
(1) for the present invention using 2- carbonyl -4- (hydroxymethyl phosphono) butyric acid and its salt as substrate, alanine is amino confession
By specific transaminase-catalyzed substrate transamination reaction occurs for body, can be fully converted into substrate as L-glufosinate-ammonium, raw material turn
Rate is high, and conversion ratio is up to 100%.
(2) the method for the present invention simplifies separating technology, and after the reaction was completed, excessive alanine is easy to and product L-glufosinate-ammonium
Separation；At the same time, by-product pyruvic acid is the intermediary of numerous biochemical reactions, it is easy to be changed into other by enzymatic reaction
Substance improves total yield of products to further simplify follow-up process for refining.
(3) raw material of the present invention is easy to get and low in cost, and excessive raw material is also recyclable to be recycled, and environmental protection pressure is small, is suitble to
Large-scale industrial production.
Detailed description of the invention
Fig. 1 is the reaction equation that L-PPT is produced by amino group donor of glutamic acid.
Fig. 2 is the reaction equation that double transaminase systems produce L-PPT.
Fig. 3 is the reaction equation that L-PPT is produced by amino group donor of aspartic acid.
Fig. 4 is the reaction equation that the present invention produces L-PPT by amino group donor of alanine.
Fig. 5 is the efficient liquid phase test map of reactants and products of the present invention (in reaction process)；
Wherein, 1: retention time 3.572min is l-Alanine；2: retention time: 4.673min is pyruvic acid；
3: retention time: 5.483min is unknown impuritie；4: retention time: 6.567min L-PPT；5: retention time::
26.441min being PPO.
Fig. 6 is the efficient liquid phase test map of reactants and products of the present invention (after reaction)；
Wherein, 1: retention time 3.569min is l-Alanine；2: retention time: 4.673min is pyruvic acid；3: retaining
Time: 5.481min and 5.727min is unknown impuritie；4: retention time: 6.560min L-PPT；
Fig. 7 is the mass spectrogram of raw material 2- carbonyl -4- (hydroxymethyl phosphono) butyric acid (abbreviation PPO)；
Wherein, the positive source mass spectrogram that A figure is PPO；The negative source mass spectrogram that B figure is PPO.
Specific embodiment
Below in conjunction with specific embodiment, the present invention will be further described.It should be understood that following embodiment is merely to illustrate this
The range of invention and is not intended to limit the present invention.
Experimental method in the present invention is conventional method unless otherwise instructed, and for details, reference can be made to J. Sas for gene cloning operation
" Molecular Cloning:A Laboratory guide " of the volumes such as nurse Brooker.
Upstream gene engineering agents useful for same: restriction enzyme and DNA ligase used in the embodiment of the present invention are purchased
From TaKaRa, precious bioengineering (Dalian) Co., Ltd；Genome extraction kit, plasmid extraction kit, DNA recovery purifying
Kit is purchased from the Hangzhou Axygen Co., Ltd；The purchase such as E.coli DH5 α, E.coli BL21 (DE3), plasmid pET-28a (+)
From Novagen company；DNA marker, FastPfu archaeal dna polymerase, low molecular weight standard protein, the purchase of agarose electrophoresis reagent
From Beijing Quanshijin Biotechnology Co., Ltd；Primer synthesis is limited by the raw work biotechnology in Shanghai with sequence work
Company completes.The above reagent application method refers to product manual.
Downstream catalytic process agents useful for same: 2- carbonyl -4- (hydroxymethyl phosphono) butyric acid (abbreviation PPO) is laboratory conjunction
At mass spectrogram is as shown in Figure 7；L-glufosinate-ammonium standard items are purchased from Sigma-Aldrich company；Other common agents are purchased from traditional Chinese medicines
Chemical reagent Co., Ltd of group.
Shown in the structural formula such as formula (1) of 2- carbonyl -4- (hydroxymethyl phosphono) butyric acid (abbreviation PPO)；L-glufosinate-ammonium (letter
Claim L-PPT) structural formula such as formula (2) shown in；It is specific as follows:
The progress that transamination reaction of the present invention is reacted by high performance liquid chromatography (HPLC) monitoring, and to each reactant and production
Object is analyzed.HPLC analysis method are as follows: liquid chromatograph: Agilent 1100；Column model:XB-
SAX,5μm,4.6mm×250mm.Mobile phase: 100mM potassium dihydrogen phosphate aqueous solution: acetonitrile=10:1.Detection wavelength: 205nm.
Flow velocity: 1.0mL/min.Column temperature: 40 DEG C.Specific each related substances appearance situation is shown in liquid chromatogram.
Embodiment 1
One, respectively from E. coli K12W3110, bacillus subtilis Bacillus subtilis 168 with
And aminotransferase gene is cloned in bacillus megaterium Bacillus magaterium YYBM1 genome, according to corresponding gene group
DNA sequence dna (GenBank accession number is respectively CP012868.1, CP010052.1 and CP001982.1) designs on corresponding PCR
Swim primer and downstream primer.
From the primer of the transaminase of E.coli:
EC-F sequence: 5 '-CCGGAATTCATGAGCAACAATGAATTCCATC-3’(EcoRI)
EC-R sequence: 5 '-CCGCTCGAGTTAATCGCTCAGCGCATCC-3’(XholI)
From the primer of the transaminase of Bacillus Subtilis:
BS-F sequence: 5 '-CCCGAGCTCATGAGTCAAACAACAGCAAGCATCA-3’(SacI)
BS-R sequence: 5 '-CCCAAGCTTTTAAGCTCGCAGGCCCGCCT-3’(HindIII)
From the primer of the transaminase of Bacillus magaterium:
BM-F sequence: 5 '-CGCGGATCCATGAGTCAAACTTTTAGCAA-3’(BamHI)
BM-R sequence: 5 '-CCCAAGCTTTTACACTTCAACCGTTTGCT-3’(HindIII)
It is separately added into restriction enzyme site in the primer of upstream and downstream, as shown in underscore, concrete restriction enzyme is shown in primer sequence
In column bracket.Respectively with E. coli K12W3110, bacillus subtilis Bacillus subtilis 168 and
Bacillus megaterium Bacillus magaterium YYBM1 genomic DNA is template, and corresponding upstream and downstream primer carries out PCR
Amplification, PCR reaction system and reaction condition are as follows:
PCR amplification system:
PCR amplification condition:
1) initial denaturation: 95 DEG C of 5min；
2) it is denaturalized: 98 DEG C of 10s；Annealing: 58 DEG C of 15s；Extend: 72 DEG C of 10s；It recycles 30 times altogether；
3) extend: 72 DEG C of 10min；
4) 4 DEG C of preservation 2.0h.
After PCR amplification, amplified production is detected with 1.0% agarose gel electrophoresis, and amplified production is as the result is shown
Single band, size are 1400bp or so.Purification and recovery, specific steps are carried out to amplified production with DNA recovery purifying kit
Referring to purification kit specification.
Two, the building of expression vector and engineering bacteria
Expression vector pET-28a (+) and pcr amplification product carry out double digestion with corresponding restriction enzyme respectively.Enzyme
The core for DNA purification kit purification and recovery being carried out to digestion products after the completion of cutting to remove restriction enzyme and digestion is got off
Thuja acid small fragment.Pcr amplification product after double digestion is connected to the expression with corresponding notch with T4DNA ligase and carries
On body pET-28a (+), linked system is as shown in table 1 below:
Table 1pET-28a (+)-gabT recombinant expression plasmid linked system
After each reagent in above-mentioned linked system is mixed, it is put in 16 DEG C of metal baths and connects 12h.By enzyme-linked product
Conversion applies plate, chooses single colonie LB Liquid Culture into E.coli DH5a competent cell, the successful sun of PCR method identification building
Property transformant, and verify by sequencing company the correctness of insetion sequence.Recombinant expression carrier is transferred to expressive host again
In E.coli BL21 (DE3), the recon of conversion is verified with PCR method, errorless genetic engineering bacterium is E.coli after verifying
BL21(DE3)/pET-28a(+)-gabT。
Embodiment 2
One, the culture of microorganism
LB liquid medium composition: peptone 10g/L, yeast powder 5g/L, NaCl10g/L are determined with after deionized water dissolving
Hold, 121 DEG C of sterilizing 20min, for use.
Genetically engineered E.coli BL21 (DE3) containing aminotransferase gene is seeded to containing 50 μ g/mL kanamycins
In 5mL LB liquid medium, 37 DEG C of shake culture 12h.It is forwarded to the fresh LB liquid training that 500mL equally contains 50 μ g/ml Kan
It supports in base, 37 DEG C of shake cultures to OD600When reaching 0.8 or so, addition IPTG to its concentration is 0.3mM, Fiber differentiation at 28 DEG C
20h.After culture, culture solution 10000rpm is centrifuged 10min, abandons supernatant, thallus is collected, is put into -70 DEG C of ultra low temperature freezers
Middle preservation, for use.
Two, the preparation of crude enzyme liquid
The thallus that will be collected into after culture, twice with the buffer washing thalline of 50mM Tris-HCl (pH 7.0).
Thallus is resuspended in Tris-HCl (50mM, pH 7.5,20mM imidazoles, 0.3M NaCl, 5mM dithiothreitol (DTT)) buffer later
In, ultrasonication bacteria suspension, centrifugation removal precipitating, obtained supernatant is the crude enzyme liquid containing transaminase.
Embodiment 3
It quantitatively weighs PPO, alanine and phosphopyridoxal pyridoxal phosphate to mix into beaker, adjusts pH value of solution=7.5 with 30% ammonium hydroxide,
It is added in volumetric flask, with deionized water constant volume, obtains the final concentration of 60mM of PPO, the final concentration of 180mM of alanine, phosphoric acid pyrrole is trembled
The mixed liquor of the final concentration of 1mM of aldehyde.
Culture can express the base of transaminase shown in SEQ ID NO.1 (from E. coli K12W3110)
Because of engineering bacteria, 1mL culture solution is taken, 10000rpm is centrifuged 10min, abandons supernatant, obtains cell 5mg；It is added to the above-mentioned acquisition of 1mL
In mixed liquor, it is resuspended, is reacted for 24 hours in 37 DEG C of metal bath oscillating reactions devices.
After reaction, HPLC detection is carried out, has L-glufosinate-ammonium generation, but do not find the apparent peak PPO, illustrates that PPO turns
Rate reaches 100%.
It quantitatively weighs PPO, alanine and phosphopyridoxal pyridoxal phosphate to mix into beaker, adjusts pH value of solution=7.5 with 30% ammonium hydroxide,
It is added in volumetric flask, with deionized water constant volume, obtains the final concentration of 60mM of PPO, the final concentration of 180mM of alanine, phosphoric acid pyrrole is trembled
The mixed liquor of the final concentration of 1mM of aldehyde.
After by the genetic engineering bacterium culture containing glutamic-pyruvic transaminase encoding gene shown in SEQ ID NO.4,1mL is taken
Medium centrifugal abandons supernatant.The solution 1mL of above-mentioned 60mM containing PPO, alanine 180mM and phosphopyridoxal pyridoxal phosphate 1mM is added, weight
Outstanding, in reacting a week in 37 DEG C of metal bath oscillating reactions devices, every 24 hours sampling analyses, HPLC detection was not found bright
Aobvious L-glufosinate-ammonium generates, and illustrates this transaminase to this reaction without catalytic effect.
Embodiment 4
Culture can express transaminase shown in SEQ ID NO.2 (from 168 (Bacillus of bacillus subtilis
Subtis 168)) genetic engineering bacterium, crude enzyme liquid is made in the cell 5g that will be collected by centrifugation.
PPO is quantitatively weighed into round-bottomed flask, pH value of solution=8.0 is adjusted with 30% ammonium hydroxide, above-mentioned crude enzyme liquid is added, makes
The final concentration of 80mM of PPO, adds the alanine of 280mM and the phosphopyridoxal pyridoxal phosphate of 1mM, with deionized water constant volume to 50mL.
Controlling reaction temperature by water-bath is 40 DEG C；Magnetic agitation, HPLC monitoring reaction, reaction time 28h.
Reaction process data are as follows:
| Reaction time (h) | Pyruvic acid (mM) | L-PPT(mM) | PPO(mM) |
| 0 | 0.0 | 0.0 | 80.0 |
| 0.5 | 0.2 | 7.1 | 73.2 |
| 1.5 | 0.9 | 19.2 | 61.3 |
| 3.0 | 2.0 | 20.8 | 60.4 |
| 6.0 | 16.1 | 50.9 | 49.7 |
| 24.0 | 21.4 | 70.1 | 10.5 |
| 28.0 | 17.4 | 80.5 | 0.0 |
Embodiment 5
Culture can express transaminase shown in SEQ ID NO.3 (from bacillus megaterium YYBM1 (Bacillus
Magaterium YYBM1)) genetic engineering bacterium, cell is collected by centrifugation.
It quantitatively weighs in PPO to 100mL reactor, adjusts pH value of solution=9.0 with 30% ammonium hydroxide, arrived with deionized water constant volume
100mL makes the final concentration of 100mM of PPO, adds the above-mentioned cell being collected by centrifugation of 25g, add 400mM alanine and
The phosphopyridoxal pyridoxal phosphate of 1mM.Controlling reaction temperature by water-bath is 50 DEG C；Magnetic agitation, HPLC detect PPO and convert situation, reaction
Time is 19h.Response data is as follows:
| Reaction time (h) | Pyruvic acid (mM) | L-PPT(mM) | PPO(mM) |
| 0 | 0.0 | 0.0 | 100.0 |
| 0.5 | 61.2 | 84.1 | 16.8 |
| 1.5 | 51.5 | 86.6 | 13.5 |
| 3.0 | 42.4 | 98.2 | 1.1 |
| 6.0 | 37.3 | 100.6 | 0.9 |
| 19.0 | 34.8 | 100.2 | 0.0 |
Comparative example 1
Culture can express transaminase shown in SEQ ID NO.3 (from bacillus megaterium YYBM1 (Bacillus
Magaterium YYBM1)) genetic engineering bacterium, cell is collected by centrifugation.
It quantitatively weighs in PPO to 100mL reactor, adjusting pH=9.0 with 30% ammonium hydroxide makes PPO with deionized water constant volume
Final concentration of 100mM, be added the above-mentioned cell being collected by centrifugation of 25g, add the glutamic acid of 400mM and the phosphoric acid pyrrole of 1mM
It trembles aldehyde.Controlling reaction temperature by water-bath is 50 DEG C；Magnetic agitation, HPLC detect PPO and convert situation, reaction time 72h.Instead
Answer data as follows:
| Reaction time (h) | Alpha Ketoglutarate (mM) | L-PPT(mM) | PPO(mM) |
| 0 | 0.0 | 0.0 | 100.0 |
| 0.5 | 39.2 | 40.1 | 59.7 |
| 1.5 | 51.5 | 46.6 | 53.5 |
| 3.0 | 62.4 | 69.2 | 28.4 |
| 6.0 | 77.3 | 79.1 | 20.8 |
| 19.0 | 74.8 | 80.2 | 19.7 |
| 72.0 | 74.0 | 80.3 | 19.4 |
Comparative example 2
It quantitatively weighs PPO, alanine and phosphopyridoxal pyridoxal phosphate to mix into beaker, adjusts pH value of solution=7.5 with 30% ammonium hydroxide,
It is added in volumetric flask, with deionized water constant volume, obtains the final concentration of 60mM of PPO, the final concentration of 180mM of alanine, phosphoric acid pyrrole is trembled
The mixed liquor of the final concentration of 1mM of aldehyde.
Culture can express third turn of ammonia transaminase of paddy shown in SEQ ID NO.4 (from e. coli k12 MG1655
(E.coli K12MG1655)) genetic engineering bacterium, crude enzyme liquid is made；It takes 1mL crude enzyme liquid to be centrifuged, abandons supernatant, obtain cell
5mg；It is added in the mixed liquor of the above-mentioned acquisition of 1mL, is resuspended, in reacting a week in 37 DEG C of metal bath oscillating reactions devices, often
Every 24 hours sampling analyses, HPLC detection did not found that apparent L-glufosinate-ammonium generates, illustrated that this transaminase urges this reaction nothing
Change effect.
Claims (8)
1. a kind of production method of L-glufosinate-ammonium, comprising: using 2- carbonyl -4- (hydroxymethyl phosphono) butyric acid and its salt the bottom of as
Object utilizes the cell catalysis substrate and ammonia of in vitro transaminase or vivoexpression transaminase under the conditions of existing for the amino group donor
Transamination reaction occurs for base donor, obtains L-glufosinate-ammonium；It is characterized in that, the amino group donor is alanine, the transaminase
Amino acid sequence is as shown in NO.1~3 SEQ ID.
2. production method as described in claim 1, which is characterized in that the cell is the engineering bacteria for expressing transaminase, described
The host cell of engineering bacteria is E.coli BL21 (DE3).
3. production method as described in claim 1, which is characterized in that the additive amount of the cell be reaction solution weight 1~
15%.
4. production method as described in claim 1, which is characterized in that the molar ratio of the alanine and substrate is 1~6:1.
5. production method as described in claim 1, which is characterized in that there are coenzyme in reaction system, the coenzyme is phosphoric acid
Pyridoxal or phosphopyridoxamine.
6. production method as claimed in claim 5, which is characterized in that based on mass fraction, the additive amount of the coenzyme is
0.01~2 ‰.
7. production method as described in claim 1, which is characterized in that the temperature of the transamination reaction is 20~70 DEG C, the time
It is 6~72 hours.
8. production method as described in claim 1, which is characterized in that the pH value for controlling transamination reaction is 6~9.
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| CN106188134B (en) * | 2016-07-01 | 2018-03-20 | 永农生物科学有限公司 | The separation of a kind of L glufosinate-ammoniums or its salt and process for purification |
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| CN108795835B (en) * | 2018-06-21 | 2020-08-04 | 浙江大学 | Genetically engineered bacterium and application thereof in preparation of L-glufosinate-ammonium |
| CN109609478B (en) * | 2019-01-14 | 2020-06-16 | 浙江工业大学 | α -transaminase and mutant thereof and application thereof in asymmetric synthesis of L-glufosinate-ammonium |
| CN109609476B (en) * | 2019-01-14 | 2020-06-19 | 浙江工业大学 | α -transaminase and mutant and application thereof in asymmetric synthesis of L-glufosinate-ammonium |
| CN109735583A (en) * | 2019-03-21 | 2019-05-10 | 浙江工业大学 | A kind of method of single transaminase-catalyzed cascade reaction asymmetric syntheses L-glufosinate-ammonium |
| CN112626142B (en) * | 2020-12-17 | 2022-10-18 | 永农生物科学有限公司 | Method for preparing L-glufosinate-ammonium by biological multi-enzyme coupling method |
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