CN108690854A

CN108690854A - A method of producing L-glufosinate-ammonium using chemo-enzymatic process

Info

Publication number: CN108690854A
Application number: CN201810338960.7A
Authority: CN
Inventors: 薛亚平; 郑裕国; 曹成浩; 徐建妙; 吴哲明
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2018-04-16
Filing date: 2018-04-16
Publication date: 2018-10-23
Anticipated expiration: 2038-04-16
Also published as: CN108690854B

Abstract

The invention discloses a kind of method producing L-glufosinate-ammonium using chemo-enzymatic process, the method is:Using racemic N- acetyl glufosinate-ammonium as substrate, the wet thallus obtained using the fermented culture of the engineering bacteria of the gene containing carboxypeptidase or the pure enzyme extracted after wet thallus ultrasonication is catalyst, the buffer solution for being 5~10 using pH value is reaction medium, it is reacted under the conditions of 45 DEG C of shaking bath 200rpm, after the reaction was complete, the reaction solution of acetyl containing D-N- glufosinate-ammonium and L-glufosinate-ammonium is obtained, reaction solution is isolated and purified, it is split after collecting D-N- acetyl glufosinate-ammonium racemizations for recycling, while obtaining L-glufosinate-ammonium.The method of the present invention does not need the coenzyme circulatory system and amino group donor similar with product structure, reaction step is simple, is easily isolated, optical selective height (ee values 99%), ion exchange column separating effect is apparent, is more easy to obtain the L-glufosinate-ammonium (purity 98%) of high-purity.

Description

A method of producing L-glufosinate-ammonium using chemo-enzymatic process

(1) technical field

The present invention relates to biological chemical field, more particularly to a kind of method of chemo-enzymatic process production L-glufosinate-ammonium.

(2) background technology

Glufosinate-ammonium, also referred to as glufosinate, English are entitled:Phosphinothricin (abbreviation PPT), the entitled 2- amino-of chemistry 4-[Hydroxyl (methyl) Lin Xianji ]Butyric acid.Glufosinate-ammonium is a kind of inner-adsorption conduction-type herbicide, has wide spectrum removing activity.Weeding Agent is widely used, and domestic and international market is huge, and glufosinate-ammonium is one of three big herbicides, in recent years due to its mechanism of action and transgenosis Technology, the market share are expected to further break through.

Now glufosinate-ammonium in the market is mainly racemic modification.There are two types of optical isomers for glufosinate-ammonium:L-glufosinate-ammonium and D- Glufosinate-ammonium.But only L-glufosinate-ammonium have activity of weeding, be twice of racemic glufosinate-ammonium, and to the toxicity of humans and animals compared with Small, effect on environment is small.But the commercialization glufosinate-ammonium mass produced now is all the form of racemic mixture.Racemic The use of glufosinate-ammonium, waste is huge, and effect on environment is more serious.In order to mitigate environmental protection pressure, lowers production cost, visit The production line of one resolution of racemic glufosinate-ammonium with industrial applications foreground of rope is with important market prospects and society Meaning.

The method for preparing L-glufosinate-ammonium now is totally broadly divided into chemical method and biological enzyme.

Wherein chemical method includes mainly chemical stereo synthetic method and chiral separation.Chemical stereo synthetic method needs to use high Expensive asymmetric syntheses reagent, mainly laboratory research scale are unfavorable for preparing on a large scale.Chemical chiral resolution method will also disappear The a large amount of expensive chiral selectors of consumption, technique is more complex, and yield is generally relatively low.

Compared with chemical method, biological enzyme has many advantages, such as that reaction condition is mild, and stereoselectivity is stringent.Prepare L- grass ammoniums Phosphine biological enzyme is divided into enzyme process asymmetric syntheses and Enzymatic Resolution.Enzyme process asymmetric syntheses theoretical yield is higher, relates generally to Transaminase and amino acid dehydrogenase.

Schulz A(Stereospecific production of the herbicide phosphinothricin (glufosinate)by transamination:isolation and characterization of a phosphinothricin-specific transaminase from Escherichia coli[J].Applied and Environmental Microbiology,1990,56(1):1-6) etc. using the transaminase cloned from Escherichia coli, with 2- Carbonyl -4- (hydroxymethyl phosphono) butyric acid is substrate, and Pidolidone is amino group donor, prepares L-glufosinate-ammonium.But it is reversible Reaction often results in yield reduction, needs that excessive amino group donor is added, this brings prodigious burden to the product separation in later stage.

Biological enzyme is split generally by chemical synthesis racemic D, L-glufosinate-ammonium or derivatives thereof, is recycled specific Enzyme selectivity is catalyzed the reaction of a certain configuration, obtains one of optical isomer, another unreacted isomers derivative By separation, again after racemization, enzymic catalytic reaction is carried out again, and theoretical yield is up to 100%.

(3) invention content

Present invention aims at a kind of method that completely new chemo-enzymatic process produces L-glufosinate-ammonium is provided, this method raw material turns Rate is high, production cost is low, high income.

The technical solution adopted by the present invention is:

The present invention provides a kind of method of chemo-enzymatic process production glufosinate-ammonium, and the method is:With racemic N- acetyl grass ammoniums Phosphine is substrate, with what is extracted after the wet thallus of the fermented culture acquisition of the engineering bacteria of the gene containing carboxypeptidase or wet thallus ultrasonication Pure enzyme is catalyst, and the buffer solution for being 5~10 with pH value (the Tris-HCl buffer solutions of preferably pH=8.9,50mM) is that reaction is situated between Matter is reacted under the conditions of 45 DEG C of shaking bath 200rpm, after the reaction was complete, obtains the glufosinate-ammonium of acetyl containing D-N- and L-glufosinate-ammonium Reaction solution isolates and purifies reaction solution, is split after collecting D-N- acetyl glufosinate-ammonium racemizations for recycling, while obtaining L- grass ammoniums Phosphine.

Further, the carboxypeptidase derives from Stenotrophomonas (Stenotrophomonas sp.) Carboxypeptidase S s1.It carries out Codon optimization after being recombinantly expressed in colibacillus engineering, has catalysis L-N- acetyl-glufosinate-ammonium production L-glufosinate-ammonium Ability.The amino acid sequence of the Carboxypeptidase S s1 is shown in SEQ ID NO.1, and the nucleotides sequence of encoding gene is classified as SEQ ID Shown in NO.2.

Further, the Final substrate concentrations are calculated as 10~100mM (preferably 100mM), the catalyst with buffer solution volume It is calculated as 5~50g/L buffer solutions (preferably 10g/L) with wet thallus weight.

Further, the racemic N- acetyl glufosinate-ammoniums are prepared as follows:Using racemic glufosinate-ammonium as raw material, add Enter acetic acid, 10-60 DEG C of (preferably 30-40 DEG C) insulated and stirred is added acetic anhydride, reaction to solution clear, 80 DEG C, Revolving removes acetic acid under the conditions of 0.075~0.085MPa, takes concentrate water dissolution, sodium hydrate aqueous solution is used in combination to be adjusted to neutrality (pH 8.5) is added ethyl alcohol standing and precipitates crystal, racemic N- acetyl glufosinate-ammoniums are obtained after dry;The acetic acid and racemic Glufosinate-ammonium weight ratio 20~30:1 (preferably 20:1), the acetic anhydride and racemic glufosinate-ammonium weight ratio are 0.7~1.5:1 (preferably 1:1).

Further, the catalyst is prepared as follows:(1) wet thallus:The engineering bacteria of the gene containing carboxypeptidase is inoculated with Into the LB liquid medium containing 50 μ g/mL kanamycins, 37 DEG C of overnight incubations, then be inoculated into 1% inoculum concentration of volumetric concentration In LB liquid medium containing 50 μ g/mL kanamycins, 37 DEG C, 150rpm cultivates to cell concentration OD₆₀₀To 0.4-0.8, it is added After the IPTG of final concentration of 0.1mM, 28 DEG C of Fiber differentiation 12h, 4 DEG C, 8000rpm centrifugation 10min collect wet thallus;(2) pure Enzyme:Step (1) wet thallus is suspended in the Tris-HCl buffer solutions of 20mM, pH 8.0, oscillation shakes up rear ultrasonic disruption (400W, 1s are crushed 1s pauses) 15min is crushed liquid and removes cell fragment in 12,000rpm centrifugations 10min, collects supernatant;It will Supernatant carries out Ni-NTA column chromatographies, after first using loading equilibration buffer Ni-NTA columns, with the rate loading of 1.5mL/min Supernatant, is eluted with loading equilibration buffer to remove unadsorbed albumen, is finally eluted with elution buffer and is collected target egg In vain, target protein is dialysed in the Tris-HCl of 20mM, pH 8.5, and it is pure enzyme to take trapped fluid;The loading equilibration buffer For the 20mM Tris-HCl, pH 8.5 of NaCl containing 500mM and 20mM imidazoles;The elution buffer be NaCl containing 500mM and The 20mM Tris-HCl, pH 8.5 of 500mM imidazoles.

Further, the method that the reaction solution isolates and purifies is:After the reaction was complete, reaction solution is centrifuged, takes supernatant tune PH value is saved to 2, filters, takes filtrate loading to Hydrogen 001x7 resin cations, ion exchange column pillar height is than 15:1, loading flow velocity For 1.0BV/h, ultrapure water 4BV is used after loading, is collected and is contained unreacted substrate D-N- acetyl glufosinate-ammonium effluxes, by D-N- It is split after acetyl glufosinate-ammonium racemization for recycling;It uses 2mol/L ammonium hydroxide to be eluted with 0.5BV/h flow velocitys again, collects and contain L- grass ammoniums The eluent of phosphine is concentrated under reduced pressure crystallization and obtains L-glufosinate-ammonium.

Further, the D-N- acetyl glufosinate-ammonium racemization method is:The decompression of D-N- acetyl glufosinate-ammonium effluxes will be contained to steam It is dry, acetic acid mixing is added, adds acetic anhydride, stirs racemization at 100-125 DEG C (preferably 120 DEG C), after reaction will Reaction solution evaporated under reduced pressure obtains racemic N- acetyl glufosinate-ammoniums, is split for recycling;The acetic anhydride and D-N- acetyl glufosinate-ammoniums Mass ratio is 0.1~3:1 (preferably 1.7:1), acetic acid and D-N- acetyl glufosinate-ammonium mass ratioes are 10~20:1 (preferably 10:1).

Beneficial effects of the present invention are embodied in:The present invention provides a kind of methods that chemo-enzymatic process produces L-glufosinate-ammonium, should Method does not need the coenzyme circulatory system and amino group donor similar with product structure, and reaction step is simple, is easily isolated.This method Catalyst carboxypeptidase optical selective is high (ee values 99%), and ion exchange column separating effect is apparent, is more easy to obtain the L- of high-purity Glufosinate-ammonium (purity 98%).

(4) it illustrates

Fig. 1 prepares the reaction equation of L-glufosinate-ammonium for the chemo-enzymatic process based on carboxypeptidase.

Fig. 2 is the liquid phase spectrogram of N- acetyl glufosinate-ammoniums in embodiment 1, retention time 9.0min.

Fig. 3 is the mass spectrogram of N- acetyl glufosinate-ammoniums in embodiment 1.

Fig. 4 is the liquid phase spectrogram of L-glufosinate-ammonium and D- glufosinate-ammoniums in embodiment 5, and L-glufosinate-ammonium retention time is 4.6min, D- glufosinate-ammonium retention times are 5.4min.

(5) specific implementation mode

With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in This:

The ultra-pure water refers to being handled by ultra-pure water instrument, in addition to hydrone (H in water₂O) outside, miscellaneous almost without what Matter, without bacterium, virus, containing organic matters and mine trace elements such as green dioxin.

Embodiment 1:The synthesis of racemic N- acetyl glufosinate-ammoniums

The racemic glufosinate-ammoniums of 5g are taken, are put into 250mL three-necked flasks, 100g acetic acid is added, 40 DEG C of insulated and stirreds are added 5g acetic anhydrides, reaction to solution clear.80 DEG C of Rotary Evaporators, 0.075~0.085MPa of vacuum degree boil off acetic acid.It is dense Contracting object water dissolution, sodium hydrate aqueous solution are adjusted to neutral (pH 8.5), and ethyl alcohol standing is added and precipitates crystal, is obtained after dry Racemic N- acetyl glufosinate-ammoniums.It is detected using liquid chromatogram (Shimadzu LC-16), liquid phase spectrogram is Fig. 2, and mass spectrum verification confirms it is N- Acetyl glufosinate-ammonium (Fig. 3).Chromatographic column isC-18column (250mm × 4.6mm, 5 μm), mobile phase 10mM Phosphoric acid solution (pH 2.1), flow velocity 0.75mL/min, ultraviolet detection wavelength 210nm, 35 DEG C of column temperature.

Embodiment 2:The structure of engineering bacteria

Stenotrophomonas (Stenotrophomonas sp.) is derived from by one plant, is annotated as the sequence of Carboxypeptidase S s1, warp Full genome synthesis, expression plasmid pET-28b are carried out after crossing codon optimization.By terminator codon rite-directed mutagenesis, make expression Recombinase tail portion (C-terminal) is containing the histidine tag in pET28b.(amino acid sequence is core shown in SEQ ID NO.1 to insetion sequence Nucleotide sequence is shown in SEQ ID NO.2) expressive host E. coli BL21 (DE3) is transferred to after sequence verification is errorless In be used for subsequent recombination enzyme expression.

Embodiment 3:The induced expression of recombinase

The engineering bacteria that embodiment 2 is built is seeded in the LB liquid medium containing 50 μ g/mL kanamycins, and 37 DEG C were cultivated Night, then be inoculated into the LB liquid medium containing 50 μ g/mL kanamycins with 1% inoculum concentration (v/v), 37 DEG C, 150rpm cultures To cell concentration OD₆₀₀To 0.6 or so, it is added the IPTG of final concentration of 0.1mM, after 28 DEG C of Fiber differentiation 12h, 4 DEG C, 8000rpm 10min is centrifuged, wet thallus cell is collected.

Embodiment 4:Enzyme isolates and purifies

The wet thallus cell that embodiment 3 is collected is suspended in 50mL Tris-HCl buffer solutions (20mM, pH 8.0), is vibrated Shake up rear ultrasonic disruption (400W, 15min, 1s are crushed 1s pauses, effective time 15min).Broken liquid is centrifuged in 12,000rpm 10min removes cell fragment, collects supernatant (crude enzyme liquid) and is isolated and purified for subsequent.Purification column is Ni-NTA, fills cylinder Product is 20mL, first loading equilibration buffer (20mM Tris-HCl, 500mM NaCl and 20mM imidazoles, pH 8.5) is used to balance Ni- NTA columns are eluted to remove unadsorbed albumen, finally with the rate loading crude enzyme liquid of 1.5mL/min with loading equilibration buffer It is eluted with elution buffer (20mM Tris-HCl, 500mM NaCl and 500mM imidazoles, pH 8.5) and collects target protein.Enzyme Liquid dialysed overnight (replacing 2 dialyzates) in 20mM, the Tris-HCl of pH 8.5, the trapped fluid for taking last time dialysis to obtain As enzyme solution.

Embodiment 5:Enzyme activity determination

Reaction system is 10mL, and the reality of final concentration of 7.8 μ g/mL is added in buffer solution containing Tris-HCl (20mM, pH 8.9) The enzyme of the preparation of example 4 is applied, the racemic N- acetyl glufosinate-ammoniums of the preparation of embodiment 1 of final concentration of 20g/L, 45 DEG C in shaking bath 10min is reacted under the conditions of 200rpm measures enzyme activity.After the completion of reaction, take 100 μ L reaction solutions that 5 μ L HCl (4M) is added to terminate anti- Answer, after pass through derivatization method and analyze conversion ratio and product ee values.Chromatographic column isC-18column(150mm× 4.6mm, 5 μm), mobile phase is methanol:Ammonium acetate solution=10 0.05mol/L:90 (v/v) flow velocitys are 1mL/min, ultraviolet detection Wavelength 215nm, 35 DEG C of column temperature.Derivatization reagent is prepared:The derivatization reagent OPA and NAC (0.015M) for weighing equivalent respectively are mixed It closes, absolute ethyl alcohol dissolving is added prepared borate buffer (20mM pH=9.8) constant volume and is placed on 4 DEG C of preservations.Derivatization Reaction system:200 μ l sample solution (s <It 2.0g/L) mixes, shakes under 400 μ l derivatization reagents (derivatization reagent is excessive) room temperature 6min is swung, the liquid phase spectrogram of L-glufosinate-ammonium is as shown in Figure 4.

Enzyme activity (U) defines:Under conditions of 45 DEG C, pH 8.9, the enzyme per minute generated needed for 1 micromole's L-glufosinate-ammonium Amount.Specific enzyme activity refers to enzyme activity possessed by every milligram of zymoprotein, U/mg.After measured, specific enzyme activity 23.1U/mg.

Embodiment 6:Influence of the metal ion to recombinase enzyme activity

Using the enzyme collected in embodiment 4 as catalyst, in Co²⁺,Cu²⁺,Fe²⁺,Ba²⁺,Mn²⁺,Mg²⁺,Fe³⁺,Ni²⁺,Al²⁺, Zn²⁺In the presence of EDTA, catalysis substrate N- acetyl glufosinate-ammonium stereo selective hydrolysis generates L-glufosinate-ammonium.Living things catalysis system Composition and operation are as follows:10mL reaction systems, system buffer solution are Tris-HCl (pH=8.9,50mM), metal ion and EDTA Final concentration of 1mM, recombinase additive amount are 8.1 μ g/mL, Final substrate concentrations 20g/L.45 DEG C in shaking bath 200rpm conditions Lower reaction measures enzyme activity using 5 method of embodiment, and the results are shown in Table 1.Enzyme activity determination shows Co²⁺Addition enzyme activity is carried Height, which has, to be significantly affected, and enzyme activity improves 1.57 times, Zn²⁺Addition to enzyme have significant inhibiting effect.

1 metal ion of table influences recombinase

Embodiment 7:Recombination bacillus coli catalysis containing recombinase generates L-glufosinate-ammonium

Using the recombination engineering wet thallus collected in embodiment 3 as biocatalyst, catalysis substrate N- acetyl glufosinate-ammoniums are vertical Body selective hydrolysis generates L-glufosinate-ammonium.Living things catalysis system forms and operation is as follows:It weighs 2.5g wet thallus and is suspended in 250mL In Tris-HCl buffer solutions (pH=8.9,50mM), the N- acetyl glufosinate-ammoniums of final concentration 100mM are added, 45 DEG C in shaking bath It is reacted under the conditions of 200rpm, after 30min, reaction solution detects conversion ratio 49%, ee values 99% using 5 method liquid phase of embodiment.

Embodiment 8:The separation of L-glufosinate-ammonium and D-N- acetyl glufosinate-ammoniums

The pretreatment of Hydrogen 001x7 resin cations:(1) column is washed with deionized water, flow velocity 1.0BV/h washes 2BV;(2) Column is washed with 2M sodium hydrate aqueous solutions, flow velocity 0.5BV/h washes 2BV;(3) column is washed with deionized water, flow velocity 1.0BV/h is washed 2BV;(4) column is washed with 2M aqueous hydrochloric acid solutions, flow velocity 0.5BV/h washes 2BV;(5) column, flow velocity 1.0BV/ is washed with deionized water H washes 2BV.

Reaction solution centrifugation removal thalline, supernatant in embodiment 7 are filtered with hydrochloric acid tune pH to 2, filtrate loading is to The Hydrogen 001x7 resin cations pre-processed, column volume 120mL, ion exchange column pillar height is than 15:1, loading flow velocity is 1.0BV/h uses ultrapure water 4BV after loading, collects and contains unreacted substrate D-N- acetyl glufosinate-ammonium effluxes.It uses again 2mol/L ammonium hydroxide is eluted with 0.5BV/h flow velocitys, collects the eluent containing L-glufosinate-ammonium.By eluent at 60 DEG C, vacuum degree Under 0.075~0.085MPa, crystallization is concentrated under reduced pressure and obtains the L-glufosinate-ammonium that purity is 98%.

Embodiment 9:The chemical racemization reaction of D-N- acetyl glufosinate-ammoniums

Efflux 100ml containing unreacted substrate D-N- acetyl glufosinate-ammoniums in embodiment 8 is concentrated under reduced pressure into 50mL, is surveyed Optical activity is -0.59, and repressurization is evaporated, and obtains D-N- acetyl glufosinate-ammoniums 3g.The mixing of 30g acetic acid is added, adds 5g acetic anhydrides, Racemization 12h is stirred at 120 DEG C, after reaction, by reaction solution evaporated under reduced pressure, is obtained racemic N- acetyl glufosinate-ammoniums, is added water-soluble 50mL is solved, it is -0.002 to survey optical activity, preserves and is split for recycling.

Comparative example 1:The separation and Extraction L-glufosinate-ammonium from enzymatic conversion liquid

By reaction solution centrifugation removal thalline in embodiment 7, evaporated under reduced pressure removal part acetic acid uses hydrogen-oxygen after water dissolution Change sodium water solution and be adjusted to neutral (pH 8.5), ethyl alcohol is added and stands, and reduce temperature to 4 DEG C, finds and nodeless mesh is precipitated.Again Acetone is added, discovery has precipitation to generate, and carries out liquid phase detection, finds to contain 30%~50% D-N- acetyl glufosinate-ammoniums in precipitation With 10%~20% L-glufosinate-ammonium.

Sequence table

<110>Zhejiang Polytechnical University

<120>A method of producing L-glufosinate-ammonium using chemical-enzymatic

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Met Ala Ala Ala Leu Pro Tyr Ile Gln Leu Glu Pro Pro Ala Val Glu

1 5 10 15

Ile Ala Met Arg Arg Ser Leu Leu Leu Ser Ala Leu Leu Leu Ala Leu

20 25 30

Pro Ala Leu Ala His Ala Gln Asp Gly Gln Arg Pro Glu Val Thr Ala

35 40 45

Ala Ala Gln Arg Leu Gln Ala Lys Val Val Glu Trp Arg Arg Asp Phe

50 55 60

His Gln His Pro Glu Leu Ser Asn Arg Glu Val Arg Thr Ser Ala Glu

65 70 75 80

Val Ala Lys Arg Leu Arg Ala Met Gly Leu Lys Pro Lys Thr Gly Ile

85 90 95

Ala His His Gly Val Val Ala Ile Ile Glu Gly Gly Lys Pro Gly Pro

100 105 110

Lys Ile Ala Leu Arg Ala Asp Met Asp Ala Leu Pro Val Thr Glu Gln

115 120 125

Thr Gly Leu Pro Phe Ala Ser Arg Ala Thr Asp Gln Tyr Arg Gly Gln

130 135 140

Thr Val Gly Val Met His Ala Cys Gly His Asp Ala His Thr Ala Thr

145 150 155 160

Leu Leu Gly Val Ala Glu Ala Leu Val Ser Met Lys Lys Asp Leu Pro

165 170 175

Gly Gln Val Met Leu Ile Phe Gln Pro Ala Glu Glu Gly Ala Pro Pro

180 185 190

Pro Glu Glu Gly Gly Ala Ala Leu Met Leu Lys Glu Gly Leu Phe Ala

195 200 205

Asp Phe Lys Pro Glu Ala Val Phe Gly Met His Val Phe Ser Ser Val

210 215 220

Gln Ala Gly Gln Ile Ala Val Arg Gly Gly Pro Leu Met Ala Ala Ser

225 230 235 240

Asp Arg Phe Gly Ile Lys Val Ile Gly Arg Gln Thr His Gly Ser Ala

245 250 255

Pro Trp Asn Gly Val Asp Pro Ile Val Ala Thr Ala Asp Leu Val Gly

260 265 270

Thr Ala Gln Thr Ile Val Ser Arg Arg Ala Asn Leu Ser Lys Gln Pro

275 280 285

Ala Val Leu Thr Phe Gly Ala Ile Asn Gly Gly Ile Arg Tyr Asn Ile

290 295 300

Ile Pro Asp Glu Val Glu Met Val Gly Thr Ile Arg Thr Phe Asp Glu

305 310 315 320

Gly Met Arg Gln Gln Ile Phe Ala Asp Leu Arg Asn Val Ala Glu His

325 330 335

Thr Ala Ala Ala His Gly Ala Lys Ala Val Thr Asp Ile Tyr Glu Ser

340 345 350

Glu Gly Asn Pro Ala Thr Val Asn Asp Pro Ala Leu Thr Ala Lys Met

355 360 365

Leu Pro Ser Leu Gln Ala Val Val Gly Lys Asp Asn Val Tyr Glu Pro

370 375 380

Pro Leu Gln Met Gly Ala Glu Asp Phe Ser Leu Tyr Ala Lys Glu Val

385 390 395 400

Pro Gly Met Phe Phe Phe Val Gly Ser Thr Ser Val Gly Ile Asp Pro

405 410 415

Ala Thr Ala Pro Ala Asn His Ser Pro Lys Phe Leu Leu Asp Glu Lys

420 425 430

Ala Leu Asp Val Gly Leu Arg Ala Leu Leu Gln Val Ser Leu Asp Tyr

435 440 445

Leu His Gly Ala Gly Thr Pro Ala Gly

450 455

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atggctgctg ctctgccgta catccagctg gaaccgccgg ctgttgaaat cgctatgcgt 60

cgttctctgc tgctgtctgc tctgctgctg gctctgccgg ctctggctca cgctcaggac 120

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gttgctaaac gtctgcgtgc tatgggtctg aaaccgaaaa ccggtatcgc tcaccacggt 300

gttgttgcta tcatcgaagg tggtaaaccg ggtccgaaaa tcgctctgcg tgctgacatg 360

gacgctctgc cggttaccga acagaccggt ctgccgttcg cttctcgtgc taccgaccag 420

taccgtggtc agaccgttgg tgttatgcac gcttgcggtc acgacgctca caccgctacc 480

ctgctgggtg ttgctgaagc tctggtttct atgaaaaaag acctgccggg tcaggttatg 540

ctgatcttcc agccggctga agaaggtgct ccgccgccgg aagaaggtgg tgctgctctg 600

atgctgaaag aaggtctgtt cgctgacttc aaaccggaag ctgttttcgg tatgcacgtt 660

ttctcttctg ttcaggctgg tcagatcgct gttcgtggtg gtccgctgat ggctgcttct 720

gaccgtttcg gtatcaaagt tatcggtcgt cagacccacg gttctgctcc gtggaacggt 780

gttgacccga tcgttgctac cgctgacctg gttggtaccg ctcagaccat cgtttctcgt 840

cgtgctaacc tgtctaaaca gccggctgtt ctgaccttcg gtgctatcaa cggtggtatc 900

cgttacaaca tcatcccgga cgaagttgaa atggttggta ccatccgtac cttcgacgaa 960

ggtatgcgtc agcagatctt cgctgacctg cgtaacgttg ctgaacacac cgctgctgct 1020

cacggtgcta aagctgttac cgacatctac gaatctgaag gtaacccggc taccgttaac 1080

gacccggctc tgaccgctaa aatgctgccg tctctgcagg ctgttgttgg taaagacaac 1140

gtttacgaac cgccgctgca gatgggtgct gaagacttct ctctgtacgc taaagaagtt 1200

ccgggtatgt tcttcttcgt tggttctacc tctgttggta tcgacccggc taccgctccg 1260

gctaaccact ctccgaaatt cctgctggac gaaaaagctc tggacgttgg tctgcgtgct 1320

ctgctgcagg tttctctgga ctacctgcac ggtgctggta ccccggctgg t 1371

Claims

1. a kind of method of chemo-enzymatic process production glufosinate-ammonium, it is characterised in that the method is:With racemic N- acetyl glufosinate-ammoniums It is pure to be extracted after the wet thallus of the fermented culture acquisition of the engineering bacteria of the gene containing carboxypeptidase or wet thallus ultrasonication for substrate Enzyme is catalyst, and the buffer solution for being 5~10 using pH value reacts, instead as reaction medium under the conditions of 45 DEG C of shaking bath 200rpm After answering completely, the reaction solution of acetyl containing D-N- glufosinate-ammonium and L-glufosinate-ammonium is obtained, reaction solution is isolated and purified, collects D-N- acetyl It is split after glufosinate-ammonium racemization for recycling, while obtaining L-glufosinate-ammonium.

2. the method for chemo-enzymatic process production glufosinate-ammonium as described in claim 1, it is characterised in that the carboxypeptidase gene nucleotide Sequence is shown in SEQ ID NO.2.

3. the method for chemo-enzymatic process production glufosinate-ammonium as described in claim 1, it is characterised in that the Final substrate concentrations are to buffer Liquid product is calculated as 10~100mM, and the catalyst amount is calculated as 5~50g/L buffer solutions with wet thallus weight.

4. the method for chemo-enzymatic process production glufosinate-ammonium as described in claim 1, it is characterised in that the racemic N- acetyl grass ammonium Phosphine is prepared as follows:Using racemic glufosinate-ammonium as raw material, acetic acid is added, acetic anhydride is added in 10-60 DEG C of insulated and stirred, To solution clear, revolving removes acetic acid under the conditions of 80 DEG C, 0.075~0.085MPa, takes concentrate water dissolution for reaction, It is used in combination sodium hydrate aqueous solution to be adjusted to neutrality, ethyl alcohol standing is added and precipitates crystal, it is dry, obtain racemic N- acetyl glufosinate-ammoniums; The acetic acid and racemic glufosinate-ammonium weight ratio 20~30:1, the acetic anhydride is 0.7 with racemic glufosinate-ammonium weight ratio ~1.5:1.

5. the method for chemo-enzymatic process production glufosinate-ammonium as described in claim 1, it is characterised in that the catalyst is as follows It prepares:(1) wet thallus:The engineering bacteria of the gene containing carboxypeptidase is seeded in the LB liquid medium containing 50 μ g/mL kanamycins, 37 DEG C of overnight incubations, then be inoculated into the LB liquid medium containing 50 μ g/mL kanamycins with 1% inoculum concentration of volumetric concentration, 37 DEG C, 150rpm cultivates to cell concentration OD₆₀₀To 0.4-0.8, the IPTG of final concentration of 0.1mM, 28 DEG C of Fiber differentiation 12h is added Afterwards, 4 DEG C, 8000rpm centrifugation 10min, collect wet thallus;(2) pure enzyme:Step (1) wet thallus is suspended in 20mM, pH's 8.0 In Tris-HCl buffer solutions, oscillation shakes up rear ultrasonic disruption 15min, is crushed liquid and removes cell in 12,000rpm centrifugations 10min Fragment collects supernatant;Supernatant is subjected to Ni-NTA column chromatographies, after first using loading equilibration buffer Ni-NTA columns, with The rate loading supernatant of 1.5mL/min is eluted with loading equilibration buffer to remove unadsorbed albumen, finally slow with elution Target protein is collected in fliud flushing elution, and target protein is dialysed in the Tris-HCl of 20mM, pH 8.5, and it is pure enzyme to take trapped fluid; The loading equilibration buffer is the 20mM Tris-HCl, pH 8.5 of NaCl containing 500mM and 20mM imidazoles;The elution buffer Liquid is the 20mM Tris-HCl, pH 8.5 of NaCl containing 500mM and 500mM imidazoles.

6. the method for chemo-enzymatic process production glufosinate-ammonium as described in claim 1, it is characterised in that the reaction solution isolated and purified Method is:After the reaction was complete, reaction solution is centrifuged, takes supernatant to adjust pH value to 2, filters, take filtrate loading to Hydrogen 001x7 Resin cation, ion exchange column pillar height is than 15:1, loading flow velocity is 1.0BV/h, uses ultrapure water 4BV after loading, collects Containing unreacted substrate D-N- acetyl glufosinate-ammonium effluxes, will be split after D-N- acetyl glufosinate-ammonium racemizations for recycling;It uses again 2mol/L ammonium hydroxide is eluted with 0.5BV/h flow velocitys, collects the eluent containing L-glufosinate-ammonium, by eluent at 60 DEG C, vacuum degree It under 0.075~0.085MPa, is concentrated under reduced pressure, crystallization obtains L-glufosinate-ammonium.

7. the method for chemo-enzymatic process production glufosinate-ammonium as claimed in claim 6, it is characterised in that the D-N- acetyl glufosinate-ammonium disappears Rotationization method is:D-N- acetyl glufosinate-ammonium efflux evaporated under reduced pressure will be contained, acetic acid mixing is added, acetic anhydride is added, in 100- 125 DEG C of stirring racemizations obtain racemic N- acetyl glufosinate-ammoniums, for recycling after reaction by reaction solution evaporated under reduced pressure It splits;The acetic anhydride is 0.1~3 with D-N- acetyl glufosinate-ammonium mass ratioes:1, acetic acid is with D-N- acetyl glufosinate-ammonium mass ratioes 10~20:1.