CN106754560B - Bacillus albus zjut528 and the application in fractionation metalaxyl - Google Patents

Bacillus albus zjut528 and the application in fractionation metalaxyl Download PDF

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CN106754560B
CN106754560B CN201710069366.8A CN201710069366A CN106754560B CN 106754560 B CN106754560 B CN 106754560B CN 201710069366 A CN201710069366 A CN 201710069366A CN 106754560 B CN106754560 B CN 106754560B
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metalaxyl
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张朝晖
张利坤
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Zhejiang University of Technology ZJUT
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Abstract

The invention discloses one plant of bacillus albus Albibacter sp.zjut528 and its splitting the application in metalaxyl；The asymmetric hydrolysis of bacterial strain catalysis racemic metalaxyl of the present invention, reacts 18h, the substrate transformation rate reaches 47.5%, eepReach 99.9%, primary product is (R)-Metalaxyl acid, then with (R)-Metalaxyl acid and methanol reaction synthesis (R)-metalaxyl.The technique splits (production racemic metalaxyl) intermediate production (R)-metalaxyl (Oh-Jin Park with commercial enzyme Lipase PS, 2006) technique is compared, and catalyst cost much lower (catalyst is somatic cells), the ee value of product are higher (the ee value of product reaches 99.3%).

Description

Bacillus albus zjut528 and the application in fractionation metalaxyl
(1) technical field
It, should the present invention relates to one plant of bacillus albus Albibacter sp.zjut528 and in the application of biocatalysis field The ability that there is bacterial strain good, chiral selectivity to split metalaxyl.Using the fractionation ability, a bioanalysis production is established The new process of R- metalaxyl.
(2) background technique
With the reinforcement of environmental protection ideas and the implementation of the strategy of sustainable development, efficient, low toxicity, high activity, low-residual are had become For the inexorable trend of Agrochemicals.In the interaction of some chipal compounds, different enantiomers often show different activities. A kind of enantiomer activity of some compounds is that efficiently, another enantiomer is inefficient or even opposite effect.Metalaxyl is A kind of white powder absorbability acid amide fungicides and a kind of Chiral pesticide with protection, therapeutic effect.Study table Bright, the activity of the R- isomers of metalaxyl is 20~30 times higher than S- isomers, and duration of efficacy is longer, therefore subtracts significantly Lack spraying times, extends spraying cycle.Compared with raceme, degradation speed faster, subtracts single metalaxyl R- isomers in the environment Effect on environment is lacked.Presently commercially available typical Metalaxyl-M is made of 97.5% R- body and 2.5% S- body.
Single enantiomter can be synthesized by chemistry, enzyme process or chemo-enzymatic process etc..Currently, a large amount of chiral compound The preparation of object is completed by chemical resolution method.Biocatalysis is better than the advantages of chemical synthesis: the usual table of enzymic catalytic reaction Existing high stereoselectivity and regioselectivity, and they can be carried out under mild conditions, therefore can be to avoid making With harsher condition, to avoid causing isomerization, racemization, epimerization and the generation for resetting problem.In addition, raw Object catalysis method can reduce environmental pollution, save cost, meet current green chemistry trend.
(3) current R- metalaxyl (product Metalaxyl-M) is mainly synthesized using chemical method.There is document report to be urged with biology Agent synthesizes R- metalaxyl (Oh-Jin Park, 2005,2006), and method is to be split to produce common metalaxyl and (disappear outside with bioanalysis Rotation) intermediate 2,6- dimethylphenylamino methyl propionate, then utilize gained R- intermediate further synthesize R- metalaxyl. Have no the report that R- metalaxyl is directly produced by resolution of racemic metalaxyl.Strain cell of the present invention can be split with direct hydrolysis Racemic metalaxyl has very high stereoselectivity and higher catalytic rate.The R- Metalaxyl acid that hydrolysis obtains can be used for giving birth to R- metalaxyl is produced, production technology is as shown in Figure 7.
(4) summary of the invention
It is an object of the present invention to provide a kind of bacterial strains with fine stereoselectivity --- bacillus albus Albibacter Sp.zjut528 and its split pesticide metalaxyl application.R- Metalaxyl acid is obtained with the bacterium hydrolysis of racemic metalaxyl, is used R- Metalaxyl acid and methanol reaction synthesis R- metalaxyl.This is the new process of a bioanalysis production R- metalaxyl, both at home and abroad still Without report.
The technical solution adopted by the present invention is that:
One plant of new strains --- bacillus albus (Albibacter sp.) zjut528 is preserved in Chinese allusion quotation for present invention offer Type culture collection, the deposit date is on October 29th, 2015, deposit number CCTCC NO:M2015650, preservation address: China, Wuhan, Wuhan University, postcode 430072.
The present invention also provides a kind of bacillus albus zjut528 to split the application in metalaxyl, and specific described answers The wet thallus obtained to the fermented culture of bacillus albus zjut528 is catalyst, using racemic modification metalaxyl as substrate, with different Propyl alcohol is cosolvent, is constituted reaction system as reaction medium using the phosphate buffer of pH7.5~8.5,30~35 DEG C, 150~ The resolution reaction that metalaxyl is carried out under the conditions of 200r/min (preferably 30 DEG C, 180rpm) after fully reacting, is contained (S)-first frost The reaction solution of spirit and (R)-Metalaxyl acid, reaction solution is isolated and purified, and obtains (R)-Metalaxyl acid.
Further, the catalyst amount is calculated as 25~40g/L reaction system, preferably 40g/L reaction with wet thallus weight System, the substrate metalaxyl additional amount are 30~50mmol/L reaction system, preferably 36mmol/L reaction system, the hydrotropy Final concentration of 10~20mL/L the reaction system of the addition of agent, preferably 20mL/L reaction system.
Further, wet thallus of the present invention the preparation method comprises the following steps:
(1) bacillus albus zjut528 is seeded to slant medium, is cultivated 2~3 days at 30 DEG C, obtain inclined-plane thalline；Institute State slant medium final concentration composition are as follows: NaCl 0.5g/L, MgSO4·7H2O 1.0g/L, K2HPO41.0g/L, NH4NO3 1.0g/L, yeast extract powder 5.0g/L, glucose 5.0g/L, solvent are water, and pH value is naturally, 115 DEG C of sterilizing 20min；
(2) inclined-plane thalline is seeded to seed culture medium, in 30 DEG C of culture 48h, obtains seed liquor；The seed culture medium Concentration composition are as follows: NaCl 0.5g/L, MgSO4·7H2O 1.0g/L, K2HPO41.0g/L, NH4NO31.0g/L, yeast leach Powder 5.0g/L, glucose 5.0g/L, solvent are water, and pH value is naturally, 115 DEG C of sterilizing 20min；
(3) seed liquor is seeded to fermentation medium with the inoculum concentration of volumetric concentration 1%~10%, in 30 DEG C, 180r/ 48h is cultivated under conditions of min, fermentation liquid is centrifuged, abandons supernatant, obtains wet thallus.The fermentation medium final concentration composition: NaCl 0.5g/L, MgSO4·7H2O1.0g/L, K2HPO41.0g/L, NH4NO31.0g/L, yeast extract powder 5.0g/L, grape Sugared 5.0g/L, metalaxyl 1g/L, methanol 5g/L (methanol is added before being inoculated with after medium sterilization), solvent is water, and pH value is certainly So, 115 DEG C of sterilizing 20min.
Further, the method that the reaction solution isolates and purifies are as follows: after reaction, with ammonium hydroxide adjust reaction solution pH value to 8-9, is added isometric ethyl acetate extracting and demixing, and removal organic phase a obtains water phase a, is with salt acid for adjusting pH value by water phase a 3~5, isometric ethyl acetate extracting and demixing is added, removal water phase b obtains organic phase b, then by organic phase b 35 DEG C~60 DEG C rotary evaporation removes ethyl acetate, obtains R- Metalaxyl acid；R- Metalaxyl acid is taken to be dissolved in anhydrous methanol (preferably anhydrous methanol Volumetric usage is calculated as 8ml/g with R- Metalaxyl acid quality), thionyl chloride is added dropwise in ice bath, rear ice bath reaction is added dropwise 10min is then refluxed for reaction 4h, and concentrated by rotary evaporation to no liquid flows out, and obtains concentrate；Concentrate is dissolved in ethyl acetate, is used Mass concentration 5%Na2CO3Aqueous solution washs 2 times, and deionized water is washed 1 time, and anhydrous magnesium sulfate water removal is added after removing cleaning solution, Revolving obtains R- metalaxyl to doing after filtering；The ratio between amount of the thionyl chloride and R- Metalaxyl acid substance is 1.1:1.Gained (R)-metalaxyl product ee value reaches 99.3%.
Organic phase a of the present invention, organic phase b, water phase a, water phase b refer to the isolated organic phase of different step and water Phase, letter itself do not have meaning.
Compared with prior art, the advantages of the present invention are mainly reflected in:
The present invention reports one plant of new strains for the first time -- and bacillus albus Albibacter sp.zjut528, it can be selective Racemization metalaxyl is split, there is very high selectivity and higher fractionation rate.The bacterial strain is catalyzed 36mmol/L racemic metalaxyl Hydrolysis, 30 DEG C of reaction 18h, the substrate transformation rate reaches 47.5%, eepReach 99.9%, primary product is (R)-Metalaxyl acid. It there is no the report with biocatalyst asymmetric hydrolysis metalaxyl both at home and abroad.
In addition, the present invention, which establishes one, makees catalyst fractionation with bacillus albus Albibacter sp.zjut528 cell Racemic metalaxyl produces the new process of (R)-metalaxyl, splits (production racemic metalaxyl with commercial enzyme Lipase PS ) technique of intermediate production (R)-metalaxyl (Oh-JinPark, 2006) compares, catalyst cost is much lower, and (catalyst is Somatic cells), the ee value of product it is higher (the ee value of product reaches 99.3%).
(5) Detailed description of the invention
Fig. 1 is the colonial morphology that bacillus albus zjut528 cultivates 3d on solid medium；
Fig. 2 is strain morphology of the bacillus albus zjut528 after Gram's staining under microscope；
Fig. 3 is the 16S rDNA Phylogenetic Analysis tree of bacillus albus zjut528；
Fig. 4 is not plus the positive HPLC of thallus catalysis resolution of racemic body metalaxyl schemes；
Fig. 5 is that thallus is added to be catalyzed resolution of racemic body metalaxyl 9h positive HPLC figure；
Fig. 6 is that thallus is added to be catalyzed resolution of racemic body metalaxyl 18h positive HPLC figure.
Fig. 7 is to make catalyst with somatic cells, and resolution of racemic metalaxyl produces the production technology figure of R- metalaxyl.
(5) specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in This:
1 bacillus albus Albibacter sp.zjut528 bacterial strain screening of embodiment
1. enrichment culture:
5g soil sample is taken to be placed in 250mL triangular flask, 100mL metalaxyl enriched medium is added, and (metalaxyl initial concentration is 20mg/L) shaken cultivation (30 DEG C, 150rpm) 1 week, takes the upper layer 5mL turbid to continue to train in 100mL fresh enriched medium It supports 1 week, repeats aforesaid operations process 2 times, the culture being inoculated with every time, which is taken from, cultivated resulting culture medium in last time.Take third Week resulting culture solution 5mL, metalaxyl concentration was increased to 50mg/L at this time in new metalaxyl enriched medium, still by above-mentioned The continuous culture of operation 2 weeks, then metalaxyl concentration is increased to 100mg/L by aforesaid operations and is continuously cultivated 2 weeks, fermentation liquid is carried out High pressure liquid chromatography (HPLC) analysis, studies the degradation situation of metalaxyl.Take the resulting culture solution 5mL of last time in new first In white spirit enriched medium, metalaxyl concentration is increased to 1g/L at this time, and continuous culture 2 weeks, HPLC analyzes metalaxyl in culture solution Degradation situation.
Metalaxyl enriched medium: K2HPO4·3H2O 2.1g/L, KH2PO40.4g/L, NaCl0.1g/L, MgSO4· 7H2O 0.2g/L, CaCl20.025g/L, NH4NO30.5g/L, liquid microelement 10mL/L, metalaxyl are (such as above-mentioned different rich 20mg/L, 50mg/L, 100mg/L or 1g/L metalaxyl is added in the collection stage), solvent is water, and adjusting pH value is 7.0,121 DEG C of sterilizings 20min。
Liquid microelement final concentration composition: CoCl20.1g/L, MnSO40.5g/L, FeSO4·7H2O 0.1g/L, CuSO4 0.1g/L, ZnSO4·7H2O 0.1g/L, H3BO30.01g/L, Al (SO4)2·12H2O 0.01g/L, Na2MoO4·2H2O 0.01g/L, EDTA2Na:1g/L, preparation method: taking 1gEDTA2Na to be dissolved in 800mL deionized water, then by other components It sequentially adds, finally adds water to 1L.
2. primary dcreening operation:
It is carried out again from taking the preferable culture solution of 1mL metalaxyl degradation effect to be added in 9ml sterile water in enriched medium Than dilution, gradient is respectively 10-4、10-5、10-6、10-7It is applied to metalaxyl primary dcreening operation solid medium, after 30 DEG C are cultivated 3~5 days, Picking single colonie carries out secondary screening.
Metalaxyl primary dcreening operation solid medium: K2HPO4·3H2O 2.1g/L, KH2PO40.4g/L, NaCl 0.1g/L, MgSO4·7H2O 0.2g/L, CaCl20.025g/L, NH4NO30.5g/L, metalaxyl 1g/L, agar 20g/L, solvent are water, Adjust pH=7.0,121 DEG C of sterilizing 20min.
3. secondary screening:
The single strain on primary dcreening operation solid plate is chosen, is inoculated into secondary screening fluid nutrient medium, 30 DEG C, revolving speed 180r/min is trained It supports, periodically sampling carries out HPLC analysis, has obtained one plant of conversion ratio 49.2% and eepReach 98.8% bacterial strain, it is preliminary to remember For bacterial strain zjut528.
Secondary screening fluid nutrient medium: NaCl 0.5g/L, MgSO4·7H2O 1.0g/L, K2HPO41.0g/L, NH4NO3 1.0g/ L, yeast extract powder 5.0g/L, glucose 5.0g/L, metalaxyl 1g/L, solvent are water, and pH value is naturally, 115 DEG C of sterilizing 20min.
2 bacterial strain zjut528 morphology of embodiment and Physiology and biochemistry are identified
Take the bacterial strain zjut528 obtained with embodiment 1 30 DEG C of constant temperature incubations in metalaxyl primary dcreening operation solid medium tablets Case culture 3 days, colony morphology characteristic is observed, and Gram's staining, microscopically observation morphological feature are carried out to it.
Colony morphology characteristic in solid medium tablets: bacterial strain zjut528 is in metalaxyl primary dcreening operation solid medium tablets Upper bacterium colony is rounded, surface elevation, neat in edge, moistens, smooth, and white, opaque, over time, bacterium colony is not Seeing has obvious color change, as shown in Figure 1.
Strain morphology feature after Gram's staining: to bacterial strain zjut528 carry out Gram's staining, microscopically observation, It is in club-shaped for Gram-negative bacteria.As shown in Figure 2.
As shown in table 1, which completes in this laboratory for the Physiology and biochemistry identification of bacterial strain.Once it sent outside complete with VITEK2 Automatic bacteria assessing instrument identifies the bacterial strain, but can not obtain qualification result, and main cause is that it can only be extremely limited several It is grown in culture medium.
1 bacterial strain zjut528 physiological and biochemical property of table
Note :+indicate positive or utilize ,-indicate negative or do not utilize
The 16S rDNA molecular biology identification of 3 bacterial strain zjut528 of embodiment
Bacterial strain zjut528, which is obtained, with embodiment 1 is sent to the identification of Shanghai biotech company, the sequence length of 16SrDNA For 1236bp (nucleotides sequence is classified as shown in SEQ ID NO.1), sequencing gained sequence is retrieved on the website NCBI with BLAST The 16SrDNA sequence of related strain in Genbank, and nucleotide homology comparison and Phylogenetic Analysis are carried out, find one plant Albibacter methylovorans strain DSM22840T similitude has reached 99%, the system hair of bacterial strain zjut528 Educate tree as shown in Figure 4.In conjunction with 16S rDNA Molecular Identification and the physiological and biochemical property of bacterial strain zjut528, by bacterial strain zjut528 It is named as bacillus albus (Albibacter sp.) zjut528.
The preparation of embodiment 4, bacillus albus zjut528 wet thallus
Inclined-plane culture: being seeded to slant medium for bacillus albus zjut528, cultivates 2-3 days at 30 DEG C, obtains inclined-plane bacterium Body.The slant medium final concentration composition are as follows: NaCl 0.5g/L, MgSO4·7H2O 1.0g/L, K2HPO41.0g/L NH4NO31.0g/L, yeast extract powder 5.0g/L, glucose 5.0g/L, solvent are water, and pH value is naturally, 115 DEG C of sterilizing 20min；
Seed culture: the thallus that inclined-plane has been grown is seeded to seed culture medium, in 30 DEG C of culture 48h, obtains seed liquor.Institute State seed culture medium concentration composition are as follows: NaCl 0.5g/L, MgSO4·7H2O 1.0g/L, K2HPO41.0g/L, NH4NO3 1.0g/L, yeast extract powder 5.0g/L, glucose 5.0g/L, solvent are water, and pH value is naturally, 115 DEG C of sterilizing 20min；
Liquid fermentation and culture: being seeded to fermentation medium for seed liquor with the inoculum concentration of volumetric concentration 5%, 30 DEG C, 48h is cultivated under conditions of 180r/min, fermentation liquid is centrifuged, abandons supernatant, obtains wet thallus.The fermentation medium final concentration Composition are as follows: NaCl 0.5g/L, MgSO4·7H2O 1.0g/L, K2HPO41.0g/L, NH4NO31.0g/L, yeast extract powder 5.0g/L, glucose 5.0g/L, metalaxyl 1g/L, methanol 5g/L (methanol is added before being inoculated with after medium sterilization), solvent is Water, pH value is naturally, 115 DEG C of sterilizing 20min.
Embodiment 5 utilizes the fractionation of Albibacter sp.zjut528 wet thallus catalysis racemic modification metalaxyl
Catalystic converter system total volume 4mL, racemic metalaxyl final concentration 100g/L, isopropanol final concentration 20mL/L, with Phosphate buffer (pH=8.0) is reaction medium, and wet thallus 0.16g prepared by embodiment 4 is added.
Catalytic reaction condition: 180rpm is reacted in shaking table under conditions of 30 DEG C, carries out substrate and product point with HPLC Analysis, reacts 9h, 18h respectively, and reaction result is shown in Table shown in 2 and Fig. 5 and Fig. 6.
2 zjut528 of table is catalyzed racemic metalaxyl asymmetric hydrolysis result
Product enantiomeric excess value (ee) and the substrate transformation rate C are calculated as follows:
Formula 1:
Formula 2:
In formula, [P]S[P]RRespectively HPLC measures the content of S and R type product in sample, eepFor the enantiomer of product Excessive value, CPFor the molar concentration of product, CSFor substrate molar concentration, C is conversion ratio.
Normal-phase HPLC analysis condition: mobile phase: n-hexane: isopropanol: trifluoroacetic acid=80:20:0.1, flow velocity: 0.8mL/ Min detects ultraviolet wavelength: 230nm, column temperature: 30 DEG C, sample volume: 10 μ l, chromatographic column: 250mm × 4mm, Daicel chirality OD Column.For analyzing the different enantiomers of substrate and product.
Embodiment 6 separates (R)-Metalaxyl acid from the reaction solution after reaction of embodiment 5
After embodiment 5 reacts 18h, isometric ethyl acetate extraction is added to 8-9 in the pH value for adjusting reaction solution with ammonium hydroxide Layering, removal organic phase a obtain water phase a, are 3~5 by water phase a salt acid for adjusting pH value, and isometric ethyl acetate extraction is added Layering is taken, removal water phase b obtains organic phase b, then organic phase b is removed ethyl acetate in 35 DEG C~60 DEG C rotary evaporations, obtains R- Metalaxyl acid 0.176g.
Embodiment 7 synthesizes (R)-metalaxyl with (R)-Metalaxyl acid separating obtained in embodiment 6 and methanol reaction
The separating obtained R- Metalaxyl acid of 6 method of 5g embodiment is taken to be dissolved in 40ml anhydrous methanol, 1.5ml dichloro is added dropwise in ice bath Sulfoxide (1.1 times of (R)-Metalaxyl acid moles) is added dropwise rear ice bath reaction 10min, is then refluxed for reaction 4h, rotates dense It is reduced to no liquid outflow, obtains concentrate.Concentrate is dissolved in ethyl acetate, with mass concentration 5%Na2CO3Aqueous solution washing 2 Secondary, deionized water is washed 1 time.Anhydrous magnesium sulfate water removal is added after removing cleaning solution, revolving obtains R- metalaxyl to doing after filtering 4.24g, gross production rate 84.6%, ee value are 99.3%.
SEQUENCE LISTING
<110>Zhejiang Polytechnical University
<120>bacillus albus zjut528 and the application in fractionation metalaxyl
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1236
<212> DNA
<213> Albibacter sp.
<400> 1
gcctggtggt acggaacaac tcagggaaac ttgagctaat accgtataag cccttttggg 60
gaaagattta tcgccaccag atcaacccgc gttggattag ctagttggtg aggtaacggc 120
tcaccaaggc gacgatccat agctggtctg agaggatgat cagccacact gggactgaga 180
cacggcccag actcctacgg gaggcagcag tggggaatat tggacaatgg gcgcaagcct 240
gatccagcca tgccgcgtga gtgatgaagg ccttagggtt gtaaagctct ttcactgggg 300
aagataatga cggtacccag agaagaagcc ccggctaact tcgtgccagc agccgcggta 360
atacgaaggg ggctagcgtt gttcggaatc actgggcgta aagcgcacgt aggcggactt 420
ttaagtcagg ggtgaaatcc caaggctcaa ccttggaact gcccttgata ctggaagtct 480
tgagttcggg agaggtgagt ggaactgcga gtgtagaggt gaaattcgta gatattcgca 540
agaacaccag tggcgaaggc ggctcactgg cccgatactg acgctgaggt gcgaaagcgt 600
ggggagcaaa caggattaga taccctggta gtccacgccg taaacgatgg acgctagccg 660
ttggccagca tgctggtcag tggcgcagct aacgctttaa gcgtcccgcc tggggagtac 720
ggtcgcaaga ttaaaactca aaggaattga cgggggcccg cacaagcggt ggagcatgtg 780
gtttaattcg aagcaacgcg cagaacctta ccagcctttg acatcctgtg acatccagag 840
agatttgggg ttcccttcgg ggacacagag acaggtgctg catggctgtc gtcagctcgt 900
gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc ctcgccccta gttgccagca 960
tttggttggg cactctaggg ggactgccgg tgataagccg cgaggaaggt ggggatgacg 1020
tcaagtcctc atggccctta cgggctgggc tacacacgtg ctacaatggc ggtgacagtg 1080
ggcagcgaag gggtgacccg gagctaatct ccaaaagccg tctcagttcg gattgcactc 1140
tgcaactcga gtgcatgaag ttggaatcgc tagtaatcgc ggatcagcat gccgcggtga 1200
atacgttccc gggccttgta cacaccgccc gtcaca 1236

Claims (7)

1. bacillus albus Albibacter sp.zjut528, is preserved in China typical culture collection center, the deposit date is On October 29th, 2015, deposit number CCTCC NO:M2015650, preservation address: China, Wuhan, Wuhan University, postcode 430072。
2. bacillus albus zjut528 described in a kind of claim 1 is splitting the application in metalaxyl.
3. application as claimed in claim 2, it is characterised in that the application is obtained with the fermented culture of bacillus albus zjut528 The wet thallus obtained is catalyst, slow with the phosphoric acid of pH7.5~8.5 using isopropanol as cosolvent using racemic metalaxyl as substrate Fliud flushing is that reaction medium constitutes reaction system, carries out catalysis reaction under the conditions of 30~35 DEG C, 150~200r/min, has reacted Quan Hou isolates and purifies reaction solution, obtains (R)-Metalaxyl acid.
4. application as claimed in claim 3, it is characterised in that the catalyst amount is calculated as 25~40g/L with wet thallus weight Reaction system, the substrate racemic metalaxyl additional amount are 30~50mmol/L reaction system, and the cosolvent is added dense eventually Degree is 10~20ml/L reaction system.
5. application as claimed in claim 3, it is characterised in that the wet thallus the preparation method comprises the following steps:
(1) bacillus albus zjut528 is seeded to slant medium, is cultivated 2~3 days at 30 DEG C, obtain inclined-plane thalline；It is described oblique Face culture medium final concentration composition: NaCl0.5g/L, MgSO4·7H2O1.0g/L, K2HPO41.0g/L, NH4NO31.0g/L, ferment Mother leaches powder 5.0g/L, glucose 5.0g/L, agar 20g/L, and solvent is water, and pH value is natural；
(2) inclined-plane thalline is seeded to seed culture medium, in 30 DEG C of culture 48h, obtains seed liquor；The seed culture medium is dense eventually Degree composition are as follows: NaCl0.5g/L, MgSO4·7H2O1.0g/L, K2HPO41.0g/L, NH4NO31.0g/L, yeast extract powder 5.0g/L, glucose 5.0g/L, solvent are water, and pH value is natural；
(3) seed liquor is seeded to fermentation medium with 1%~10% inoculum concentration of volumetric concentration, in 30 DEG C, the item of 180r/min 48h is cultivated under part, fermentation liquid is centrifuged, abandons supernatant, obtains wet thallus；The fermentation medium final concentration composition: NaCl0.5g/L, MgSO4·7H2O1.0g/L, K2HPO41.0g/L, NH4NO31.0g/L, yeast extract powder 5.0g/L, grape Sugared 5.0g/L, racemic metalaxyl 1.0g/L, methanol 5.0g/L, methanol is after medium sterilization, addition before being inoculated with, and solvent is Water, pH value are natural.
6. application as claimed in claim 3, it is characterised in that the method that the reaction solution isolates and purifies are as follows: after reaction, To 8-9 isometric ethyl acetate extracting and demixing is added, removal organic phase a obtains water phase in the pH value for adjusting reaction solution with ammonium hydroxide Water phase a salt acid for adjusting pH value is 3~5 by a, and isometric ethyl acetate extracting and demixing is added, and removal water phase b obtains organic Phase b, then organic phase b is removed into ethyl acetate in 35 DEG C~60 DEG C rotary evaporations, obtain (R)-Metalaxyl acid.
7. application as claimed in claim 6, it is characterised in that the method that (the R)-Metalaxyl acid prepares (R)-metalaxyl Are as follows: take (R)-Metalaxyl acid to be dissolved in anhydrous methanol, thionyl chloride is added dropwise in ice bath, rear ice bath reaction 10min is added dropwise, then Back flow reaction 4h, concentrated by rotary evaporation to no liquid flow out, and obtain concentrate；Concentrate is dissolved in ethyl acetate, with mass concentration 5% Na2CO3Aqueous solution washs 2 times, and deionized water is washed 1 time, and anhydrous magnesium sulfate water removal is added after removing cleaning solution, rotates after filtering To doing, (R)-metalaxyl is obtained；The ratio between amount of the thionyl chloride and (R)-Metalaxyl acid substance is 1.1:1.
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