CN111139271A - Method for asymmetrically synthesizing L-glufosinate-ammonium by single transaminase catalytic cascade reaction - Google Patents

Abstract

The invention discloses a method for asymmetrically synthesizing L-glufosinate-ammonium by single transaminase catalytic cascade reaction, which comprises the following steps: 4- (methyl hydroxyl phosphoryl) -2-carbonyl-butyric acid or salt thereof is taken as a substrate, and in a reaction system with an amino donor, the substrate is catalyzed by using in vitro transaminase or cells expressing the transaminase in vitro to carry out a transamination reaction to obtain L-glufosinate-ammonium; the amino donor is L-glutamic acid and L-aspartic acid; only one transaminase is added into the reaction system, and the amino acid sequence of the transaminase is shown in SEQ ID No. 4. The process for producing L-glufosinate-ammonium by transaminase ATA117-rd11 catalytic cascade reaction solves the problem that the dosage of an amino donor is too high in the existing single transaminase process, and also solves the technical problems that the cost of a biocatalyst is higher, the fermentation process is complicated and the reaction time is longer in the existing two transaminase processes.

Description

Method for asymmetrically synthesizing L-glufosinate-ammonium by single transaminase catalytic cascade reaction

Technical Field

The invention relates to the technical field of bioengineering, in particular to a method for asymmetrically synthesizing L-glufosinate-ammonium by single transaminase catalysis cascade reaction.

Background

Glufosinate, also called glufosinate, is named Phsophithotricin (PPT for short) in English, and is named 2-amino-4- [ hydroxy (methyl) phosphonyl ] -butyric acid in chemical name, so that the glufosinate is a non-selective herbicide with high efficiency, broad spectrum and low toxicity, is an ideal herbicide for transgenic resistant crops at present, and has very wide application prospect. Glufosinate has two enantiomers, but only the L-configuration has phytotoxicity, is easy to decompose in soil, has low toxicity to human and animals, and has low destructive power to the environment. Currently, glufosinate-ammonium is generally marketed as a racemic mixture. If the glufosinate-ammonium product can be used in the form of an L-configuration optical pure isomer, the using amount of the glufosinate-ammonium can be reduced by 50 percent, which has important significance for improving atom economy, reducing cost and relieving environmental pressure.

The method for producing L-glufosinate-ammonium includes chemical synthesis, fermentation and biological synthesis. The enzymes involved in the biosynthesis method mainly include: proteases, deacetylases, transaminases, amidases, ester hydrolases and nitrile hydratases. In a plurality of enzymatic synthesis routes of glufosinate-ammonium, ketocarbonyl of a keto acid intermediate is a latent chiral functional group, a chiral center can be constructed through an enzymatic synthesis route, and the keto acid route is suitable for industrial development and production of L-glufosinate-ammonium due to the fact that raw materials are cheap and easy to obtain and virulent cyanides can be avoided.

Transaminases (Amine transaminases, ATA, ec2.6.1.x) belong to the transferase class, and are enzymes that catalyze the transfer of the amino group from 1 amino donor (amino acid or simple Amine) to a prochiral acceptor ketone to yield a chiral Amine and a byproduct ketone or α -keto acid, and the reaction process requires the participation of pyridoxal phosphate (PLP), and is reversible, and transaminases can be divided into α -Transaminase and ω -Transaminase (EC2.6.1.1) reactions, wherein the first reaction transfers the amino group from the amino donor to the carbonyl group of PLP under the action of ω -Transaminase to form pyridoxamine 5-phosphate (PMP) and the corresponding ketone from the amino donor, and the second reaction transfers the amino group from PMP to the amino acceptor under the action of ω -Transaminase to realize the PLP cycle.

Schulz A et al, using transaminase isolated from E.coli K-12, 2-carbonyl-4- (hydroxymethylphosphonamide) butyric acid as a substrate and L-glutamic acid as an amino donor, produced L-glufosinate (stereospecification of the recombinant phosphinothricin (gluf synthase) by transamination, with isolation and catalysis of a phospho protein-specific transaminase from Escherichia coli [ J ]. Applied and environmental Microbiology,1990,56(1):1-6), with a maximum product concentration of 76.1 g/L. The transaminase is immobilized and then installed in a bioreactor, the highest product generation rate of the reactor is 50g/(L.h), and the e.e. value of the glufosinate-ammonium exceeds 99.9%. However, the process has two defects, one is that the raw material PPO can not be completely converted into L-PPT, and the conversion rate is only 90% at most; the second is to make the reversible reaction go on to the direction of producing L-PPT, need more than 4 times of equivalent L-glutamic acid as amino donor, the excessive glutamic acid brings great trouble to the separation, the key point of the problem lies in that enzymatic transamination reaction is a reversible reaction when taking L-glutamic acid as amino donor.

The patent with the publication number of CN105603015B reports that the conversion rate can reach 100% by using transaminase derived from Bacillus subtilis 168(Bacillus subtilis 168), taking 2-carbonyl-4- (hydroxymethyl phosphonamide) butyric acid as a substrate and taking L-alanine as an amino hydrogen donor when the substrate concentration is 100mM and producing L-glufosinate-ammonium by using transamination. The reaction raw materials are easy to obtain, the cost is low, and the water phase is used as a reaction system, so that the method is green and environment-friendly; however, the amount of the amino donor used in the reaction is still large, and further optimization is required in order to reduce the production cost of L-glufosinate-ammonium.

Bartsch K (U.S. Pat. No. 3, 6335186, 1) adds an oxaloacetate transaminase to the single-enzyme reaction system provided by Schulz A et al to form a double-transaminase coupled reaction system (FIG. 2). However, the activity of oxaloacetate transaminase is not high, resulting in a limited overall coupling reaction rate, resulting in a longer reaction time and a low space-time yield, and the conversion of PPO in this process is only 85% at the maximum.

Therefore, there is a need for further improvement of the above process to solve the problems of increased fermentation cost and excessively long reaction time.

Disclosure of Invention

The invention provides a method for asymmetrically synthesizing L-glufosinate-ammonium by a single transaminase catalytic cascade reaction, which adopts a single transaminase, can specifically catalyze 2-carbonyl-4- (hydroxymethyl phosphonamide) butyric acid to generate L-2-amino-4- [ hydroxy (methyl) phosphonyl ] -butyric acid and α -ketoglutaric acid by taking L-glutamic acid as an amino donor, can also specifically catalyze α -ketoglutaric acid to react to obtain oxaloacetic acid and L-glutamic acid by taking L-aspartic acid as an amino donor, and solves the problems of overhigh dosage of the amino donor when single transaminase is adopted, overhigh fermentation cost, overlong reaction time and the like when two transaminases are adopted in the prior art.

The specific technical scheme is as follows:

a method for asymmetrically synthesizing L-glufosinate-ammonium by a single transaminase-catalyzed cascade reaction, which comprises the following steps: 4- (methyl hydroxyl phosphoryl) -2-carbonyl-butyric acid or salt thereof is taken as a substrate, and in a reaction system with an amino donor, the substrate is catalyzed by using in vitro transaminase or cells expressing the transaminase in vitro to carry out a transamination reaction to obtain L-glufosinate-ammonium; the amino donor consists of two amino acids, namely L-glutamic acid and L-aspartic acid; only one transaminase is added into the reaction system, and the amino acid sequence of the transaminase is shown in SEQ ID No. 4.

The equation for the above reaction is as follows:

as can be seen from the above reaction formulae, the transaminase ATA117- -rd11 employed in the present invention participates in two transamination reactions simultaneously, namely:

(1) transaminase ATA117-rd11 catalyzes a substrate 4- (methyl hydroxyl phosphoryl) -2-carbonyl-butyric acid (PPO for short) to have a transamination reaction with amino donor L-Glu to obtain 2-amino-4- [ hydroxyl (methyl) phosphonyl ] -butyric acid (PPT for short) and α -ketoglutaric acid (α -KG for short);

(2) transaminase ATA117-rd11 catalyzes substrate α -ketoglutaric acid to carry out transamination reaction with amino donor L-aspartic acid to obtain oxaloacetic acid and L-glutamic acid.

Because the transaminase ATA117-rd11 can participate in two transamination reactions at the same time, the application amount of the transaminase and the application amount of an amino donor in the whole reaction system are reduced.

Further, the cell is an engineering bacterium for expressing transaminase, and the host cell of the engineering bacterium is E.coli BL21(DE 3).

Preferably, in the reaction system, the addition amount of the engineering bacteria is 20-200 g/L of reaction liquid based on the weight of wet bacteria; more preferably, the addition amount of the engineering bacteria is 20-100 g/L of reaction liquid; most preferably, the addition amount of the engineering bacteria is 20g/L of reaction liquid.

Further, the buffer solution in the reaction system is Tris/HCl buffer solution, and the pH value is 8-9. The buffer solution is screened, and the conversion rate of the method is higher when the buffer solution is carried out in a Tris/HCl buffer solution with the pH value of 8-9; in borax-boric acid buffer and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, the conversion rate is low regardless of the pH range.

Theoretically, only a small amount of L-glutamic acid in the amino donor is needed to ensure that the reaction process for generating PPT can be carried out, and experiments show that the molar ratio of the L-glutamic acid to the L-aspartic acid is 1: 4-1: 8, so that the amino donor investment is low and the conversion rate is high.

Further, the molar ratio of the amino donor to the substrate is preferably 1.2 to 1.6: 1.

Preferably, the temperature of the transamination reaction is 35-45 ℃ and the time is 24-48 h.

Preferably, the reaction system further comprises a coenzyme, the coenzyme is pyridoxal phosphate, and the molar ratio of the coenzyme to the substrate is 1: 10-1: 200.

In the reaction system, the dosage of the catalyst is 20-200 g/L calculated by the weight of wet bacteria, the initial concentration of a substrate is 10-500 mM, the dosage of the coenzyme is 0.02-25 mM, and the dosage of an amino donor is 10-700 mM, wherein the dosage of L-glutamic acid is 1-100 mM, and the dosage of L-aspartic acid is 6-600 mM.

Furthermore, the amount of the catalyst is 20-100 g/L of the reaction solution based on the weight of the wet bacteria, the initial concentration of the substrate is 100-200 mM, the amount of the coenzyme is 2-10 mM, and the amount of the amino donor is 100-300 mM, wherein the amount of the L-glutamic acid is 10-50 mM, and the amount of the L-aspartic acid is 90-250 mM.

Compared with the prior art, the invention has the following beneficial effects:

the transaminase ATA117-rd11 adopted by the method not only can specifically catalyze the reaction of 2-carbonyl-4- (hydroxymethyl phosphonamide) butyric acid to generate L-2-amino-4- [ hydroxyl (methyl) phosphonyl ] -butyric acid and α -ketoglutaric acid by taking L-glutamic acid as an amino donor, but also can specifically catalyze α -ketoglutaric acid to react to obtain oxaloacetic acid and L-glutamic acid by taking L-aspartic acid as an amino donor, thereby not only solving the problem of overhigh dosage of the amino donor in the existing single transaminase process, but also solving the technical problems of higher cost of biocatalysts, fussy fermentation process and longer reaction time in the existing two transaminase processes.

Drawings

FIG. 1 is a schematic diagram showing the reaction equation for asymmetric synthesis of L-glufosinate-ammonium by a single transaminase-catalyzed cascade reaction of the present invention.

FIG. 2 is a schematic diagram showing the reaction equation for the co-production of L-glufosinate-ammonium by transaminase and oxaloacetate transaminase.

FIG. 3 is a physical map of the recombinant plasmid pET-28b-ATA117-rd11 in example 7.

FIG. 4 is a low melting point agarose gel electrophoresis of the transaminase gene ATA117-rd11 in example 7; wherein, Lane 1 is DL2000DNA Marker; lane 2 shows the gene fragments for transaminases ATA117-rd 11.

FIG. 5 is an SDS-PAGE pattern of the transaminase in example 7; wherein, Lane 1 is the protein molecular weight Marker, Lane 2 is the transaminase ATA117-rd 11.

Detailed Description

The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.

Example 1 amplification of transaminase Gene ATA117

Based on the sequencing information of transaminase gene from Arthrobacter (Arthrobacter sp) KNK168 recorded in Genbank, total genomic DNA of Arthrobacter (Arthrobacter sp) KNK168 was extracted with a nucleic acid rapid extractor, and PCR amplification was performed with the genomic DNA as a template and the primers.

Primer 1: 5'-ATGGCGTTCTCAGCGGACACCCCTG-3', respectively;

primer 2: 5'-TTAGTACTGTACCGGGGTCAGCAG-3' are provided.

PCR reaction system (total volume 50. mu.L): 10 Xpfu DNA Polymerase Buffer 5. mu.L, 10mM dNTPmix (2.5 mM each of dATP, dCTP, dGTP and dTTP) 1. mu.L, 50. mu.M each of clone primer 1 and primer 2, 1. mu.L of genomic DNA, 1. mu.L of Pfu DNA Polymerase, and 40. mu.L of nucleic acid-free water.

PCR conditions using a BioRad PCR instrument: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 65 deg.C for 45s, extension at 72 deg.C for 1min for 30 cycles, and final extension at 72 deg.C for 10 min.

The PCR reaction solution was subjected to 0.9% agarose gel electrophoresis, and the fragment was recovered and purified by cutting the gel, and then a base A was introduced into the 5' end of the fragment using Taq DNA polymerase. The fragment is connected with pMD18-T vector under the action of T4 DNA ligase to obtain the cloned recombinant plasmid pMD18-T-ATA 117.

The recombinant plasmid is transformed into Escherichia coli JM109, a basket white spot screening system is used for screening, white clone sequencing is randomly selected, a software analysis sequencing result shows that: the length of the nucleotide sequence amplified by the primer 1 and the primer 2 is 990bp (the nucleotide sequence of the ATA117 gene is shown as SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO: 2), and the sequence encodes a complete open reading frame.

Example 2 construction of recombinant E.coli BL21(DE3)/pET28b-ATA117

Primers were designed based on the ATA117 gene sequence of example 1,

primer 3: 5' -CCGCATATGGCGTTCTCAGCGGACA-3’；

Primer 4: 5' -TTGCTCGAGGTAGTGCACAGGCGTC-3’；

And Nde I and Xho I restriction sites were introduced into primer 3 and primer 4, respectively. Under the initiation of primer 3 and primer 4, amplification was performed using high fidelity Pfu DNA polymerase, using recombinant plasmid pMD18-T-ATA117 as a template (obtained in example 1), the ATA117 gene sequence was obtained, the amplified fragment was treated with Nde I and Xhol I restriction enzymes (TaKaRa) after sequencing, and the fragment was ligated with commercial vector pET28b (Invitrogen) treated with the same restriction enzymes using T4 DNA ligase (TaKaRa), to construct expression vector pET28b-ATA 117.

The constructed expression vector pET28b-ATA117 is transformed into Escherichia coli BL21(DE3) (Invitrogen) (42 ℃, 90s), spread on LB plate containing 50. mu.g/ml kanamycin resistance, cultured for 8-12h at 37 ℃, randomly picked clone extraction plasmid for sequencing identification, and screened to obtain recombinant Escherichia coli BL21(DE3)/pET28b-ATA117 containing expression recombinant plasmid pET28b-ATA 117.

Example 3 inducible expression of transaminase (ATA117)

The recombinant Escherichia coli BL21(DE3)/pET28b-ATA117 obtained in example 2 was inoculated into LB liquid medium containing 50. mu.g/ml kanamycin resistance, cultured at 37 ℃ at 200rpm for 12 hours, further inoculated into fresh LB liquid medium containing 50. mu.g/ml kanamycin resistance in an inoculum size of 1% (v/v), and cultured at 37 ℃ at 150rpm until the microbial OD₆₀₀Reaching 0.6-0.8, adding IPTG with final concentration of 0.1mM, and inducing culture at 25 deg.CAfter culturing for 18h, centrifuging at 8000rpm for 20min at 4 ℃, discarding supernatant, and collecting precipitate to obtain recombinant Escherichia coli BL21/pET28b-ATA117 wet thallus containing recombinant plasmid. The thallus can be directly used as a biocatalyst.

EXAMPLE 4 isolation and purification of transaminase (pET28b-ATA117)

After the wet cells obtained in example 3 (i.e., recombinant Escherichia coli BL21(DE3)/pET28b-ATA117 wet cells) were resuspended in a binding buffer (50mM, pH7.4 sodium phosphate buffer), performing ultrasonication, centrifuging at 12000rpm for 40min, incubating the supernatant with Ni affinity chromatography resin balanced by the above binding buffer, washing with washing buffer (50mM, pH7.4 sodium phosphate buffer) until no foreign protein exists, eluting with elution buffer (50mM, pH7.4 sodium phosphate buffer) and collecting target protein, combining the target proteins after electrophoretic purity determination, dialyzing with dialysis buffer (50mM, pH sodium phosphate buffer) for 48h, taking the retentate, determining the protein content to be 2mg/mL by Coomassie brilliant blue method, diluting the enzyme solution to the final concentration of 0.5mg/mL, subpackaging, freezing and storing at-80 ℃ to obtain the transaminase ATA117 pure enzyme.

Example 5 Activity assay of recombinant transaminase ATA117

The transaminase ATA117 whole cell isolated and purified by the method of example 3 is used for catalyzing the substrate 4- (methyl hydroxyl phosphoryl) -2-carbonyl-butyric acid.

The composition of the catalytic system and the catalytic conditions are as follows: 10ml of Tris/HCl buffer (100mM, pH8.0) was added with transaminase ATA117 whole cells (cell mass 50g/L buffer), L-aspartic acid (final concentration 20mM) and L-glutamic acid (final concentration 4mM), 4- (methylhydroxyphosphoryl) -2-carbonyl-butyric acid (final concentration 20mmol/L buffer), PLP (final concentration 1mmol/L buffer) to constitute a reaction system. Reacting for 24 hours at 35 ℃ and the rotating speed of 600r/min, and sampling to detect the conversion rate of the substrate. In the same manner, the control was prepared by dialyzing disrupted supernatants of E.coli BL21(DE3) and E.coli BL21(DE3)/pET28a cells.

TABLE 1 determination of the enzymatic Activity of the transaminase ATA117

Strain/plasmid	Substrate conversion of enzyme
		Escherichia coli BL21(DE3)	0
Escherichia coli BL21(DE3)/pET28b	0
		Escherichia coli BL21(DE3)/pET28b-ATA117	35.91％

Example 6 creation of ATA117 Gene mutation library

Error-prone PCR was performed using the plasmid pET28b-ATA117 constructed in example 2 as a template.

Error-prone PCR was performed with primers 1 (5'-ATGGCGTTCTCAGCGGACACCCCTG-3') and 2 (5'-TTAGTACTGTACCGGGGTCAGCAG-3').

PCR reaction system (total volume 50. mu.L): 10 Xpfu DNA Polymerase Buffer 5. mu.L, 10mM dNTPmix (2.5 mM each of dATP, dCTP, dGTP and dTTP) 1. mu.L, 50. mu.M each of clone primer 1, primer 2 0.5. mu.M, plasmid template 0.8 ng/. mu.L, TaqDNA Polymerase 2.5U, MnCl₂0.2mM, deionized water to make up 50. mu.L.

PCR conditions using a BioRad PCR instrument: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 65 deg.C for 45s, extension at 72 deg.C for 1min for 30 cycles, and final extension at 72 deg.C for 10 min.

After the error-prone PCR product was purified, a large primer PCR product (i.e., mutant library 1) was obtained by performing large primer PCR using the error-prone PCR product as a primer and the plasmid pET28b-ATA117 constructed in example 2 as a template.

And (3) PCR system: 10 ng/. mu.L of large primer, 1 ng/. mu.L of plasmid template and 2.5U of Pfu DNA Polymerase. And (3) PCR reaction conditions: removing A tail at 72 deg.C for 5min, pre-denaturing at 96 deg.C for 2min, denaturing at 96 deg.C for 30s, annealing at 60 deg.C for 45s, extending at 72 deg.C for 4min for 25 cycles, and extending at 72 deg.C for 10 min.

Example 7 screening of Gene mutation library to obtain mutant ATA117-rd11

7017 recombinant E.coli wet cells containing mutant genes, i.e., mutant wet cells, were obtained by transferring the mutant library 1 of example 6 into competent cells of E.coli BL21(DE3), subjecting the competent cells to a heat shock at 42 ℃ for 90 seconds, selecting a single clone on an LB-resistant plate containing 50. mu.g/ml kanamycin, and inoculating the single clone into LB medium containing 50. mu.g/ml kanamycin, respectively, for induction expression under the conditions of example 3.

After obtaining Escherichia coli containing the mutant protein, 0.36g of glufosinate-precursor ketone PPO is subjected to biotransformation, and the final concentration composition and the catalysis conditions of a catalytic system (10ml) are as follows: coli BL21(DE3)/pET28b-ATA117-rd11 wet cell 0.075g, Tris-HCl buffer (pH8.0), pyridoxal phosphate 0.2mM, L-alanine 70mM, 20mM4- (methylhydroxyphosphoryl) -2-carbonyl-butyric acid.

Reaction conditions are as follows: the temperature is 35 ℃, the stirring speed is 150r/min, and the reaction time is 24 h. Under the same conditions, the reaction solution with added sterile vector was used as a blank control, and the mutant wet cells were replaced with the empty vector-containing wet cells of Escherichia coli BL21/pET28b as a negative control.

After the reaction was completed, samples were taken for HPLC detection (50:50 acetonitrile: water, 10mM ammonium acetate, 0.8ml/min flow rate, 268nm detection wavelength), and from 7017 muteins, 6198 muteins had lower viability than the original protein, 778 muteins had viability comparable to the original protein, and only 41 muteins had viability significantly higher than the original protein (increased by 20%), wherein the one mutant with the highest substrate conversion rate, pET28b-ATA117-rd11, had a conversion rate of 96% and ee > 99%.

The nucleotide sequence and the amino acid sequence of the mutant pET28b-ATA117-rd11 are shown as SEQ ID No. 3 and SEQ ID No.4 in the sequence table. The mutant pET28b-ATA117-rd11 is a mutant of SEQ ID NO: 2, the serine at the 8 th site of the amino acid shown in the figure is replaced by proline, the histidine at the 26 th site is replaced by valine, the tyrosine, the leucine and the histidine at the 60 th, the 61 th and the 62 th sites are replaced by phenylalanine, tyrosine and threonine, the valine at the 65 th site is replaced by alanine, the valine at the 69 th site is replaced by threonine, the aspartic acid at the 81 th site is replaced by glycine, the methionine and the isoleucine at the 94 th and the 96 th sites are replaced by isoleucine and leucine, the phenylalanine, the serine and the serine at the 122 th, 124 th and the 126 th sites are replaced by methionine, threonine and threonine, the glycine and the glutamic acid at the 136 th and the 137 th sites are replaced by phenylalanine and threonine, the tyrosine and the valine at the 150 th and the 152 th sites are replaced by serine and cysteine, the alanine at the 169 th site is replaced by leucine, the threonine at the 178 th site is replaced by serine, the valine at the 199 th site is replaced by isoleucine, alanine at position 209 is substituted with leucine, glycine and glycine at positions 215 and 217 are substituted with cysteine and asparagine, serine at position 223 is substituted with proline, leucine at position 269 is substituted with proline, leucine at position 273 is substituted with tyrosine, threonine and alanine at positions 282 and 284 are substituted with serine and glycine, proline at position 297 is substituted with serine, and serine at position 321 is substituted with proline.

Example 8 testing of the transaminase ATA117-rd11 with different amino donors in the transamination procedure

The recombinant Escherichia coli BL21/pET28b-ATA117-rd11 wet cells containing the recombinant plasmid for expression obtained in example 7 was used as a biocatalyst, and 4- (methylhydroxyphosphoryl) -2-carbonyl-butyric acid was used as a substrate.

Respectively taking L-aspartic acid, L-glutamic acid, L-aspartic acid, L-glutamic acid and L-alanine as amino donors.

Transformation system: 90mL of tris-HCl buffer was added with 0.36g (20mM) of PPO (glufosinate-ammonium), 0.3192g (24mM) of L-aspartic acid, and 0.0588g (4mM) of L-glutamic acid. 1.2g of Tris was added to adjust the pH to 8.0. The volume is 100mL, 0.0247g PLP (1mM) is added, and the amount of ATA117-rd11 wet bacteria is 50 g/L.

The amino donor was replaced with 0.3724g (28mM) L-aspartic acid, 0.4116g (28mM) L-glutamic acid, 0.6237g (70mM) L-alanine, respectively. Meanwhile, the reaction is carried out for 24 hours at 35 ℃, samples are respectively taken, diluted by 10 times and subjected to derivatization, and the following results can be obtained according to standard curves:

amino donor	Conversion rate%
		L-aspartic acid and L-glutamic acid	63.95
L-aspartic acid	26.70
		L-glutamic acid	61.97
L-alanine	43.22

To investigate the reason why the conversion rates were higher when using L-aspartic acid and L-glutamic acid as mixed amino donors than when using two amino acids as amino donors, respectively, to determine whether the transaminases ATA117-rd11 could catalyze two-step reactions simultaneously, the following supplementary experiments were performed:

(1) 90mL of tris-HCl buffer was added with 0.353g (20mM) of L-glutamic acid and 2mM, 4mM, 6mM, 8mM, 10mM, 20mM, and 30mM MPPO, respectively. Tris was added to adjust the pH to 8.0. The volume is 100mL, 0.0247g PLP (1mM) is added, and the amount of ATA117-rd11 wet bacteria is 50 g/L. After 24 hours of reaction at 35 ℃, sampling, diluting by 10 times, performing derivatization, and obtaining the following results according to standard curves:

substrate concentration (mM)	Conversion (%)
		2	100
4	100
		6	95.44
8	90.16
		10	77.48
20	60.58
		30	43.15

(2) Preparing 90mL of Tris-HCl buffer solution, adding 0.266g (20mM) of L-aspartic acid, adding 2mM, 4mM, 6mM, 8mM, 10mM, 20mM and 30mM α -ketoglutaric acid respectively, supplementing Tris, adjusting the pH to 8.0, diluting to 100mL, adding 0.0247g of PLP (1mM), adjusting the amount of ATA117-rd11 wet bacteria to 50g/L, reacting for 24h at 35 ℃, sampling and diluting 10 times respectively, performing derivatization, and obtaining the following results according to the standard:

Α-KG concentration (mM)	Conversion (%)
		2	53.67％
4	51.42％
		6	55.50％
8	61.61％
		10	60.96％
20	36.97％
		30	36.22％

Thus, it is demonstrated that the transaminase ATA117-rd11 can catalyze two reactions simultaneously.

Example 9 optimization of the conditions of the transaminase ATA117-rd11 in the transamination Process

(1) Selection of buffer system

Disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, Tris/HCl buffer solution, borax-boric acid buffer solution and sodium hydroxide are respectively used as buffer systems.

Transformation System 1:

① disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution

Reaction system: 40mL of a disodium hydrogenphosphate-sodium dihydrogenphosphate buffer solution was prepared by mixing 0.18g of PPO (glufosinate-ammonium), 0.16g L-aspartic acid, and 0.0294g L-glutamic acid, respectively. The pH was adjusted to 6.0 and 7.0, respectively. To 50mL volume, 0.0125g PLP (1mM) was added. The wet bacterial count of ATA117-rd11 was 50 g/L. The reaction is carried out for 6h, 12h and 24h at the temperature of 35 ℃, samples are taken for dilution, and 200 mu L of the solution is respectively taken for derivatization.

② Tris/HCl buffer

Reaction system: Tris/HCl buffer 40mL × 2 was added with 0.18g of PPO (glufosinate acid), 0.16g L-aspartic acid, and 0.0294g L-glutamic acid, respectively. The pH was adjusted to 8.0 and 9.0, respectively. To 50mL volume, 0.0125g PLP (1mM) was added. The wet bacterial count of ATA117-rd11 was 50 g/L. The reaction is carried out for 6h, 12h and 24h at the temperature of 35 ℃, samples are taken for dilution, and 200 mu L of the solution is respectively taken for derivatization.

③ Potassium chloride-sodium hydroxide as buffer

Reaction system: 40mL of potassium chloride-sodium hydroxide buffer solution was added with 0.18g of PPO (glufosinate-ammonium), 0.16g of L-aspartic acid, and 0.0294g L-glutamic acid. Adjusted to pH 10.0. To 50mL volume, 0.0125g PLP (1mM) was added. The wet bacterial count of ATA117-rd11 was 50 g/L. The reaction is carried out for 6h, 12h and 24h at the temperature of 35 ℃, samples are taken for dilution, and 200 mu L of the solution is respectively taken for derivatization.

According to the standard, the following results can be obtained:

transformation System 2:

① is mixed with 40mL × 4 borax-boric acid buffer solution, 0.18g PPO (glufosinate-ammonium), 0.16g L-aspartic acid and 0.0294g L-glutamic acid are respectively added, the pH is respectively adjusted to 7.5, 8.0, 8.5 and 9.0, the solution is diluted to 50mL, 0.0125g PLP (1mM), ATA117-rd11 wet bacteria amount is 50g/L, the reaction is carried out for 4h, sampling and dilution are carried out, and 200 μ L are respectively taken for derivatization.

② is matched with 40mL multiplied by 3 disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, 0.18g PPO (glufosinate-ammonium), 0.16g L-aspartic acid and 0.0294g L-glutamic acid are respectively added, the pH is respectively adjusted to 6.0, 6.5 and 7.0, the volume is adjusted to 50mL, 0.0125g PLP (1mM), ATA117-rd11 wet bacteria amount is 50 g/L.35 ℃, the reaction is carried out for 4h, sampling dilution is carried out, and 200 mu L is respectively taken for derivatization.

According to the standard, the following results can be obtained:

(2) temperature optimization

Transformation system: 40mL of Tris/HCl buffer was prepared, and 0.18g of PPO (glufosinate-ammonium), 0.16g L-aspartic acid, and 0.0294g L-glutamic acid were added. The pH was adjusted to 8.0 with additional Tris solution. The volume is 50mL, 0.0125g PLP (1mM) is added, and the ATA117-rd11 wet bacterial dose is 50 g/L. Reacting for 4h in a water bath at 30 ℃, 35 ℃, 40 ℃, 45 ℃ and 50 ℃, sampling and diluting, and derivatizing 200 mu L of the solution respectively to obtain the following results according to standard curves:

(3) bacterial load optimization

Transformation system: 40mL of Tris/HCl buffer was prepared, and 0.9g of PPO (glufosinate-ammonium), 0.8g of L-aspartic acid, and 0.2352g of L-glutamic acid were added. The pH was adjusted to 8.0 with additional Tris solution. The volume was 50mL and 0.0625g PLP was added. The following results were obtained from the standard samples by weighing 0.05g, 0.1g, 0.2g, 0.4g, 0.6g, 0.8g, and 1.0g of microbial cells per 10mL of the reaction system, reacting for 4 hours in a 40 ℃ water bath, sampling and diluting, and derivatizing 200. mu.L of each of the samples:

(4) optimization of coenzyme PLP dosage

Transformation system: to 40mL of Tris/HCl buffer, 0.18g of PPO (glufosinate-ammonium), 0.16g of L-aspartic acid, and 0.0294g of L-glutamic acid were added. The pH was adjusted to 8.0 with additional Tris solution. A gradient of 0.02mM to 2mM PLP was weighed to 50mL each, and the amount of ATA117-rd11 wet mycelia was 50 g/L. Reacting for 4 hours in a water bath at 40 ℃, sampling and diluting, and respectively taking 200 mu L of the solution for derivatization, wherein the following results can be obtained according to standard curves:

example 10 Synthesis of L-Glufosinate-ammonium from the transaminase ATA117-rd11

0.36g (20mM) PPO (glufosinate-ammonium), 0.3192g (24mM) L-aspartic acid, 0.0588g (4mM) L-glutamic acid were weighed out. The pH was adjusted to 8.0 with Tris buffer. The volume is 100mL, 0.0247g PLP (1mM) is added, and the ATA117-rd11 wet bacterial weight is 50 g/L. The reaction is carried out at 40 ℃, samples are diluted, 200 mu L of the diluted solution is respectively taken for derivatization, and the following results can be obtained according to standard curves:

reaction time/h	Conversion rate
			1	28.51％
2	39.35％
		4	48.75％
6	61.27％
		12	70.54％
24	67.20％

Example 11

30mL of Tris/HCl buffer was prepared, the pH was adjusted to 8.0, and 0.36g (20mM) of PPO (glufosinate-ammonium), 0.399g (30mM) of L-aspartic acid, and 0.0588g (4mM) of L-glutamic acid were added. Tris was added to adjust pH to 8.0. The volume is adjusted to 30mL, 0.0247g PLP (1mM) is added, and the ATA117-rd11 wet bacterial weight is 50 g/L. The reaction is carried out at 40 ℃, samples are taken for dilution, 200uL of the solution is respectively used for derivatization, and the following results can be obtained according to standard curves:

example 12

8mL of Tris/HCl buffer was prepared, the pH was adjusted to 8.0, and 0.18g (100mM) of PPO (glufosinate-ammonium), 0.16g (120mM) of L-aspartic acid, and 0.0294g (20mM) of L-glutamic acid were added. Tris was added to adjust pH to 7.0. The volume is adjusted to 10mL, 0.0125g PLP is added, and the ATA117-rd11 wet bacterial quantity is 60 g/L. The reaction is carried out at 40 ℃, samples are diluted, 200 mu L of the diluted solution is respectively taken for derivatization, and the following results can be obtained according to standard curves:

reaction time/h	Conversion rate
		6	64.63％
12	78.87％
		24	86.82％
36	88.69％
		48	91.71％

Example 13

8mL of Tris/HCl buffer was prepared, the pH was adjusted to 8.0, and 0.36g (200mM) of PPO (glufosinate-ammonium), 0.32g (600mM) of L-aspartic acid, and 0.0588g (100mM) of L-glutamic acid were added. Tris was added to adjust pH to 7.0. The volume is adjusted to 10mL, 0.025g PLP is added, and the amount of ATA117-rd11 wet bacteria is 60 g/L. The reaction is carried out at 40 ℃, samples are diluted, 200 mu L of the diluted solution is respectively taken for derivatization, and the following results can be obtained according to standard curves:

reaction time/h	Conversion rate
		6	59.37％
12	73.61％
		24	77.06％
36	82.45％
		48	83.33％

Example 14

8mL of Tris/HCl buffer was prepared, the pH was adjusted to 8.0, and 0.9g (500mM) of PPO (glufosinate-ammonium), 0.8g (600mM) of L-aspartic acid, and 0.147g (100mM) of L-glutamic acid were added. Tris was added to adjust pH to 7.0. The volume is adjusted to 10mL, 0.0625g PLP is added, and the amount of ATA117-rd11 wet bacteria is 60 g/L. The reaction is carried out at 40 ℃, samples are diluted, 200 mu L of the diluted solution is respectively taken for derivatization, and the following results can be obtained according to standard curves:

reaction time/h	Conversion rate
		6	24.57％
12	32.56％
		24	53.82％
36	62.75％
		48	68.87％

From the above experimental results, the recombinant Escherichia coli containing transaminase gene obtained by the present invention has strong transaminase ability, and can directly perform biocatalysis or transformation reaction by using enzyme-containing bacterial cells as enzyme source. Transaminase (BL21(DE3)/pET28a-ATA117-rd11) is used as a conversion enzyme, 4- (methyl hydroxyl phosphoryl) -2-carbonyl-butyric acid is used as a substrate, and a biotransformation reaction is carried out to prepare the high-optical-purity pesticide L-glufosinate-ammonium.

Sequence listing

<110> Zhejiang industrial university

<120> method for asymmetrically synthesizing L-glufosinate-ammonium by single transaminase catalytic cascade reaction

<150>201910216011.6

<151>2020-03-13

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Claims (8)

1. A method for asymmetrically synthesizing L-glufosinate-ammonium by a single transaminase-catalyzed cascade reaction, which comprises the following steps: 4- (methyl hydroxyl phosphoryl) -2-carbonyl-butyric acid or salt thereof is taken as a substrate, and in a reaction system with an amino donor, the substrate is catalyzed by using in vitro transaminase or cells expressing the transaminase in vitro to carry out a transamination reaction to obtain L-glufosinate-ammonium;

characterized in that the amino donor consists of two amino acids, namely L-glutamic acid and L-aspartic acid; only one transaminase is added into the reaction system, and the amino acid sequence of the transaminase is shown in SEQ ID No. 4.

2. The method for asymmetrically synthesizing L-glufosinate-ammonium by a single transaminase-catalyzed cascade reaction of claim 1, wherein the cell is an engineering bacterium for expressing transaminase, and a host cell of the engineering bacterium is E.coli BL21(DE 3).

3. The method for asymmetrically synthesizing L-glufosinate-ammonium by single transaminase catalysis cascade reaction according to claim 2, wherein the addition amount of the engineering bacteria is 20-200 g/L of reaction liquid based on the weight of wet bacteria in a reaction system.

4. The method for asymmetrically synthesizing L-glufosinate-ammonium by a single transaminase-catalyzed cascade reaction of claim 1, wherein the buffer solution in the reaction system is Tris/HCl buffer solution, and the pH value is 8-9.

5. The method for asymmetrically synthesizing L-glufosinate-ammonium by a single transaminase-catalyzed cascade reaction of claim 1, wherein the molar ratio of L-glutamic acid to L-aspartic acid is 1:4 to 1: 8.

6. The method for asymmetrically synthesizing L-glufosinate-ammonium by a single transaminase-catalyzed cascade reaction of claim 5, wherein the molar ratio of the amino donor to the substrate is 1.2-1.6: 1.

7. The method for asymmetrically synthesizing L-glufosinate-ammonium by a single transaminase-catalyzed cascade reaction of claim 1, wherein the temperature of the transaminase-catalyzed cascade reaction is 35-45 ℃ and the time is 24-48 h.

8. The method for asymmetrically synthesizing L-glufosinate-ammonium by single transaminase catalysis cascade reaction according to claim 1, wherein the reaction system further comprises a coenzyme, the coenzyme is pyridoxal phosphate, and the molar ratio of the coenzyme to the substrate is 1: 10-1: 200.

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