CN109777845A - A kind of preparation method of C4H9NO2 - Google Patents
A kind of preparation method of C4H9NO2 Download PDFInfo
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- CN109777845A CN109777845A CN201910246388.6A CN201910246388A CN109777845A CN 109777845 A CN109777845 A CN 109777845A CN 201910246388 A CN201910246388 A CN 201910246388A CN 109777845 A CN109777845 A CN 109777845A
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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
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention belongs to enzyme catalysis fields, more particularly to a kind of preparation method of C4H9NO2, enzyme catalyst and zymoexciter are added in substrate, the substrate is threonine and ammonium formate, the enzyme catalyst is threonine deaminase, leucine dehydrogenase and hydrogenlyase, and the zymoexciter is phosphate.The above-mentioned method for preparing C4H9NO2, using phosphate as zymoexciter, it is used cooperatively with leucine dehydrogenase, the intermediate product of reaction is converted rapidly, it is mutually matched the catalytic rate of three kinds of enzymes, improves the transformation efficiency of enzymatic, avoids the packing phenomenon of by-product, the yield for not only increasing product is more conducive to the separating-purifying of product.
Description
Technical field
The invention belongs to enzyme catalysis field more particularly to a kind of preparation methods of C4H9NO2.
Background technique
C4H9NO2 is a kind of Non-natural chiral amino acid, has and inhibits the transmitting of human nerve information, reinforces grape
The activity of sugar phosphate enzyme and the effect for promoting brain cell to be metabolized are a kind of important industrial chemicals and medicine intermediate,
It is widely used in pharmaceutical synthesis, such as antibacterial antituberculotic ethambutol hydrochloride, antiepileptic Levetiracetam and cloth
Wa Xitan etc..
Currently, the production method of C4H9NO2 is divided into chemical method and biological enzyme both at home and abroad, chemical method be compare through
The method of allusion quotation, mainly there is chemical synthesis and chemical resolution.Chemical synthesis is mainly using n-butyric acie and bromine as starting material, by α
α-bromo-butyric acid is made in halogenating reaction, the synthesizing amino butyric acid in ammonium hydroxide, and chemical resolution method is to use hand-type resolving agent, but because make
Expensive hand-type resolution reagent is used, cost is relatively high, and industrial production should not use, and it is dirty to generate a large amount of organic solvent slop
Contaminate environment.
Biological enzyme includes microbe fermentation method and enzymatic conversion method, microbe fermentation method reaction product complicated component,
Separate it is cumbersome, enzymatic conversion be then a kind of highly selective reaction, thus achieve the purpose that orientation conversion.
Enzymatic conversion method is prepared there are mainly two types of C4H9NO2s: first is that the butyrine to racemization carries out enzyme process
Preparation is split, aceticanhydride acetylated amino butyric acid is mainly used, is then split again with amino-acylase, reaction step is more, receives
Rate is relatively low；Second is that this method reaction substrate concentration is low, high production cost using alpha-carbonyl butyric acid as raw material.
Application No. is 201010146920.6, the China of entitled " a kind of method of preparing L-2-aminobutyric acid by enzyme " is specially
Benefit, disclosed scheme is: using L-threonine for starting material, so that it is converted into 2- ketone fourth by threonine deaminase first
Then acid makes 2- ketone butyric acid be converted into C4H9NO2, is added in reaction and is used for regenerating coenzyme using leucine dehydrogenase
Hydrogenlyase.This method prepares C4H9NO2, and product separation is carried out in a manner of crystallization, substantially increases product
Purity, but the problem of be not directed to the conversion ratio of C4H9NO2.
Summary of the invention
The purpose of the present invention is to provide a kind of high conversion, low cost and the L-2- amino fourths for being easy to industrialized production
The preparation method of acid.
To achieve the above object, the technical solution adopted by the present invention are as follows: a kind of preparation method of C4H9NO2, the bottom of at
Enzyme catalyst and zymoexciter are added in object, the substrate is threonine and ammonium formate, and the enzyme catalyst is threonine deaminase
Enzyme, leucine dehydrogenase and hydrogenlyase, the zymoexciter are phosphate.
The above-mentioned method for preparing C4H9NO2 is cooperated using phosphate as zymoexciter with leucine dehydrogenase
It uses, the intermediate product of reaction is converted rapidly, the catalytic rate of three kinds of enzymes is mutually matched, improve the conversion effect of enzymatic
Rate avoids the packing phenomenon of by-product, not only increases the yield of product, is more conducive to the separating-purifying of product.
Preferably, the preparation method of the C4H9NO2, the specific steps are that:
A threonine and ammonium formate) are added into water, is configured to conversion fluid A；
B phosphate) is added into conversion fluid and obtains solution B；
C threonine deaminase, leucine dehydrogenase and hydrogenlyase) are added into solution B, it is anti-under the conditions of (30 ± 5) DEG C
16~20h is answered, after reaction up to L- aminobutyric acid solution.
Preferably, the mass ratio of threonine and ammonium formate is 2:(1~2).Specifically, after threonine and ammonium formate are added,
The two concentration is respectively 100~200g/L and 50~200g/L.Water used is tap water in synthesis process, without steaming to water
It evaporates or the processing such as deionization, therefore method of the invention reduces the requirement to reaction environment, while also reducing and being produced into
This investment.
Preferably, phosphate additional amount are as follows: phosphorus acid ion concentration is 0.5mmol~20mmol/L.Phosphate anion is made
For zymoexciter, enzymatic activity can be improved, cooperation leucine dehydrogenase uses, and rapid conversion intermediate product further promotes
The catalytic rate of the rate of initial reaction, three kinds of enzymes is mutually matched, and is improved the transformation efficiency of enzymatic, is avoided by-product
Packing phenomenon.But removal difficulty is increased after largely introducing phosphate anion, it finally will affect product purity, therefore realizing
While the effect of zymoexciter, introducing phosphate anion as few as possible is needed.Specifically, the phosphate is biphosphate
Ammonium, disodium hydrogen phosphate, sodium dihydrogen phosphate, diammonium hydrogen phosphate, calcium monohydrogen phosphate, calcium phosphate, calcium pyrophosphate, potassium dihydrogen phosphate, phosphoric acid
At least one of hydrogen dipotassium, sodium acid pyrophosphate, sodium phosphate, sodium pyrophosphate.After phosphate is added, pH adjusting agent is added and adjusts
For the pH value of mixed solution to 7~8, the pH adjusting agent is at least one of ammonium hydroxide, sodium hydroxide, potassium hydroxide.
Preferably, after the microbial inoculum for producing threonine deaminase, leucine dehydrogenase and hydrogenlyase is added, total enzyme
Concentration is 3~6U.Wherein, enzyme activity is defined as: the C4H9NO2 that every milliliter of conversion fluid generates 1 μm of ol per minute is an enzyme
Unit living.
The source of enzyme in the present invention program are as follows: (nucleotide sequence is by the L-threonine deaminase of Escherichia coli
The upper Gene ID:948287 of NCBI), it is the L-Leu dehydrogenase (the upper Gene ID:1206507 of NCBI) in bacillus source, rich
The hydrogenlyase (the upper GenBank:KM454879.1 of NCBI) of Yi Ding Candida, is imported by the method for genetic engineering bacterium
In Escherichia coli, building while the genetic engineering bacterium for producing L-threonine dehydrogenase, L-Leu dehydrogenase, hydrogenlyase are kept away
The purification step for exempting from enzyme, using more convenient.
Detailed description of the invention
Fig. 1 is the line chart that conversion ratio changes over time in the reaction process of embodiment 1.
Specific embodiment
One, it prepares
Embodiment 1
1200g threonine, 1200g ammonium formate, ammonium dihydrogen phosphate are added into 10L reaction kettle, adds water and stirs dissolution, then
PH to 7.5 is adjusted with 20-25% ammonium hydroxide, is warming up to 30 DEG C, is then added and produces threonine deaminase, leucine dehydrogenase and formic acid
The bacterium mud of dehydrogenase, wherein phosphorus acid ion concentration 20mmol/L, total enzyme concentration is 6U in mixed liquor, and mixed liquor is placed in
It is stirred to react under 30 DEG C of constant temperatures, reaction terminates after 16h, obtains L- aminobutyric acid solution.
Embodiment 2
1200g threonine, 600g ammonium formate, diammonium hydrogen phosphate are added into 10L reaction kettle, adds water and stirs dissolution, then
PH to 7.5 is adjusted with 20-25% ammonium hydroxide, is warming up to 30 DEG C, is then added and produces threonine deaminase, leucine dehydrogenase and formic acid
The bacterium mud of dehydrogenase, wherein phosphorus acid ion concentration 10mmol/L, total enzyme concentration is 4U in mixed liquor, and mixed liquor is placed in
It is stirred to react under 30 DEG C of constant temperatures, reaction terminates after 20h, obtains L- aminobutyric acid solution.
Embodiment 3
1200g threonine, 800g ammonium formate, sodium pyrophosphate are added into 10L reaction kettle, adds water and stirs dissolution, then uses
20-25% ammonium hydroxide adjusts pH to 7.7, is warming up to 30 DEG C, and it is de- that production threonine deaminase, leucine dehydrogenase and formic acid is then added
The bacterium mud of hydrogen enzyme, wherein phosphorus acid ion concentration 15mmol/L, total enzyme concentration is 6U in mixed liquor, and mixed liquor is placed in 30
It is stirred to react under DEG C constant temperature, reaction terminates after 18h, obtains L- aminobutyric acid solution.
Two, it detects
By in 1 reaction process of embodiment, threonine and L- ammonia in reaction system were detected with liquid chromatograph every two hours
The content of base butyric acid, and calculate conversion ratio.
Detection method is as follows: the borate buffer 300ul of PH=9.5, conversion fluid sample are sequentially added in EP pipe
250ul, derivating agent 200ul (take 0.3430g o-phthalaldehyde+5ml dehydrated alcohol+0.1472gN- acetyl-L cysteine, use
0.1mol/L borate buffer solution (PH=9.5) is fixed molten to 25ml, is protected from light spare), it is waited 2 minutes after mixing, the strict control time
With reagent additive amount, then sample introduction.Required compound 0.05mol/L sodium-acetate buffer: methanol=63: 35 are washed
It is de-, flow velocity 1.0ml/min, acquisition time 10min.Chromatographic condition is XDB-C8 (150mm) neutral column, 30 DEG C of column temperature, is detected
Wavelength 334nm.
The resulting conversion ratio of various time points is recorded into table 1, and makes the line chart that conversion ratio changes over time, and is seen attached
Fig. 1.
The conversion ratio at each time point of 1 embodiment of table 1
| Reaction time/h | 2 | 4 | 6 | 8 | 10 | 12 | 14 | 16 | 18 | 20 |
| Conversion ratio/% | 25.2 | 40.0 | 55.3 | 70.1 | 80.5 | 87.2 | 93.4 | 98.0 | 98.4 | 98.6 |
As shown in Table 1, the L- aminobutyric acid solution prepared using the preparation method of embodiment 1, threonine are converted into L- ammonia
The conversion ratio of base butyric acid is 98.6%.
According to the method described above, the conversion ratio of system after embodiment 2 and 3 reaction process neutralization reaction of embodiment is detected,
Obtain final result: the conversion ratio reacted in embodiment 2 is 98.1%, and the conversion ratio reacted in embodiment 3 is 98.4%, can
See, preparation method of the invention prepares L- aminobutyric acid, and high conversion rate is up to 98% or more.
Claims (8)
1. a kind of preparation method of C4H9NO2, it is characterised in that: enzyme catalyst and zymoexciter, institute are added in substrate
Stating substrate is threonine and ammonium formate, and the enzyme catalyst is threonine deaminase, leucine dehydrogenase and hydrogenlyase, institute
Stating zymoexciter is phosphate；
The phosphate be ammonium dihydrogen phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, diammonium hydrogen phosphate, calcium monohydrogen phosphate, calcium phosphate,
At least one of calcium pyrophosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium acid pyrophosphate, sodium phosphate, sodium pyrophosphate.
2. the preparation method of C4H9NO2 according to claim 1, the specific steps are that:
A threonine and ammonium formate) are added into water, is configured to conversion fluid A；
B phosphate) is added into conversion fluid and obtains solution B；
C threonine deaminase, leucine dehydrogenase and hydrogenlyase) are added into solution B, it is anti-under the conditions of (30 ± 5) DEG C
16~20h is answered, after reaction up to L- aminobutyric acid solution.
3. the preparation method of C4H9NO2 according to claim 1 or 2, it is characterised in that: threonine and ammonium formate
Mass ratio be 2:(1~2).
4. the preparation method of C4H9NO2 according to claim 1 or claim 2, it is characterised in that: phosphate additional amount are as follows: phosphorus
Acid ion concentration is 0.5mmol~20mmol/L.
5. the preparation method of C4H9NO2 according to claim 2, it is characterised in that: the step B) in, phosphorus is added
After hydrochlorate, the pH value of pH adjusting agent adjusting mixed solution is added to 7~8, the pH adjusting agent is ammonium hydroxide, sodium hydroxide, hydrogen-oxygen
Change at least one of potassium.
6. the preparation method of C4H9NO2 according to claim 2, it is characterised in that: the step C) in, it is added and produces
After the microbial inoculum of threonine deaminase, leucine dehydrogenase and hydrogenlyase, in mixed solution total enzyme concentration be 3~
6U。
7. the preparation method of C4H9NO2 according to claim 1 or claim 2, it is characterised in that: threonine and ammonium formate add
After entering, the two concentration is respectively 100~200g/L and 50~200g/L.
8. the preparation method of C4H9NO2 according to claim 1 or claim 2, it is characterised in that: water used in preparation process
For tap water.
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Cited By (3)
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| CN109999016A (en) * | 2019-05-23 | 2019-07-12 | 济宁医学院附属医院 | Application of the ebutol in treatment intractable epilepsy |
| CN112680487A (en) * | 2021-02-03 | 2021-04-20 | 江南大学 | Method for synthesizing L-2-aminobutyric acid by catalyzing long-side-chain aromatic amine and 2-ketobutyric acid |
| WO2021143356A1 (en) * | 2020-01-18 | 2021-07-22 | 江南大学 | Method for preparing l-2-aminobutyric acid by means of double enzyme series connection |
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| CN112680487A (en) * | 2021-02-03 | 2021-04-20 | 江南大学 | Method for synthesizing L-2-aminobutyric acid by catalyzing long-side-chain aromatic amine and 2-ketobutyric acid |
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