CN111909979A - Preparation method of L-glufosinate-ammonium - Google Patents
Preparation method of L-glufosinate-ammonium Download PDFInfo
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Abstract
The invention discloses a preparation method of a compound shown in formula I, which is obtained by reacting a compound shown in formula V, wherein the reaction formula is as follows:
R1selected from the group consisting of alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl, substituted heteroaryl; r2Selected from the group consisting of hydrogen, alkyl, silyl ether protecting groups, benzyl ether protecting groups, alkoxymethyl, alkoxy-substituted methyl, C2-C10 linear or branched alkenylalkyl, C2-C10 linear or branched alkynylalkyl, 3-8 membered cycloaliphatic, aryl, heteroaryl, substituted aryl,Ar(CH2)n-one of the groups, Ar represents an aryl or heteroaryl group, and n is from 1 to 6. The method has the advantages of simple process flow, no special requirements on equipment, suitability for industrial production, environmental friendliness and great reduction of cost.
Description
Technical Field
The invention relates to a preparation method of a pesticide, in particular to a preparation method of L-glufosinate-ammonium.
Background
Glufosinate, chemical name 4- [ hydroxy (methyl) phosphono ] -DL-homoalanine, developed and produced by hester (bayer, germany) in the last 80 th century, is a phosphonic acid herbicide, is a glutamine synthesis inhibitor, a non-selective contact herbicide, which was registered for use as a herbicide in 1984. Glufosinate technical material registration is only available in 2004 and product registration is only available in 2005.
Since the wide application of glyphosate, glyphosate-resistant weeds are increasing and the harm is gradually aggravated. Paraquat is a strong weed-killing herbicide and has strong toxic effect on human and livestock. 7, 1 month in 2014, China cancels paraquat water registration and production permission and stops production; the water aqua is sold and used in China after 2016, 7 months and 1 day. Glufosinate is a world wide large tonnage pesticide variety and is also a herbicide tolerant for the second largest transgenic crop in the world. The glufosinate-ammonium has low toxicity, is relatively safe, is easy to degrade in soil, is safe to crops, is not easy to drift, has a wide weeding spectrum, is high in activity, small in dosage, small in environmental pressure, can quickly kill more than 100 gramineous weeds and broadleaf weeds, can use water as a base agent, is safe and convenient to use, and has the characteristic that the product is superior to other herbicides, so that the glufosinate-ammonium can still be sold after a plurality of high-efficiency and super-high-efficiency products appear.
At present, the synthetic technical routes of glufosinate-ammonium at home and abroad are more, and the review reports are made on Yanghai Chang et al (pesticides, 2002, 41(9), 46-48), but the problems of more reaction steps and high production cost exist in each route generally. Bayer, U.S. Pat. Nos. 4,193,521 and 6359162, disclose a process for synthesizing glufosinate-ammonium by a series of reactions from dichloromethylphosphine and methyl phosphite. The method has high yield and low cost, but the starting route has active physicochemical properties of the raw materials and products for synthesizing the dichloromethylphosphine, and is flammable and explosive. The synthesis process is controlled at 500-600 ℃, and if the control is unstable, yellow phosphorus and phosphine which are very easy to be natural are easily generated, so that the danger is very high. In addition, the materials have strong corrosivity and have strict requirements on material selection of reaction equipment and processing and manufacturing processes of the reaction equipment, and the current domestic processing and manufacturing level is difficult to meet the production requirements.
These methods are all methods for preparing glufosinate-ammonium, which is an L/D mixed type, wherein L-type is mainly used; l-glufosinate-ammonium can be degraded by microorganisms in soil, while D-glufosinate-ammonium is difficult to degrade, and finally soil hardening can be caused. Therefore, the L-glufosinate-ammonium is more efficient, lower in cost and safer than the traditional glufosinate-ammonium.
Therefore, it is necessary to develop a biosynthetic preparation method of L-glufosinate-ammonium, which can improve the utilization rate of raw materials, reduce the production cost and avoid the danger in the industrial process.
Disclosure of Invention
The invention aims to overcome the defects of the background technology and provide a preparation method of L-glufosinate-ammonium, which can improve the utilization rate of raw materials and reduce the production cost and is suitable for industrial production.
The invention provides a preparation method of a compound shown in formula I, wherein the compound shown in formula II is reacted under the action of E1 and E2 to prepare the compound shown in formula I, and the reaction formula is as follows:
e1 is a substance capable of hydrolyzing a compound of formula II to a compound of formula I;
e2 is a substance capable of racemizing the compound of the formula III;
wherein R is1Selected from the group consisting of alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl, substituted heteroaryl.
R2Selected from amino OR-OR0；R0Selected from the group consisting of hydrocarbyl, silyl, benzyl, alkyl methyl, alkyl-substituted alkoxy, 3-8 membered cycloaliphatic, aryl, heteroaryl, substituted aryl, substituted heteroaryl, Ar (CH)2)nOne of O-groups, Ar represents aryl or heteroaryl, and n is 1-6.
Preferably, the preparation method of the L-glufosinate-ammonium comprises the following steps of reacting a compound shown in a formula IV under the action of E1 and E2 to prepare a compound shown in a formula V, deprotecting the compound shown in the formula V to obtain the L-glufosinate-ammonium,
wherein R is3Selected from the group consisting of hydrogen, alkyl, silyl ether protecting groups, benzyl ether protecting groups, alkoxymethyl, alkoxy-substituted methyl, C2-C10 linear or branched alkenylalkyl, C2-C10 linear or branched alkynylalkyl, 3-8 membered cycloaliphatic, aryl, heteroaryl, amino, substituted amino, Ar (CH)2)a-one of the groups, Ar represents an aryl, heteroaryl group, a takes 1 to 6.
Further, E1 is a biological enzyme, and E1 gene sources include Bacillus subtilis, Streptomyces ramulosus, Pseudomonas azotoformans, Bacillus licheniformis, Mycobacterium tuberculosis, pork liver, Candida antartica, Pseudomonas wadanswerensis, Pseudomonas sp, Brevundimonas diminuta.
Further, E2 is one or more aldehydes or ketones or organic acids, wherein the aldehydes include linear aldehydes, branched aldehydes, aromatic aldehydes, substituted aromatic aldehydes, heterocyclic aldehydes, substituted heterocyclic aldehydes.
Further, R3Is hydrogen, R2Is ethyl or propyl or isopropyl, and the pH of the reaction system is controlled to be 6.5-10.
Further, E2 is selected from one or more of glutaraldehyde, pyridoxal phosphate, salicylaldehyde, benzaldehyde, o-carboxybenzaldehyde, m-carboxybenzaldehyde, p-carboxybenzaldehyde, 9-fluorenone, 3, 5-dinitrosalicylaldehyde, 3, 5-dichlorosalicylaldehyde, and 5-bromo-2-hydroxy-3-nitrobenzaldehyde.
Further, enzyme E1 is a polypeptide having the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 or SEQ ID NO: 17, or the protein of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 or SEQ ID NO: 9 is a protein having the activity of hydrolyzing the compound of formula V to the compound of formula I after substitution, deletion or addition of one or more amino acid residues, or is SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 or SEQ ID NO: 9 has homology of more than 80 percent and is protein with the activity of hydrolyzing the compound shown in the formula V into the compound shown in the formula I, and the enzyme E1 is a whole cell, broken enzyme liquid, freeze-dried powder or immobilized enzyme or immobilized cell of a genetically engineered bacterium.
Further, the reaction is carried out in the presence of a solvent.
Further, the solvent is water or a mixed solvent composed of a buffer solution and an organic solvent.
Further, the buffer solution is selected from one or more of disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate-sodium hydroxide buffer solution, carbonate buffer solution, Tri-HCl buffer solution, citric acid-sodium citrate buffer solution, glycine-sodium hydroxide buffer solution or MOPS buffer solution; the organic solvent is one or more selected from DMSO, ethyl acetate, butyl acetate, isopropanol, DMF, TBME, dichloromethane, tert-butyl alcohol, n-butyl alcohol, vinyl acetate, benzene and toluene.
The advantages of the invention are mainly embodied in the following aspects:
firstly, the process flow is simple, no special requirements are required on equipment, and the method is suitable for industrial production;
secondly, the invention has high utilization rate of the substrate, mild reaction condition and environmental protection;
thirdly, the preparation process of the invention is an upgrade and update of the traditional chemical synthesis method, thus greatly reducing the cost.
Detailed Description
The present invention is described in further detail below with reference to specific examples, which should not be construed as limiting the invention.
Example 1
1. Expression of the enzyme E1 in E.coli
Respectively selecting enzymes from bacillus subtilis, Streptomyces ramulosus, Pseudomonas azotoformans, bacillus licheniformis, Mycobacterium tuberculosis, pork liver, Candida antartica, Pseudomonas wadensswellensis, Pseudomonas sp, Brevundimonas diminuta and the like, and a plurality of sequences, wherein the gene sequences are expressed in escherichia coli through codon optimization, cloned into an expression vector pET28a through artificial synthesis and put into an expression strain host bacterium E.coli BL21(DE3), and obtaining a recombinant expression vector after picking a transformant for positive sequencing and identification; transferring the recombinant expression vector into an E.coli BL21(DE3) strain;
coli BL21(DE3) strain containing cloned gene was cultured overnight at 37 ℃ in LB medium with appropriate antibiotics to obtain seed culture solution; inoculating the seed culture solution into TB culture medium containing proper antibiotics, wherein the inoculation amount is 1 percent of the volume of the TB culture medium containing kanamycin; culturing at 37 deg.C for 2-5h, adding sterile IPTG to make the final concentration of IPTG reach 0.1mM, and culturing at 25 deg.C for 20 h.
Example 2
Racemic 2-aminopropionamide reacts under the action of enzymes E1 and E2 to prepare the S-2-aminopropionic acid with single chirality.
Enzyme E1 expression the reaction (10mL) was carried out as described in example 1 using enzyme E1(50g wet cells/L), butyl acetate (0.25mL), tert-butanol (5.5mL), E2(1mM) and racemic 2-aminopropionamide (30g/L) in disodium hydrogenphosphate-potassium dihydrogenphosphate buffer, pH controlled at 8.0, conversion temperature at 37 ℃ and conversion for 12h to give S-2-aminopropionic acid of single chirality. The derivatization assay, conversion and EE value are shown in the table below:
| serial number | Origin of enzyme E1 | Enzyme E1 amino acid sequence | E2 | Conversion rate | EE value |
| 1 | Streptomyces ramulosus | SEQ ID NO：1 | Glutaraldehyde | 96.4％ | 89.1％ |
| 2 | Pseudomonas azotoformans | SEQ ID NO：2 | Pyridoxal phosphate | 96.1％ | 91.2％ |
| 3 | Mycobacterium tuberculosis | SEQ ID NO：3 | Salicylaldehyde | 93.5％ | 95.3％ |
| 4 | Bacillus subtilis | SEQ ID NO：4 | Benzaldehyde | 96.7％ | 93.1％ |
| 5 | Candida antartica | SEQ ID NO：5 | O-carboxybenzaldehyde | 92.6％ | 96.8％ |
| 6 | Bacillus licheniformis | SEQ ID NO：6 | 3, 5-dinitrosalicylaldehyde | 97.4％ | 96.3％ |
| 7 | Pseudomonas sp | SEQ ID NO：7 | 9-fluorenones | 89.1％ | 92.8％ |
| 8 | Brevundimonas diminuta | SEQ ID NO：8 | 5-bromo-2-hydroxy-3-nitrobenzaldehyde | 91.6％ | 89.8％ |
Example 3
Racemic methyl 2-aminobutyric acid is reacted under the action of enzymes E1 and E2 to prepare the S-2-aminobutyric acid with single chirality.
Enzyme E1 expression the reaction (50mL) was carried out using the enzymes E1(60g wet cells/L), DMSO (2.5mL), n-butanol (38mL), E2(1mM) and racemic methyl 2-aminobutyric acid (20g/L) in glycine-sodium hydroxide buffer solution, pH controlled at 10.0, conversion temperature 30 ℃ to give S-2-aminobutyric acid of single chirality after 12h conversion, as described in example 1. The derivatization assay, conversion and EE value are shown in the table below:
| serial number | Origin of enzyme E1 | Enzyme E1 amino acid sequence | E2 | Conversion rate | EE value |
| 1 | Streptomyces ramulosus | SEQ ID NO：1 | Glutaraldehyde | 96.9％ | 85.6％ |
| 2 | Pseudomonas azotoformans | SEQ ID NO：2 | Pyridoxal phosphate | 91.6％ | 95.8％ |
| 3 | Mycobacterium tuberculosis | SEQ ID NO：3 | Salicylaldehyde | 95.7％ | 97.5％ |
| 4 | Bacillus subtilis | SEQ ID NO：4 | Benzaldehyde | 96.8％ | 95.9％ |
| 5 | Candida antartica | SEQ ID NO：5 | O-carboxybenzaldehyde | 89.7％ | 92.8％ |
| 6 | Bacillus licheniformis | SEQ ID NO：6 | 3, 5-dinitrosalicylaldehyde | 96.9％ | 98.1％ |
| 7 | Pseudomonas sp | SEQ ID NO：7 | 9-fluorenones | 89.7％ | 93.2％ |
| 8 | Brevundimonas diminuta | SEQ ID NO：8 | 5-bromo-2-hydroxy-3-nitrobenzaldehyde | 88.4％ | 93.2％ |
Example 4
Racemic benzyl 2-amino-4-methyl-pentanoate is reacted under the action of enzymes E1 and E2 to prepare the S-2-amino-4-methyl-pentanoic acid with single chirality.
Enzyme E1 expression the reaction (20mL) was carried out as described in example 1 using the enzymes E1(40g wet cells/L), ethyl acetate (12mL), E2(5mM) and racemic benzyl 2-amino-4-methyl-pentanoate (10g/L) in disodium hydrogenphosphate-monosodium phosphate buffer, controlling pH at 7.5, conversion temperature at 25 ℃ and after 12h conversion, single chiral S-2-amino-4-methyl-pentanoic acid was obtained, with the conversion and EE values determined as shown in the following table:
| serial number | Origin of enzyme E1 | Enzyme E1 amino acid sequence | E2 | Conversion rate | EE value |
| 1 | Streptomyces ramulosus | SEQ ID NO：1 | Glutaraldehyde | 98.1％ | 92.6％ |
| 2 | Pseudomonas azotoformans | SEQ ID NO：2 | Pyridoxal phosphate | 88.9％ | 88.2％ |
| 3 | Mycobacterium tuberculosis | SEQ ID NO：3 | Salicylaldehyde | 93.6％ | 89.8％ |
| 4 | Bacillus subtilis | SEQ ID NO：4 | Benzaldehyde | 92.6％ | 89.2％ |
| 5 | Candida antartica | SEQ ID NO：5 | O-carboxybenzaldehyde | 96.9％ | 91.6％ |
| 6 | Bacillus licheniformis | SEQ ID NO：6 | 3, 5-dinitrosalicylaldehyde | 91.7％ | 96.4％ |
| 7 | Pseudomonas sp | SEQ ID NO：7 | 9-fluorenones | 88.5％ | 88.9％ |
| 8 | Brevundimonas diminuta | SEQ ID NO：8 | 5-bromo-2-hydroxy-3-nitrobenzaldehyde | 93.6％ | 85.7％ |
Example 5
Racemic 2-amino-3-phenyl-propionic acid methyl ester reacts under the action of enzymes E1 and E2 to prepare the S-2-amino-3-phenyl-propionic acid with single chirality.
Enzyme E1 expression the reaction (100mL) was carried out as described in example 1 using enzyme E1(40g wet cells/L), TBME (4.5mL), isopropanol (69mL), E2(3mM) and racemic methyl 2-amino-3-phenyl-propionate (45g/L) in potassium dihydrogen phosphate-sodium hydroxide buffer, pH controlled at 7.5, conversion temperature 25 ℃ to give S-2-amino-3-phenyl-propionic acid of single chirality after 12h of conversion, with the derivation measurements of conversion and EE values as shown in the following table:
| serial number | Origin of enzyme E1 | Enzyme E1 amino acid sequence | E2 | Conversion rate | EE value |
| 1 | Streptomyces ramulosus | SEQ ID NO：1 | Glutaraldehyde | 96.4％ | 87.6％ |
| 2 | Pseudomonas azotoformans | SEQ ID NO：2 | Pyridoxal phosphate | 89.5％ | 94.6％ |
| 3 | Mycobacterium tuberculosis | SEQ ID NO：3 | Salicylaldehyde | 93.6％ | 91.8％ |
| 4 | Bacillus subtilis | SEQ ID NO：4 | Benzaldehyde | 86.8％ | 92.6％ |
| 5 | Candida antartica | SEQ ID NO：5 | O-carboxybenzaldehyde | 89.5％ | 87.3％ |
| 6 | Bacillus licheniformis | SEQ ID NO：6 | 3, 5-dinitrosalicylaldehyde | 92.7％ | 95.6％ |
| 7 | Pseudomonas sp | SEQ ID NO：7 | 9-fluorenones | 91.5％ | 88.9％ |
| 8 | Brevundimonas diminuta | SEQ ID NO：8 | 5-bromo-2-hydroxy-3-nitrobenzaldehyde | 92.8％ | 93.8％ |
Example 6
Racemic 2-amino-3-chloro-butyric acid methyl ester reacts under the action of enzymes E1 and E2 to prepare the S-2-amino-3-chloro-butyric acid with single chirality.
Enzyme E1 expression the reaction (30mL) was carried out as described in example 1 using the enzymes E1(60g wet cells/L), butyl acetate (3mL), vinyl acetate (20mL), E2(2mM) and racemic methyl 2-amino-4-methyl-valerate (35g/L) in citrate-sodium citrate buffer solution, controlling the pH at 6.0, the conversion temperature at 28 ℃ and after 12h of conversion, obtaining S-2-amino-3-chloro-butyric acid of single chirality, the derivatization assay conversions and EE values are shown in the following table:
| serial number | Origin of enzyme E1 | Enzyme E1 amino acid sequence | E2 | Conversion rate | EE value |
| 1 | Streptomyces ramulosus | SEQ ID NO：1 | Glutaraldehyde | 92.9％ | 95.7％ |
| 2 | Pseudomonas azotoformans | SEQ ID NO：2 | Pyridoxal phosphate | 88.6％ | 95.6％ |
| 3 | Mycobacterium tuberculosis | SEQ ID NO：3 | Salicylaldehyde | 89.7％ | 91.6％ |
| 4 | Bacillus subtilis | SEQ ID NO：4 | Benzaldehyde | 91.3％ | 89.8％ |
| 5 | Candida antartica | SEQ ID NO：5 | O-carboxybenzaldehyde | 95.7％ | 88.9％ |
| 6 | Bacillus licheniformis | SEQ ID NO：6 | 3, 5-dinitrosalicylaldehyde | 91.8％ | 91.6％ |
| 7 | Pseudomonas sp | SEQ ID NO：7 | 9-fluorenones | 92.7％ | 94.3％ |
| 8 | Brevundimonas diminuta | SEQ ID NO：8 | 5-bromo-2-hydroxy-3-nitrobenzaldehyde | 90.4％ | 94.8％ |
Example 7
Racemic 2-amino-4- (hydroxymethyl phosphonyl) -butyramide is reacted under the action of enzymes E1 and E2 to prepare the L-2-amino-4- (methoxymethylphosphonyl) -butyric acid with single chirality.
Enzyme E1 expression the reaction (50mL) was carried out as described in example 1 using the enzymes E1(35g wet cells/L), DMF (4mL), dichloromethane (40mL), E2(4mM) and racemic 2-amino-4- (hydroxymethylphosphono) -butyramide (50g/L) in MOPS buffer, pH controlled at 6.5, conversion temperature controlled at 37 ℃ to give, after 12h of conversion, a single chiral L-2-amino-4- (hydroxymethylphosphono) -butyric acid, i.e.L-glufosinate, with derivatization to determine conversion and EE values as shown in the following table:
| serial number | Origin of enzyme E1 | Enzyme E1 amino acid sequence | E2 | Conversion rate | EE value |
| 1 | Streptomyces ramulosus | SEQ ID NO：1 | Glutaraldehyde | 96.1％ | 94.9％ |
| 2 | Pseudomonas azotoformans | SEQ ID NO：2 | Pyridoxal phosphate | 89.3％ | 98.4％ |
| 3 | Mycobacterium tuberculosis | SEQ ID NO：3 | Salicylaldehyde | 91.1％ | 92.1％ |
| 4 | Bacillus subtilis | SEQ ID NO：4 | Benzaldehyde | 88.7％ | 92.5％ |
| 5 | Candida antartica | SEQ ID NO：5 | O-carboxybenzaldehyde | 91.2％ | 98.9％ |
| 6 | Bacillus licheniformis | SEQ ID NO：6 | 3, 5-dinitrosalicylaldehyde | 95.3％ | 98.4％ |
| 7 | Pseudomonas sp | SEQ ID NO：7 | 9-fluorenones | 93.9％ | 91.8％ |
| 8 | Brevundimonas diminuta | SEQ ID NO：8 | 5-bromo-2-hydroxy-3-nitrobenzaldehyde | 87.7％ | 92.4％ |
Example 8
Racemic 2-amino-4- (isopropoxymethylphosphono) -butyric acid tert-butyl ester reacts under the action of enzymes E1 and E2 to prepare the L-2-amino-4- (isopropoxymethylphosphono) -butyric acid with single chirality.
The enzyme E1 was expressed as described in example 1, and the reaction (100mL) was carried out using the enzyme E1(80g wet cells/L), toluene (1mL), t-butanol (70mL), E2(6mM) and racemic tert-butyl 2-amino-4- (isopropoxymethylphosphono) -butyrate (40g/L) in disodium hydrogenphosphate-potassium dihydrogenphosphate buffer solution, pH controlled at 8.0, conversion temperature controlled at 35 ℃ after 12h of conversion, hydrochloric acid at a concentration of 10% to 37% was added to the conversion solution to deprotect the same to give L-2-amino-4- (isopropoxymethylphosphono) -butyric acid of single chirality, chemical deprotection to give L-2-amino-4- (hydroxymethyl) -butyric acid of single chirality, i.e., L-glufosinate, the derivatization assay conversion and EE value are shown in the table below:
| serial number | Origin of enzyme E1 | Enzyme E1 amino acid sequence | E2 | Conversion rate | EE value |
| 1 | Streptomyces ramulosus | SEQ ID NO：1 | Glutaraldehyde | 91.3％ | 88.9％ |
| 2 | Pseudomonas azotoformans | SEQ ID NO：2 | Pyridoxal phosphate | 92.6％ | 79.5％ |
| 3 | Mycobacterium tuberculosis | SEQ ID NO：3 | Salicylaldehyde | 94.2％ | 92.4％ |
| 4 | Bacillus subtilis | SEQ ID NO：4 | Benzaldehyde | 93.6％ | 83.4％ |
| 5 | Candida antartica | SEQ ID NO：5 | O-carboxybenzaldehyde | 88.9％ | 84.6％ |
| 6 | Bacillus licheniformis | SEQ ID NO：6 | 3, 5-dinitrosalicylaldehyde | 93.7％ | 91.4％ |
| 7 | Pseudomonas sp | SEQ ID NO：7 | 9-fluorenones | 93.4％ | 87.9％ |
| 8 | Brevundimonas diminuta | SEQ ID NO：8 | 5-bromo-2-hydroxy-3-nitrobenzaldehyde | 85.7％ | 88.6％ |
Example 9
Racemic 2-amino-4- (methoxymethylphosphonyl) -butyric acid methyl ester reacts under the action of enzymes E1 and E2 to prepare the L-2-amino-4- (methoxymethylphosphonyl) -butyric acid with single chirality.
Enzyme E1 expression the reaction (50mL) was carried out as described in example 1 using the enzymes E1(30g wet cells/L), TBME (10mL), isopropanol (20mL), E2(8mM) and racemic methyl 2-amino-4- (methoxymethylphosphonyl) -butyrate (90g/L) in glycine-sodium hydroxide buffer solution, pH 8.5 controlled, conversion temperature 37 ℃ controlled, after 12h of conversion to give mono-chiral L-2-amino-4- (methoxymethylphosphonyl) -butyric acid, chemical deprotection to give mono-chiral L-2-amino-4- (hydroxymethylphosphono) -butyric acid, L-glufosinate, conversion of derivatization and EE values as shown in the following table:
| serial number | Origin of enzyme E1 | Enzyme E1 amino acid sequence | E2 | Conversion rate | EE value |
| 1 | Streptomyces ramulosus | SEQ ID NO：1 | Glutaraldehyde | 96.4％ | 97.4％ |
| 2 | Pseudomonas azotoformans | SEQ ID NO：2 | Pyridoxal phosphate | 92.5％ | 94.3％ |
| 3 | Mycobacterium tuberculosis | SEQ ID NO：3 | Salicylaldehyde | 93.6％ | 92.9％ |
| 4 | Bacillus subtilis | SEQ ID NO：4 | Benzaldehyde | 91.6％ | 82.7％ |
| 5 | Candida antartica | SEQ ID NO：5 | O-carboxybenzaldehyde | 85.9％ | 93.4％ |
| 6 | Bacillus licheniformis | SEQ ID NO：6 | 3, 5-dinitrosalicylaldehyde | 92.3％ | 94.6％ |
| 7 | Pseudomonas sp | SEQ ID NO：7 | 9-fluorenones | 86.7％ | 91.3％ |
| 8 | Brevundimonas diminuta | SEQ ID NO：8 | 5-bromo-2-hydroxy-3-nitrobenzaldehyde | 93.6％ | 86.4％ |
Example 10
Racemic 2-amino-4- (ethoxymethylphosphono) -isopropyl butyrate is reacted under the action of enzymes E1 and E2 to prepare the L-2-amino-4- (ethoxymethylphosphono) -butyric acid with single chirality.
Expression of enzyme E1 As described in example 1, the reaction (200mL) was carried out using enzyme E1(90g wet cells/L), DMF (15mL), n-butanol (125mL), E2(7mM) and racemic isopropyl 2-amino-4- (ethoxymethylphosphono) -butyrate (70g/L) in Tris-hydrochloric acid buffer, pH 8.5 controlled, conversion temperature 40 ℃ controlled, after 12h of conversion, to give L-2-amino-4- (ethoxymethylphosphono) -butyric acid of single chirality, chemical deprotection to give L-2-amino-4- (hydroxymethylphosphono) -butyric acid of single chirality, i.e.L-glufosinate, with the measured conversions and EE values as shown in the following table:
| serial number | Origin of enzyme E1 | Enzyme E1 amino acid sequence | E2 | Conversion rate | EE value |
| 1 | Streptomyces ramulosus | SEQ ID NO：1 | Glutaraldehyde | 96.4％ | 91.7％ |
| 2 | Pseudomonas azotoformans | SEQ ID NO：2 | Pyridoxal phosphate | 94.5％ | 92.4％ |
| 3 | Mycobacterium tuberculosis | SEQ ID NO：3 | Salicylaldehyde | 92.6％ | 93.4％ |
| 4 | Bacillus subtilis | SEQ ID NO：4 | Benzaldehyde | 95.1％ | 86.7％ |
| 5 | Candida antartica | SEQ ID NO：5 | O-carboxybenzaldehyde | 92.4％ | 89.4％ |
| 6 | Bacillus licheniformis | SEQ ID NO：6 | 3, 5-dinitrosalicylaldehyde | 93.5％ | 95.2％ |
| 7 | Pseudomonas sp | SEQ ID NO：7 | 9-fluorenones | 93.4％ | 88.7％ |
| 8 | Brevundimonas diminuta | SEQ ID NO：8 | 5-bromo-2-hydroxy-3-nitrobenzaldehyde | 93.4％ | 87.5％ |
Example 11
Racemic 2-amino-4- (hydroxymethyl phosphonyl) -butyric acid benzyl ester reacts under the action of enzymes E1 and E2 to prepare the L-2-amino-4- (hydroxymethyl phosphonyl) -butyric acid with single chirality.
Enzyme E1 expression the reaction (100mL) was carried out as described in example 1 using enzyme E1(55g wet cells/L), vinyl acetate (10mL), tert-butanol (60mL), E2(6mM) and racemic benzyl 2-amino-4- (hydroxymethylphosphono) -butyrate (60g/L) in potassium dihydrogen phosphate-sodium hydroxide buffer solution, pH controlled at 7.0, conversion temperature controlled at 35 ℃ and after 12h of conversion, single chiral L-2-amino-4- (hydroxymethylphosphono) -butyric acid was obtained, with derivatisation to determine conversion and EE values as shown in the following table:
| serial number | Origin of enzyme E1 | Enzyme E1 amino acid sequence | E2 | Conversion rate | EE value |
| 1 | Streptomyces ramulosus | SEQ ID NO：1 | Glutaraldehyde | 95.4％ | 93.9％ |
| 2 | Pseudomonas azotoformans | SEQ ID NO：2 | Pyridoxal phosphate | 94.7％ | 79.1％ |
| 3 | Mycobacterium tuberculosis | SEQ ID NO：3 | Salicylaldehyde | 89.6％ | 95.7％ |
| 4 | Bacillus subtilis | SEQ ID NO：4 | Benzaldehyde | 91.8％ | 95.2％ |
| 5 | Candida antartica | SEQ ID NO：5 | O-carboxybenzaldehyde | 96.4％ | 91.2％ |
| 6 | Bacillus licheniformis | SEQ ID NO：6 | 3, 5-dinitrosalicylaldehyde | 91.6％ | 95.4％ |
| 7 | Pseudomonas sp | SEQ ID NO：7 | 9-fluorenones | 92.3％ | 82.7％ |
| 8 | Brevundimonas diminuta | SEQ ID NO：8 | 5-bromo-2-hydroxy-3-nitrobenzaldehyde | 87.6％ | 86.7％ |
Example 12
Racemic ethyl 2-amino-4- (hydroxymethylphosphono) -butyrate is reacted under the action of an enzyme E1 variant and an enzyme E2 variant to prepare the L-2-amino-4- (hydroxymethylphosphono) -butyric acid with single chirality.
Variant enzyme E1 and variant enzyme E2 the reactions (50mL) were carried out at 30 ℃ using the enzymes E1(70g wet cells/L), benzene (35mL), E2(4mM) and racemic ethyl 2-amino-4- (hydroxymethylphosphono) -butyrate (80g/L) in glycine-sodium hydroxide buffer solution, pH 9.0, conversion temperature 37 ℃ to give, after 12h of conversion, a single chiral ethyl L-2-amino-4- (hydroxymethylphosphono) -butyrate, the derivatization assay conversions and EE values are shown in the following table:
| serial number | Variant amino acid sequence of enzyme E1 | E2 | Conversion rate | EE value |
| 1 | SEQ ID NO：9 | 3, 5-dinitrosalicylaldehyde | 99.8％ | 99.6％ |
Wherein the variant of enzyme E1 in example 12 is a variant of SEQ ID NO: mutation of 6 amino acids on the basis of 1.
Details not described in the present specification belong to the prior art known to those skilled in the art.
Sequence listing
<110> Wuhanyingkai Biotechnology research institute Co., Ltd
Preparation method of <120> L-glufosinate-ammonium
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 268
<212> PRT
<213> Streptomyces ramulosus
<400> 1
Met Arg Leu Ser Arg Arg Ala Ala Thr Ala Ser Ala Leu Leu Leu Thr
1 5 10 15
Pro Ala Leu Ala Leu Phe Gly Ala Ser Ala Ala Val Arg Ala Pro Arg
20 25 30
Ile Gln Ala Thr Asp Tyr Val Ala Leu Gly Asp Ser Tyr Ser Ser Gly
35 40 45
Val Gly Ala Gly Ser Tyr Asp Ser Ser Ser Gly Ser Cys Lys Arg Ser
50 55 60
Thr Lys Ser Tyr Pro Ala Leu Trp Ala Ala Ser His Thr Gly Thr Arg
65 70 75 80
Phe Asn Phe Thr Ala Cys Ser Gly Ala Arg Thr Gly Asp Val Leu Ala
85 90 95
Lys Gln Leu Thr Pro Val Asn Ser Gly Thr Asp Leu Val Ser Ile Thr
100 105 110
Ile Gly Gly Asn Asp Ala Gly Phe Ala Asp Thr Met Thr Thr Cys Asn
115 120 125
Asp Gln Gly Glu Ser Ala Cys Leu Ala Arg Ile Ala Lys Ala Arg Ala
130 135 140
Tyr Ile Gln Gln Thr Leu Pro Ala Gln Leu Asp Gln Val Tyr Asp Ala
145 150 155 160
Ile Asp Ser Arg Ala Pro Ala Ala Gln Val Val Val Leu Gly Tyr Pro
165 170 175
Arg Phe Tyr Lys Leu Gly Gly Ser Cys Ala Val Gly Leu Ser Glu Lys
180 185 190
Ser Arg Ala Ala Ile Asn Ala Ala Ala Asp Asp Ile Asn Ala Val Thr
195 200 205
Ala Lys Arg Ala Ala Asp His Gly Phe Ala Phe Gly Asp Val Asn Thr
210 215 220
Thr Phe Ala Gly His Glu Leu Cys Ser Gly Ala Pro Trp Leu His Ser
225 230 235 240
Val Thr Leu Pro Val Glu Asn Ser Tyr His Val Thr Ala Asn Gly Gln
245 250 255
Ser Lys Gly Tyr Leu Pro Val Leu Asn Ser Ala Thr
260 265
<210> 2
<211> 310
<212> PRT
<213> Pseudomonas azotoformans
<400> 2
Met Glu Phe Ile Glu Lys Ile Arg Glu Gly Tyr Ala Ala Phe Gly Ala
1 5 10 15
Tyr Gln Thr Trp Tyr Arg Val Thr Gly Asp Leu Ser Ser Gly Arg Thr
20 25 30
Pro Leu Val Val Ile His Gly Gly Pro Gly Cys Thr His Asp Tyr Val
35 40 45
Asp Ala Phe Lys Asp Val Ala Ala Ser Gly His Ala Val Ile His Tyr
50 55 60
Asp Gln Leu Gly Asn Gly Arg Ser Arg His Leu Pro Asp Lys Asp Pro
65 70 75 80
Ser Phe Trp Thr Val Gly Leu Phe Leu Glu Glu Leu Asn Asn Leu Leu
85 90 95
Asp His Leu Gln Ile Ser Asp Asn Tyr Ala Ile Leu Gly Gln Ser Trp
100 105 110
Gly Gly Met Leu Gly Ser Glu His Ala Ile Leu Gln Pro Lys Gly Leu
115 120 125
Arg Ala Phe Ile Pro Ala Asn Ser Pro Thr Cys Met Arg Thr Trp Val
130 135 140
Ser Glu Ala Asn Arg Leu Arg Lys Leu Leu Pro Glu Gly Val His Glu
145 150 155 160
Thr Leu Leu Lys His Glu Thr Ala Gly Thr Tyr Gln Asp Pro Glu Tyr
165 170 175
Leu Ala Ala Ser Arg Val Phe Tyr Asp His Asn Val Cys Arg Val Ile
180 185 190
Pro Trp Pro Glu Glu Val Ala Arg Thr Phe Ala Ala Val Asp Ala Asp
195 200 205
Pro Thr Val Tyr His Ala Met Ser Gly Pro Thr Glu Phe His Val Ile
210 215 220
Gly Ser Leu Lys Asp Trp Lys Ser Thr Gly Arg Leu Ser Ala Ile Asn
225 230 235 240
Val Pro Thr Leu Val Ile Ser Gly Arg His Asp Glu Ala Thr Pro Leu
245 250 255
Val Val Lys Pro Phe Leu Asp Glu Ile Ala Asp Val Arg Trp Ala Leu
260 265 270
Phe Glu Asp Ser Ser His Met Pro His Val Glu Glu Arg Gln Ala Cys
275 280 285
Met Gly Thr Val Val Lys Phe Leu Asp Glu Val Cys Ser Ala Lys Tyr
290 295 300
Lys Val Leu Lys Ala Ser
305 310
<210> 3
<211> 277
<212> PRT
<213> Mycobacterium tuberculosis
<400> 3
Met Arg Ala Pro Gly Val Arg Ala Ala Asp Gly Ala Gly Arg Val Val
1 5 10 15
Leu Tyr Leu His Gly Gly Ala Phe Val Met Cys Gly Pro Asn Ser His
20 25 30
Ser Arg Ile Val Asn Ala Leu Ser Gly Phe Ala Glu Ser Pro Val Leu
35 40 45
Ile Val Asp Tyr Arg Leu Ile Pro Lys His Ser Leu Gly Met Ala Leu
50 55 60
Asp Asp Cys His Asp Ala Tyr Gln Trp Leu Arg Ala Arg Gly Tyr Arg
65 70 75 80
Pro Glu Gln Ile Val Leu Pro Gly Asp Ser Ala Gly Gly Tyr Leu Ala
85 90 95
Leu Ala Leu Ala Gln Arg Leu Gln Cys Asp Asp Glu Lys Pro Ala Ala
100 105 110
Ile Val Ala Ile Ser Pro Leu Leu Gln Leu Ala Lys Gly Pro Lys Gln
115 120 125
Asp His Pro Asn Ile Gly Thr Asp Ala Met Phe Pro Ala Arg Ala Phe
130 135 140
Asp Ala Leu Ala Ala Trp Val Arg Ala Ala Ala Ala Lys Asn Met Val
145 150 155 160
Asp Gly Arg Pro Glu Asp Leu Tyr Glu Pro Leu Asp His Ile Glu Ser
165 170 175
Ser Leu Pro Pro Thr Leu Ile His Val Ser Gly Ser Glu Val Leu Leu
180 185 190
His Asp Ala Gln Leu Gly Ala Val Lys Leu Ala Ala Ala Gly Val Cys
195 200 205
Ala Glu Val Arg Val Trp Pro Gly Gln Ala His Leu Phe Gln Leu Ala
210 215 220
Thr Pro Leu Val Pro Glu Ala Thr Arg Ser Leu Arg Gln Ile Gly Gln
225 230 235 240
Phe Ile Arg Asp Ala Thr Ala Asp Ser Ser Leu Ser Ile Val His Arg
245 250 255
Ser Arg Tyr Val Ala Gly Ser Pro Arg Ala Ala Ser Arg Gly Ala Phe
260 265 270
Gly Gln Ser Pro Ile
275
<210> 4
<211> 382
<212> PRT
<213> Bacillus subtilis
<400> 4
Met Arg Gly Lys Lys Val Trp Ile Ser Leu Leu Phe Ala Leu Ala Leu
1 5 10 15
Ile Phe Thr Met Ala Phe Gly Ser Thr Thr Ser Ala Gln Ala Ala Gly
20 25 30
Lys Ser Asn Gly Glu Lys Lys Tyr Ile Val Gly Phe Lys Gln Thr Met
35 40 45
Ser Thr Met Ser Ala Ala Lys Lys Lys Asp Val Ile Ser Glu Lys Gly
50 55 60
Gly Lys Val Glu Lys Gln Phe Lys Tyr Val Asp Ala Ala Ser Ala Thr
65 70 75 80
Leu Asn Glu Lys Ala Val Lys Glu Leu Lys Lys Asp Pro Ser Val Ala
85 90 95
Tyr Val Glu Glu Asp His Val Ala Gln Ala Tyr Ala Gln Ser Val Pro
100 105 110
Tyr Gly Val Ser Gln Ile Lys Ala Pro Ala Leu His Ser Gln Gly Phe
115 120 125
Thr Gly Ser Asn Val Lys Val Ala Val Ile Asp Ser Gly Ile Asp Ser
130 135 140
Ser His Pro Asp Leu Lys Val Ala Gly Gly Ala Ser Met Val Pro Ser
145 150 155 160
Glu Thr Asn Pro Phe Gln Asp Asn Asn Ser His Gly Thr His Val Ala
165 170 175
Gly Thr Val Ala Ala Leu Val Asn Ser Val Gly Val Leu Gly Val Ala
180 185 190
Pro Ser Ala Ser Leu Tyr Ala Val Lys Val Leu Gly Ala Asp Gly Ser
195 200 205
Gly Gln Tyr Ser Trp Ile Ile Asn Gly Ile Glu Trp Ala Ile Ala Asn
210 215 220
Asn Met Asp Val Ile Asn Met Ser Leu Gly Gly Pro Ser Gly Ser Ala
225 230 235 240
Ala Leu Lys Ala Ala Val Asp Lys Ala Val Ala Ser Gly Val Val Val
245 250 255
Val Ala Ala Ala Gly Asn Glu Gly Thr Ser Gly Gly Ser Ser Thr Val
260 265 270
Gly Tyr Pro Gly Lys Tyr Pro Ser Val Ile Ala Val Gly Ala Val Asn
275 280 285
Ser Ser Asn Gln Arg Ala Ser Phe Ser Ser Val Gly Ser Glu Leu Asp
290 295 300
Val Met Ala Pro Gly Val Ser Ile Gln Tyr Thr Leu Pro Gly Asn Lys
305 310 315 320
Tyr Gly Ala Tyr Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly
325 330 335
Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Trp Thr Asn Thr Gln
340 345 350
Val Arg Ser Ser Leu Glu Asn Thr Ala Thr Lys Leu Gly Asp Ala Phe
355 360 365
Tyr Tyr Gly Lys Gly Leu Ile Asn Val Gln Ala Ala Ala Gln
370 375 380
<210> 5
<211> 342
<212> PRT
<213> Candida antarctica
<400> 5
Met Lys Leu Leu Ser Leu Thr Gly Val Ala Gly Val Leu Ala Thr Cys
1 5 10 15
Val Ala Ala Thr Pro Leu Val Lys Arg Leu Pro Ser Gly Ser Asp Pro
20 25 30
Ala Phe Ser Gln Pro Lys Ser Val Leu Asp Ala Gly Leu Thr Cys Gln
35 40 45
Gly Ala Ser Pro Ser Ser Val Ser Lys Pro Ile Leu Leu Val Pro Gly
50 55 60
Thr Gly Thr Thr Gly Pro Gln Ser Phe Asp Ser Asn Trp Ile Pro Leu
65 70 75 80
Ser Thr Gln Leu Gly Tyr Thr Pro Cys Trp Lys Ser Pro Pro Pro Phe
85 90 95
Met Leu Asn Asp Thr Gln Val Asn Thr Glu Tyr Met Val Asn Ala Ile
100 105 110
Thr Ala Leu Tyr Ala Gly Ser Gly Asn Asn Lys Leu Pro Val Leu Thr
115 120 125
Trp Ser Gln Gly Gly Leu Val Ala Gln Trp Gly Leu Thr Phe Phe Pro
130 135 140
Ser Ile Arg Ser Lys Val Asp Arg Leu Met Ala Phe Ala Pro Asp Tyr
145 150 155 160
Lys Gly Thr Val Leu Ala Gly Pro Leu Asp Ala Leu Ala Val Ser Ala
165 170 175
Pro Ser Val Trp Gln Gln Thr Thr Gly Ser Ala Leu Thr Thr Ala Leu
180 185 190
Arg Asn Ala Gly Gly Leu Thr Gln Ile Val Pro Thr Thr Asn Leu Tyr
195 200 205
Ser Ala Thr Asp Glu Ile Val Val Pro Gln Val Ser Asn Ser Pro Leu
210 215 220
Asp Ser Ser Tyr Leu Phe Asn Gly Lys Asn Val Gln Ala Gln Ala Val
225 230 235 240
Cys Gly Pro Leu Phe Val Ile Asp His Ala Gly Ser Leu Thr Ser Gln
245 250 255
Phe Ser Tyr Val Val Gly Arg Ser Ala Leu Arg Ser Thr Thr Gly Gln
260 265 270
Ala Arg Ser Ala Asp Tyr Gly Ile Thr Asp Cys Asn Pro Leu Pro Ala
275 280 285
Asn Asp Leu Thr Pro Glu Gln Lys Val Ala Ala Ala Ala Leu Leu Ala
290 295 300
Pro Ala Ala Ala Ala Ile Val Ala Gly Pro Lys Gln Asn Cys Glu Pro
305 310 315 320
Asp Leu Met Pro Tyr Ala Arg Pro Phe Ala Val Gly Lys Arg Thr Cys
325 330 335
Ser Gly Ile Val Thr Pro
340
<210> 6
<211> 273
<212> PRT
<213> Bacillus licheniformis
<400> 6
Ala Gln Thr Val Pro Tyr Gly Ile Pro Leu Ile Lys Ala Asp Lys Val
1 5 10 15
Gln Ala Gln Gly Phe Lys Gly Ala Asn Val Lys Val Ala Val Leu Asp
20 25 30
Thr Gly Ile Gln Ala Ser His Pro Asp Leu Asn Val Val Gly Gly Ala
35 40 45
Ser Phe Val Ala Gly Glu Ala Tyr Ile Thr Asp Gly Asn Gly His Gly
50 55 60
Thr His Val Ala Gly Thr Val Ala Ala Leu Asp Asn Thr Thr Gly Val
65 70 75 80
Leu Gly Val Ala Pro Ser Val Ser Leu Tyr Ala Val Lys Val Leu Asn
85 90 95
Ser Ser Gly Ser Gly Ser Tyr Ser Gly Ile Val Ser Gly Ile Glu Trp
100 105 110
Ala Thr Thr Asn Gly Met Asp Val Ile Asn Met Ser Leu Gly Gly Ala
115 120 125
Ser Gly Ser Thr Ala Met Lys Gln Ala Val Asp Asn Ala Tyr Ala Lys
130 135 140
Arg Val Val Val Val Ala Val Gly Asn Ser Gly Ser Ser Gly Asn Thr
145 150 155 160
Asn Thr Ile Gly Tyr Pro Ala Lys Tyr Glu Ser Val Ile Ala Val Gly
165 170 175
Ala Val Asp Ser Asn Ser Asn Arg Ala Ser Phe Ser Ser Val Gly Ala
180 185 190
Glu Leu Glu Val Met Ala Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro
195 200 205
Thr Ser Thr Tyr Ala Thr Leu Asn Gly Thr Ser Met Ala Ser Pro His
210 215 220
Val Ala Gly Ala Ala Ala Leu Ile Leu Ser Lys His Pro Asn Leu Ser
225 230 235 240
Ala Ser Gln Val Arg Asn Arg Leu Ser Ser Ala Ala Thr Tyr Leu Gly
245 250 255
Ser Ser Phe Tyr Tyr Gly Lys Gly Leu Ile Asn Val Glu Ala Ala Ala
260 265 270
Gln
<210> 7
<211> 299
<212> PRT
<213> Pseudomonas sp
<400> 7
Met Asp Leu Pro Val Thr His Glu Gly Phe Ala Pro Phe Gly Pro Tyr
1 5 10 15
Gln Thr Trp Tyr Arg Ile Thr Gly Asp Phe Ser Ser Gly Arg Thr Pro
20 25 30
Leu Leu Ile Leu His Gly Gly Pro Gly Cys Thr His Asp Tyr Val Asp
35 40 45
Ala Phe Lys Asp Val Ala Asn Ser Gly Tyr Pro Val Ile His Tyr Asp
50 55 60
Gln Leu Gly Asn Gly Arg Ser Thr His Leu Pro Glu Lys Asp Pro Ser
65 70 75 80
Phe Trp Asn Val Ala Leu Phe Leu Asp Glu Leu Glu Asn Leu Leu Asp
85 90 95
His Leu Gly Ile Trp Asp Asn Tyr Ala Leu Leu Gly Gln Ser Trp Gly
100 105 110
Gly Met Leu Ala Ser Glu His Ala Val Leu Gln Pro Ala Gly Leu Arg
115 120 125
Val Leu Ile Val Ala Asn Ser Pro Ala Asp Met Ser Thr Trp Leu Leu
130 135 140
Glu Val Asn Arg Leu Arg Gln Leu Leu Pro Ile Glu Val Gln Gln Thr
145 150 155 160
Leu Leu Val His Glu Arg Ala Gly Thr Leu Thr Ser Thr Glu Tyr Phe
165 170 175
Glu Ala Ala Arg Val Phe Tyr Asp Arg His Val Cys Arg Val Thr Pro
180 185 190
Trp Pro Asp Glu Val Ala Arg Thr Phe Ala Gln Ile Asp Ala Asp Pro
195 200 205
Thr Val Tyr His Ala Met Ala Gly Pro Thr Glu Phe His Val Ile Gly
210 215 220
Ser Met Lys Asp Trp Ser Ile Thr Asp Arg Leu Pro Arg Ile Ser Val
225 230 235 240
Pro Thr Leu Leu Ile Ser Gly Arg Tyr Asp Glu Ala Thr Pro Leu Val
245 250 255
Val Lys Pro Tyr Val Asp His Val Pro Asp Ile Arg Trp Ala Leu Phe
260 265 270
Glu His Ser Ser His Met Pro His Ile Glu Glu Arg Met Ala Cys Met
275 280 285
Gly Thr Val Val Ser Phe Leu Asp Glu Cys Leu
290 295
<210> 8
<211> 493
<212> PRT
<213> Brevundimonas diminuta
<400> 8
Met Gly Met Lys Ile Glu Phe Val Ala Ala Ser Gly Ala Ala Glu Ile
1 5 10 15
Leu Ala Leu Leu Val His Glu Asp Arg Ala Leu Ala Gly Thr Gly Pro
20 25 30
Ala Leu Asp Ala Ala Ala Ser Gly Ala Leu Val Lys Ala Met Lys Lys
35 40 45
Ser Arg Phe Val Gly Ala Ala Ser Ser Ser Leu Asn Val Ala Ala Pro
50 55 60
Ser Gly Ile Asp Ala Asn Ala Val Leu Leu Val Gly Ala Gly Ala Ala
65 70 75 80
Asp Lys Leu Asp Asp Leu Ala Val Glu Thr Phe Gly Ala Ala Ala Val
85 90 95
Gln Ala Thr Lys Leu Ser Gly Ala Glu Val Leu Thr Ile Asp Val Ser
100 105 110
Gly Leu Ser Pro Glu Leu Ala Ala Arg Ala Gly Phe Ala Ala Arg Leu
115 120 125
Ala Ala Tyr Arg Phe Ala Lys Tyr Leu Thr Lys Gln Lys Ala Asp Lys
130 135 140
Ile Pro Ser Val Thr Ala Val Arg Val Val Thr Ser Asp Val Lys Ala
145 150 155 160
Ala Glu Ala Ala Leu Gln Pro Leu Ser Ala Val Ala Asp Ala Val Leu
165 170 175
Phe Ala Arg Asp Leu Val Ser Glu Pro Ala Asn Ile Leu Tyr Pro Ala
180 185 190
Glu Phe Ala Arg Arg Val Lys Glu Leu Glu Ala Leu Gly Ala Lys Val
195 200 205
Glu Ile Leu Gly Glu Ala Glu Met Gln Lys Leu Gly Met Gly Ser Leu
210 215 220
Leu Gly Val Gly Gln Gly Ser Val Arg Glu Ser Gln Leu Ala Val Ile
225 230 235 240
Gln Trp Asn Gly Gly Glu Glu Gly Glu Ala Pro Ile Ala Phe Val Gly
245 250 255
Lys Gly Val Cys Phe Asp Thr Gly Gly Ile Ser Leu Lys Ala Ala Asp
260 265 270
Gly Met Glu Glu Met Lys Trp Asp Met Gly Gly Ala Ala Ala Val Thr
275 280 285
Gly Leu Met His Ala Leu Val Gly Arg Lys Ala Lys Val Asn Val Val
290 295 300
Gly Val Leu Gly Leu Val Glu Asn Met Pro Asp Gly Asn Ala Gln Arg
305 310 315 320
Pro Gly Asp Val Val Thr Ser Met Ser Gly Gln Thr Val Glu Val Leu
325 330 335
Asn Thr Asp Ala Glu Gly Arg Leu Val Leu Ala Asp Ala Leu Trp Tyr
340 345 350
Thr Gln Gln Arg Phe Lys Pro Lys Phe Met Ile Asp Leu Ala Thr Leu
355 360 365
Thr Gly Ala Met Ile Ile Ser Leu Gly Leu Glu Tyr Ala Gly Val Phe
370 375 380
Thr Asn Ser Asp Ala Leu Ala Ala Asn Ile Ala Asp Ala Gly Pro Lys
385 390 395 400
Val Gly Glu Asn Ser Trp Arg Met Pro Ile Pro Ala Glu Tyr Asp Gln
405 410 415
His Ile Asp Ser Pro Ile Ala Asp Val Lys Asn Met Gly Asn Gly Arg
420 425 430
Ala Gly Gly Ser Ile Thr Ala Ala Leu Phe Leu Gln Arg Phe Thr Asn
435 440 445
Gly Thr Pro Trp Ala His Ile Asp Ile Ala Pro Thr Ala Trp Val Lys
450 455 460
Asp Ser Lys Asn Pro Thr Val Pro Asp Gly Gly Val Gly Tyr Gly Val
465 470 475 480
Arg Leu Leu Asp Arg Met Val Ala Asp His Tyr Glu Gly
485 490
<210> 9
<211> 268
<212> PRT
<213> Streptomyces ramulosus
<400> 9
Met Arg Leu Ser Arg Arg Ala Ala Thr Ala Ser Ala Leu Leu Leu Thr
1 5 10 15
Pro Ala Leu Ala Leu Phe Gly Ala Ser Ser Ala Val Arg Ala Pro Arg
20 25 30
Ile Gln Ala Thr Asp Tyr Val Ala Leu Gly Asp Ser Tyr Ser Ser Gly
35 40 45
Val Gly Ala Gly Ser Tyr Asp Ser Ser Ser Gly Ser Cys Lys Arg Ser
50 55 60
Thr Ile Ser Tyr Pro Ala Leu Trp Ala Ala Ser His Thr Gly Thr Arg
65 70 75 80
Phe Asn Phe Thr Ala Cys Ser Gly Ala Arg Thr Gly Asp Val Leu Ala
85 90 95
Lys Gln Leu Thr Pro Val Asn Ala Gly Thr Asp Leu Val Ser Ile Thr
100 105 110
Ile Gly Gly Asn Asp Ala Gly Phe Ala Asp Thr Met Thr Thr Cys Asn
115 120 125
Asp Gln Gly Glu Ser Ala Cys Leu Ala Arg Ile Ala Lys Ala Arg Ala
130 135 140
Tyr Ile Gln Gln Thr Leu Pro Ala Gln Leu Asp Gln Val Tyr Asp Ala
145 150 155 160
Ile Asp Ser Asn Ala Pro Ala Ala Gln Val Val Val Leu Gly Tyr Pro
165 170 175
Arg Phe Tyr Lys Leu Gly Gly Ser Cys Ala Val Gly Leu Ser Glu Lys
180 185 190
Ser Arg Ala Ala Ile Asn Ala Ala Ala Asp Val Ile Asn Ala Val Thr
195 200 205
Ala Lys Arg Ala Ala Asp His Gly Phe Ala Phe Gly Asp Val Asn Thr
210 215 220
Thr Phe Ala Gly His Glu Leu Cys Phe Gly Ala Pro Trp Leu His Ser
225 230 235 240
Val Thr Leu Pro Val Glu Asn Ser Tyr His Val Thr Ala Asn Gly Gln
245 250 255
Ser Lys Gly Tyr Leu Pro Val Leu Asn Ser Ala Thr
260 265
Claims (10)
1. The preparation method of the compound of the formula I is characterized in that the compound of the formula II is reacted under the action of E1 and E2 to prepare the compound of the formula I, wherein the reaction formula is as follows:
e1 is a substance capable of hydrolyzing a compound of formula II to a compound of formula I;
e2 is a substance capable of racemizing the compound of the formula III;
wherein R is1Selected from the group consisting of alkyl, aryl, heteroaryl, substituted alkyl, substituted aryl, substituted heteroaryl;
R2selected from amino OR-OR0；R0Selected from the group consisting of hydrocarbyl, silyl, benzyl, alkyl methyl, alkyl-substituted alkoxy, 3-8 membered cycloaliphatic, aryl, heteroaryl, substituted aryl, substituted heteroaryl, Ar (CH)2)nOne of O-groups, Ar represents aryl or heteroaryl, and n is 1-6.
2. The preparation method of claim 1, wherein the preparation method of L-glufosinate-ammonium comprises reacting the compound of formula IV with E1 and E2 to obtain the compound of formula V, and deprotecting the group to obtain L-glufosinate-ammonium,
wherein R is3Selected from the group consisting of hydrogen, alkyl, silyl ether protecting groups, benzyl ether protecting groups, alkoxymethyl, alkoxy-substituted methyl, C2-C10 linear or branched alkenylalkyl, C2-C10 linear or branched alkynylalkyl, 3-8 membered cycloaliphatic, aryl, heteroaryl, amino, substituted amino, Ar (CH)2)a-one of the groups, Ar represents an aryl, heteroaryl group, a takes 1 to 6.
3. The method of claim 1, wherein the E1 is a biological enzyme, and the E1 gene source comprises Bacillus subtilis, Streptomyces ramulosus, Pseudomonas azotoformans, Bacillus licheniformis, Mycobacterium tuberculosis, pork liver, Candida antartica, Pseudomonas wadanswerensis, Pseudomonas sp, Brevundimonas diminuta.
4. The preparation method according to claim 1, wherein E2 is one or more aldehydes or ketones or organic acids, and the aldehyde compounds include linear aldehydes, branched aldehydes, aromatic aldehydes, substituted aromatic aldehydes, heterocyclic aldehydes, and substituted heterocyclic aldehydes.
5. The process for producing L-glufosinate according to claim 2, wherein R3 is hydrogen, R2 is ethyl, propyl or isopropyl, and the pH of the reaction system is controlled to 6.5 to 10.
6. The method according to claim 1, wherein E2 is one or more selected from glutaraldehyde, pyridoxal phosphate, salicylaldehyde, benzaldehyde, o-carboxybenzaldehyde, m-carboxybenzaldehyde, p-carboxybenzaldehyde, 9-fluorenone, 3, 5-dinitrosalicylaldehyde, 3, 5-dichlorosalicylaldehyde, and 5-bromo-2-hydroxy-3-nitrobenzaldehyde.
7. The process according to claim 3, wherein the enzyme E1 is a polypeptide having the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 or SEQ ID NO: 17, or the protein of SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 or SEQ ID NO: 9 is a protein having the activity of hydrolyzing the compound of formula V to the compound of formula I after substitution, deletion or addition of one or more amino acid residues, or is SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 or SEQ ID NO: 9 has homology of more than 80 percent and is protein with the activity of hydrolyzing the compound shown in the formula V into the compound shown in the formula I, and the enzyme E1 is a whole cell, broken enzyme liquid, freeze-dried powder or immobilized enzyme or immobilized cell of a genetically engineered bacterium.
8. The method according to claim 1, wherein the reaction is carried out in the presence of a solvent.
9. The biosynthesis method according to claim 8, wherein the solvent is water or a mixed solvent of a buffer solution and an organic solvent.
10. The method according to claim 9, wherein the buffer solution is selected from one or more of a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, a potassium dihydrogen phosphate-sodium hydroxide buffer solution, a carbonate buffer solution, a Tri-HCl buffer solution, a citric acid-sodium citrate buffer solution, a glycine-sodium hydroxide buffer solution, or a MOPS buffer solution; the organic solvent is one or more selected from DMSO, ethyl acetate, butyl acetate, isopropanol, DMF, TBME, dichloromethane, tert-butyl alcohol, n-butyl alcohol, vinyl acetate, benzene and toluene.
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| CN112940031A (en) * | 2021-02-01 | 2021-06-11 | 河北威远生物化工有限公司 | N-naphthyl-acetyl-glufosinate-ammonium, synthesis method thereof and synthesis method for synthesizing L-glufosinate-ammonium by using N-naphthyl-acetyl-glufosinate-ammonium |
| WO2022100011A1 (en) * | 2020-11-14 | 2022-05-19 | 山西大学 | 2709 alkaline protease mutant modified on the basis of molecular dynamics calculation and use thereof |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2022100011A1 (en) * | 2020-11-14 | 2022-05-19 | 山西大学 | 2709 alkaline protease mutant modified on the basis of molecular dynamics calculation and use thereof |
| CN112940031A (en) * | 2021-02-01 | 2021-06-11 | 河北威远生物化工有限公司 | N-naphthyl-acetyl-glufosinate-ammonium, synthesis method thereof and synthesis method for synthesizing L-glufosinate-ammonium by using N-naphthyl-acetyl-glufosinate-ammonium |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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