CN107118977B

CN107118977B - Serratia marcescens and application thereof

Info

Publication number: CN107118977B
Application number: CN201710207986.3A
Authority: CN
Inventors: 薛亚平; 郑裕国; 吕胜芝; 徐建妙; 柳志强
Original assignee: Zhejiang University of Technology ZJUT
Current assignee: Zhejiang University of Technology ZJUT
Priority date: 2017-03-31
Filing date: 2017-03-31
Publication date: 2020-02-21
Anticipated expiration: 2037-03-31
Also published as: CN107118977A

Abstract

The invention discloses Serratia marcescens and application thereof in catalyzing α -aminonitrile substrate (2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile) to prepare L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid serving as an important chiral precursor of L-glufosinate-ammonium, provides a strain capable of producing nitrilase, Serratia marcescens (Serratia marcocens) ZJB-16006, and also provides a method for preparing L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid serving as an important chiral precursor of L-glufosinate-ammonium by catalyzing 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile by using wet bacteria obtained by fermenting and culturing Serratia marcescens ZJB-16006 as a biocatalyst, and has important application prospect.

Description

Serratia marcescens and application thereof

(I) technical field

The invention relates to a bacterial strain for producing nitrilase and application of the bacterial strain in preparing L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid serving as an important chiral precursor of L-glufosinate-ammonium by biocatalysis of α -aminonitrile substrate (2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile).

(II) background of the invention

Nitrilase (Nitrilase, EC 3.5.5.1) can directly convert cyano in nitrile compounds into carboxyl to prepare carboxylic acid, has unique chemoselectivity, stereoselectivity and regioselectivity for nitrile compounds, and can be used for synthesizing carboxylic acid with low synthesis efficiency or difficult synthesis by various chemical methods. The method has the advantages of good selectivity and high conversion rate, meanwhile, compared with the traditional process, the method has the advantages of mild reaction conditions, no need of high temperature and high pressure, strong acid and strong base, less pollution, incomparable superiority of a chemical method, accordance with the development direction of atom economy and green chemistry, great application potential in chemical synthesis, great development in application quantity and variety, and wide application in the fields of medicines and intermediates thereof, pesticides and intermediates thereof, foods and feed additives and the like: preparing key intermediate (R) -3-hydroxy glutaric acid ethyl ester of Rosuvastatin (Rosuvastatin) by nitrilase catalysis; preparing a key intermediate R-type o-chloromandelic acid of an anti-platelet aggregation medicament Clopidogrel (Clopidogrel); the key intermediate iminodiacetic acid for preparing herbicide glyphosate in the IDA route, and the like.

DL-glufosinate-ammonium, the active ingredient is phosphinothricin (PPT for short), the chemical name is 4- [ hydroxymethyl phosphonyl ] -DL-homoalanine (alias: D, L-glufosinate amine salt), the herbicide is a broad-spectrum, contact type, sterilant and non-residue herbicide, which is developed in the 20 th century 80 th generation by German Herster company (currently, Bayer company), besides the herbicidal activity, the herbicide has the characteristics of high efficiency, low toxicity, easy degradation and the like, the glufosinate-ammonium is a racemic mixture, only the L-type has phytotoxicity, the herbicidal activity is 2 times of that of the racemic mixture, the commercially available glufosinate-ammonium is a racemic mixture, and if the L-type glufosinate-ammonium can replace the L-type glufosinate-ammonium, the L-amino-4 (hydroxyethyl methylphosphonyl) -butyric acid can be used for preparing L-glufosinate-ammonium, the synthesis method can reduce the cost by using a chiral amino acid-phospho-ethyl-transferase, the L-glufosinate-amino-4 (L-glufosinate-ethyl-amino-4) is an important chiral precursor for preparing L-glufosinate, the L-glufosinate-L-homoamino acid, the L-homoamino acid is a novel sterilant herbicide developed by a broad-ketolase, the novel sterilant herbicide developed in the 20 th 80 th ages of 20 th ages of the German-Herschel, the German-Herber, the German-one, the German-Hertz, the German, the modern Hertz, the L-Carpine, the same as the same.

The method for industrially producing glufosinate-ammonium can be summarized as the following three routes, namely, Bayer company prepares D, L-glufosinate-ammonium by using a Michael free radical addition method, the D, L-glufosinate-ammonium is synthesized by using a Srecker method, and L-glufosinate-ammonium is synthesized by using an asymmetric catalytic hydrogenation method in a bright control fruit preparation process, wherein the D synthesized by the Srecker method and the L-glufosinate-ammonium route have mild reaction conditions and are a route adopted by the current industrial large production.

Disclosure of the invention

The invention aims to provide a nitrilase-producing strain-Serratia marcescens (Serratiamarecens) ZJB-16006 and application thereof in preparing L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid by biocatalysis of α -aminonitrile substrate (2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile).

The technical scheme adopted by the invention is as follows:

the invention provides a new bacterial strain with nitrilase production performance, namely Serratia marcescens (Serratiamarcescens) ZJB-16006, which is preserved in China center for type culture Collection with the preservation number of CCTCC No: m2017033, date of deposit 2017, month 01, day 13, address: wuhan, Wuhan university, zip code 430072.

The invention also relates to an application of the Serratia marcescens (Serratia marcocens) ZJB-16006 to catalyze α -aminonitrile (2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile) to prepare L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid which is an important chiral precursor of L-glufosinate-ammonium, wherein the application is that wet thalli obtained by fermentation culture of the Serratia marcescens ZJB-16006 is used as a catalyst, 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile is used as a substrate, distilled water or 100mM phosphate buffer solution with the pH value of 7.0 is used as a reaction medium, conversion reaction is carried out at the temperature of 30-50 ℃ and the speed of 100-200rpm, and after the reaction is finished, the reaction liquid is separated and purified to obtain the L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid.

The nitrilase biocatalysis of α -aminonitrile (2-amino-4 (hydroxyethylmethylphosphoryl) -butyronitrile) to L-2-amino-4 (hydroxyethylmethylphosphoryl) -butyric acid was as follows:

furthermore, the dosage of the catalyst is 10-100g/L of reaction medium, preferably 35g/L, calculated by the weight of wet bacteria, and the final concentration of the substrate initially added is 2-100g/L of reaction medium, preferably 5 g/L.

Further, the catalyst is prepared by the following method: (1) slant culture: inoculating serratia marcescens ZJB-16006 to a slant culture medium, and culturing at 30 deg.C for 48 hr to obtain slant thallus; the final concentration of the slant culture medium is as follows: 1-20.0 g/L of mannitol, 1-10.0 g/L of sodium glutamate, 1-5.0 g/L of yeast extract and K₂HPO₄0.1～1.0g/L，KH₂PO₄0.1～1.0g/L，MgSO₄0.1-1.0 g/L, 0.1-2.0 g/L caprolactam, 20.0g/L agar, deionized water as solvent and pH 7.0-7.5; preferably, the final concentration composition of the slant culture medium is as follows: 10.0g/L of mannitol, 7.0g/L of sodium glutamate, 3.0g/L of yeast extract and K₂HPO₄0.75g/L，KH₂PO₄0.75g/L，MgSO₄0.5g/L, caprolactam 1.0g/L, agar 20.0g/L, deionized water as solvent, and pH 7.0-7.5;

(2) seed culture: selecting slant thallus, inoculating to seed culture medium, and culturing at 30 deg.C for 24 hr to obtain seed solution; the final concentration of the seed culture medium is as follows: 1-20.0 g/L of mannitol, 1-10.0 g/L of sodium glutamate, 1-5.0 g/L of yeast extract and K₂HPO₄0.1～1.0g/L，KH₂PO₄0.1～1.0g/L，MgSO₄0.1-1.0 g/L, 0.1-2.0 g/L caprolactam, deionized water as solvent and pH 7.0-7.5; preferably, the final concentration composition of the seed culture medium is as follows: 10.0g/L of mannitol, 7.0g/L of sodium glutamate, 3.0g/L of yeast extract and K₂HPO₄0.75g/L，KH₂PO₄0.75g/L，MgSO₄0.5g/L of caprolactam, 1.0g/L of caprolactam and deionized water as a solvent, wherein the pH value is 7.0-7.5;

(3) fermentation culture: inoculating the seed solution into a fermentation culture medium in an inoculation amount with the volume concentration of 1-10%, carrying out shaking culture at 30 ℃ and 150rpm for 48h, centrifuging at 12000g for 10min, and collecting wet thalli; the final concentration of the fermentation medium is as follows: 1-20.0 g/L of mannitol, 1-10.0 g/L of sodium glutamate, 1-5.0 g/L of yeast extract and K₂HPO₄0.1～1.0g/L，KH₂PO₄0.1～1.0g/L，MgSO₄0.1-1.0 g/L, 0.1-2.0 g/L caprolactam, deionized water as solvent, and pH 7.0-7.5. Preferably, the final concentration of the fermentation medium consists of: 10.0g/L of mannitol, 7.0g/L of sodium glutamate, 3.0g/L of yeast extract and K₂HPO₄0.75g/L，KH₂PO₄0.75g/L，MgSO₄0.5g/L, caprolactam 1.0g/L and deionized water as solvent, and the pH value is 7.0-7.5.

The method for separating and purifying the reaction liquid comprises the following steps: adjusting the pH value of the reaction liquid to 2.5, performing column chromatography by adopting anion exchange resin 201 multiplied by 7, loading the sample at the flow rate of the column of 1-6.0 BV/h (preferably 4BV/h), washing with deionized water, eluting with 0.5-2.0M (preferably 1M) of ammonia water at the elution speed of 1.0-4.0 BV/h (preferably 2BV/h), and collecting the eluent containing the target substance; distilling the eluent under reduced pressure until no solvent flows out, adding methanol for dissolving, stirring in ice bath, recrystallizing, filtering, and drying the filter cake to obtain the product L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid.

In the invention, 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile is selectively catalyzed and hydrolyzed by nitrilase to prepare L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid serving as an important chiral precursor of L-glufosinate-ammonium, and a basis is provided for synthesizing L-glufosinate-ammonium by a Srecker method with a mature industrial route. Meanwhile, the acidolysis reaction in the domestic industrial Strecker route is replaced, and the acidic wastewater pollution generated by the acidolysis reaction and the corrosion damage of the acidolysis reaction to equipment are reduced.

The invention has the following beneficial effects: the invention provides a bacterial strain capable of producing nitrilase, namely Serratia marcescens (Serratia marcescens) ZJB-16006, and also provides a method for preparing L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid serving as an important chiral precursor of L-glufosinate-ammonium by catalyzing 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile with a biocatalyst by using wet thallus obtained by fermentation culture of the Serratia marcescens ZJB-16006, and the method has important application prospect.

(IV) description of the drawings

FIG. 1 is a liquid phase result spectrum of D-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid and L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid under the condition of pre-column derivatization reversed phase high performance liquid chromatography.

FIG. 2 is a standard curve of ammonium ion concentration.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1: screening and identification of nitrilase-producing strains

1. Screening of nitrilase-producing strains

(1) Taking soil samples from all over the country, weighing 1g of the soil samples, suspending the soil samples by using 0.85% physiological saline, standing the soil samples, adding 1mL of supernatant into 50mL of enrichment medium added with 1g/L α -aminonitrile substrate (2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile) as a unique carbon source, culturing the mixture for 3 days on a shaking table at the temperature of 30 ℃ and the speed of 150rpm, taking 1mL of culture solution after the culture medium is turbid, transferring the culture solution into a fresh enrichment medium added with 1 g/L2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile, and culturing the mixture for 3 days again, so circularly enriching the mixture for 3-4 cycles;

(2) diluting the final enrichment culture solution with sterile water for 10 gradients step by step, and selecting 10^-4、10^-5、10^-6、10^-7、10^-80.1mL of each of the five dilutions was obtainedUniformly coating on a plate culture medium added with 1 g/L2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile, culturing for 2-3 days in a 30 ℃ constant temperature incubator, picking up a single colony on the plate, inoculating on a slant culture medium, culturing for 2-3 days in a 30 ℃ constant temperature incubator, and preserving at 4 ℃;

(3) the strain preserved on the slant was inoculated into a seed medium and cultured at 30 ℃ for 24 hours. The seed solution was inoculated into the fermentation medium at an inoculum size of 1% by volume concentration and cultured at 30 ℃ for 48 hours with shaking at 150 rpm. Centrifuging at 12000g for 10min, collecting wet thallus, suspending certain amount of thallus and substrate (2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile) in distilled water, making into reaction system with bacterium concentration of 0.2g/10mL and substrate concentration of 10g/L for conversion, and placing in a water bath shaker at 30 ℃ for conversion for 24 h. Sampling, carrying out primary screening by a phenol-hypochlorite method, detecting the concentration and optical purity of a product L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid by high performance liquid chromatography, and finally screening to obtain the strain ZJB-16006, wherein the nitrilase activity is relatively good.

The final concentration composition of the enrichment medium is (g/L): glucose 5.0, sodium chloride 1.0, K₂HPO₄·3H₂O 0.8，KH₂PO₄3.3，MgSO₄·7H₂O0.2, deionized water as solvent, pH7.0, and 1 g/L2-amino-4 (hydroxyethyl methylphosphoryl) -butyronitrile as the sole carbon source.

The plate screening culture medium is (g/L): glucose 5.0, sodium chloride 1.0, K₂HPO₄·3H₂O 0.8，KH₂PO₄3.3，MgSO₄·7H₂O0.2, agar 20.0, solvent deionized water, pH 7.0. Sterilizing at 115 deg.C for 30 min. After cooling to room temperature, 1.0g/L of 2-amino-4- (hydroxymethyl-phosphoryl) -butyronitrile was added.

The final concentration composition of the slant culture medium is (g/L): 10.0 percent of mannitol, 7.0 percent of sodium glutamate, 3.0 percent of yeast extract and K₂HPO₄0.75，KH₂PO₄0.75，MgSO₄0.5, caprolactam 1.0, agar 20.0 and deionized water as solvent, and the pH value is 7.0-7.5.

The fermentation medium isThe concentration composition is (g/L): 10.0 percent of mannitol, 7.0 percent of sodium glutamate, 3.0 percent of yeast extract and K₂HPO₄0.75，KH₂PO₄0.75，MgSO₄0.5, caprolactam 1.0 and deionized water as solvent, and the pH value is 7.0-7.5.

(4) Primary screening by phenol-hypochlorite method

2ml of the solution A is taken by a pipette and placed in a 10ml centrifuge tube, 4. mu.L of the solution to be tested is added, a cover is covered, and the mixture is vigorously mixed. Then 2ml of solution B was added by pipette, capped and mixed again. And (3) heating in a water bath at 37 ℃ for 5min until the color in the centrifugal tube does not change any more, placing 200 mu L of the solution in a 96-well plate, measuring the absorbance at 620nm, and comparing the absorbance with a standard curve to obtain the concentration of ammonium in the solution to be measured, wherein the concentration of the ammonium is equal to the concentration of the converted 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid, so that the yield of the 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid in the conversion process can be detected.

The solution A comprises: 5g of phenol and 25mg of sodium nitrosoferricyanide were weighed, placed in a 500mL volumetric flask, added with ultrapure water to a constant volume, and then stored in a brown bottle in a refrigerator at 4 ℃.

And the liquid B: 2.5g of NaOH was weighed into a 500mL volumetric flask, and 4.4mL of an aqueous sodium hypochlorite solution containing 5.2% available chlorine was added by a pipette, and then the volume was fixed with ultrapure water, and the mixture was stored in a brown bottle and stored in a refrigerator at 4 ℃.

Preparation of a standard curve: the phenol-hypochlorite method can measure the concentration of ammonium ions at room temperature by using a cuvette and an ultraviolet-visible spectrophotometer or a 96-well plate and a universal microplate spectrophotometer, and the OD620 value has a good linear relation with the concentration of ammonium ions (shown in figure 2) at the wavelength of 620nm, R is²0.9982, which shows that the method has high feasibility and accuracy.

(5) Pre-column derivatization reversed-phase high performance liquid chromatography rescreening

And (3) taking 500 mu L of reaction liquid with a better result obtained after primary screening, adding an equal volume of derivatization reagent, uniformly mixing, carrying out derivatization reaction for 5min, then injecting 15 mu L of sample, and carrying out HPLC analysis.

The derivatization reagent is as follows: 10mg of o-phthalaldehyde (OPA) and 12mg of N-acetyl-L-cysteine (NAC) are weighed respectively, dissolved in L mL of absolute ethanol, and then diluted to 5mL with 0.2mol/L boric acid buffer solution (pH value of 9.8) and stored in a refrigerator at-4 ℃ for standby (the storage time is not longer than 3 d).

The analysis conditions of the high performance liquid chromatography are as follows: the adopted gas chromatograph is a U3000 liquid chromatograph (provided with a fluorescence detector) of Daian in America; a chromatographic column: daran C18(4.6 mm. times.250 mm, silicon hydroxyl filler); mobile phase: methanol: 0.05mol/L ammonium acetate solution (volume ratio 10:90, pH value 5.7); flow rate: 1 mL/min; column temperature: 35 ℃; sample introduction amount: 15 mu L of the solution; fluorescence detection wavelength: excitation wavelength Ex 350nm, emission wavelength Em 450 nm.

Under the condition, the peak-off time of L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid and D-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid is 9.2min and 11.1min respectively, as shown in figure 1.

2. Molecular identification of Strain ZJB-16006

Extracting the total DNA of the strain ZJB-16006 obtained by screening in the step 1 by using a molecular kit, amplifying the 16S rDNA gene (shown in SEQ ID NO: 1) of the strain by using the total DNA of the strain ZJB-16006 as a template and using primers P1:5'-AGAGTTTGATCCTGGCTCAG-3' and P2:5'-AAGGAGGTGATCCAGCCGCA-3', connecting the gene product with a T vector, entrusting Shanghai worker to amplify and sequence the 16S rDNA of the strain to obtain the 16S rDNA sequence of the strain, searching the 16S rDNA gene sequence of related strains in GenBank by using BLAST on an NCBI website, and carrying out homology comparison. The microorganism has the highest homology (homology, 100 percent, base on16S rDNA) with Serratia marcocens strain FZSF01, and the identified strain basically belongs to a control strain based on the homology of a 16S rDNA sequence higher than 95 percent according to the identification principle of microbial molecular genetics. Therefore, the strain ZJB-16006 identified in this experiment is Serratia marcescens (Serratia marcescens) and is supposed to be named as Serratia marcescens (Serratia marcescens) ZJB-16006.

Any pair of SEQ ID NOs: 1 by substitution, deletion or insertion of one or more nucleotides, provided that it has more than 90% homology with the nucleotide, is within the scope of the present invention.

Example 2: preparation of Wet cells

(1) Slant culture

Serratia marcescens (Serratia marcocens) ZJB-16006 was inoculated to slant culture medium and cultured at 30 deg.C for 48h to obtain slant thallus.

(2) Seed culture

Selecting bacteria from the inclined plane bacteria, inoculating the bacteria to a seed culture medium, and culturing at 30 ℃ for 24h to obtain a seed solution; the final concentration composition of the seed culture medium is (g/L): 10.0 percent of mannitol, 7.0 percent of sodium glutamate, 3.0 percent of yeast extract and K₂HPO₄0.75，KH₂PO₄0.75，MgSO₄0.5, 1.0 caprolactam and deionized water as solvent, wherein the pH value is 7.0-7.5;

(3) fermentation culture

Inoculating the seed solution into a fermentation medium at a volume concentration of 1%, performing shaking culture at 30 deg.C and 150rpm for 48h, centrifuging at 12000g for 10min, and collecting wet thallus.

The final concentration composition of the fermentation medium is (g/L): 10.0 percent of mannitol, 7.0 percent of sodium glutamate, 3.0 percent of yeast extract and K₂HPO₄0.75，KH₂PO₄0.75，MgSO₄0.5, caprolactam 1.0 and deionized water as solvent, and the pH value is 7.0-7.5.

EXAMPLE 3 bioconversion reaction Using α -aminonitrile (2-amino-4 (hydroxyethylmethylphosphoryl) -butyronitrile) as substrate

(1) 0.2g of wet cells prepared in example 2 and a final concentration of 2-amino-4 (hydroxyethylmethylphosphoryl) -butyronitrile of 2g/L as a substrate were added to 10mL of phosphate buffer (100mM, pH 7.0), and the mixture was transferred for 24 hours at 150rpm in a shaker 30 ℃.1ml of the transformation solution is put into an EP tube and centrifuged at 12000r/min for 2min, and the supernatant is taken for chiral liquid phase detection. The result shows that the optical purity of the L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid obtained by the conversion of the strain ZJB-16005 on 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile is 99.9%.

(2) The conversion solution containing the product L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid obtained by the enzyme reaction is separated on an ion exchange column by adopting anion exchange resin 201 x 7.

1) Resin pretreatment

Soaking in warm water at 50 deg.C to fully expand and remove fine particles (inclined or flotation method); soaking in l.0M NaOH aqueous solution for 3 hr, washing with deionized water to neutrality, soaking in l.0M HCl aqueous solution for 3 hr, washing with deionized water to neutrality, soaking in l.0M NaOH aqueous solution for 3 hr, and converting into OH^-And finally washing the mixture to be neutral by deionized water for later use.

2) Column mounting

The method comprises the steps of filling a column (with the inner diameter of 1.5cm and the height of 40cm) by a wet method, firstly adding deionized water (generally 1/3 with the length of the column) with a certain height into an ion exchange column, filling 30mL of wet resin 201 multiplied by 7 into a glass cup, adding 50mL of deionized water, slowly stirring, pouring suspended resin into the ion exchange column, and naturally settling the resin, wherein the resin must be uniformly distributed in the column, no obvious boundary line is required, and no air bubbles are generated.

3) Loading and eluting

Adjusting the pH value of the conversion solution obtained in the step (1) to 2.5, sampling at the column flow rate of 4.0BV/h, taking the effluent liquid at intervals to perform liquid phase detection, when the adsorption reaches the maximum value, washing the column with deionized water, then eluting with 1.0M ammonia water at the elution speed of 2.0BV/h, collecting the eluent, and detecting the L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid contained in the eluent at intervals. After the elution is finished, the ion exchange column is washed by deionized water, and the resin 201 multiplied by 7 is converted into OH^-Used for next separation and extraction.

4) Purification of

Distilling the eluate under reduced pressure to obtain yellow viscous substance, dissolving in methanol, stirring in ice bath, recrystallizing to obtain white solid, and filtering to obtain L-2-amino-4 (hydroxyethyl methylphosphoryl) -butyric acid.

Preparing 1L of conversion solution under the reaction condition of the step (1), and finally obtaining 0.8g of L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid solid with optical purity of 99.9 percent after the separation and purification.

Example 4:

0.2g of wet cells prepared in example 2 and a final concentration of 10g/L of the substrate 2-amino-4 (hydroxyethylmethylphosphoryl) -butyronitrile were added to 10mL of phosphate buffer (100mM, pH 7.0), and the mixture was transferred for 24 hours at 150rpm in a shaker at 30 ℃.1ml of the transformation solution is put into an EP tube and centrifuged at 12000r/min for 2min, and the supernatant is taken for chiral liquid phase detection. 1L of the converted solution was prepared under the same reaction conditions, and after separation and purification by the method of example 3, 4g of L-2-amino-4 (hydroxyethylmethylphosphoryl) -butyric acid was obtained as a product with an optical purity of 99.9%.

Example 5:

1g of the wet cell prepared in example 2 was added to 10mL of phosphate buffer (100mM, pH 7.0) to give a final substrate concentration of 10g/L, and the mixture was transferred at 150rpm for 24 hours in a shaker 30 ℃.1ml of the transformation solution is put into an EP tube and centrifuged at 12000r/min for 2min, and the supernatant is taken for chiral liquid phase detection. The preparation of the above strain ZJB-16006 under the same reaction conditions was performed to convert 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile to obtain 1L of the conversion solution, which was separated and purified by the method of example 3 to obtain 5.1g of L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid with an optical purity of 99.9%.

Example 6:

0.2g of wet cells prepared in example 2 and a final concentration of 10g/L of the substrate 2-amino-4 (hydroxyethylmethylphosphoryl) -butyronitrile were added to 10mL of phosphate buffer (100mM, pH 7.0), and the mixture was transferred for 24 hours at 150rpm in a shaker at 40 ℃ in a water bath. 1ml of the transformation solution is put into an EP tube and centrifuged at 12000r/min for 2min, and the supernatant is taken for chiral liquid phase detection. The preparation of the above strain ZJB-16006 under the same reaction conditions was performed to convert 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile to obtain 1L of the conversion solution, which was separated and purified by the method of example 3 to obtain 4.1g of L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid with an optical purity of 99.9%.

Example 7:

5g of the wet cell prepared in example 2 was added to 100mL of phosphate buffer (100mM, pH 7.0) to give a final substrate concentration of 10g/L, and the mixture was transferred at 150rpm for 48 hours in a shaker 30 ℃.1ml of the transformation solution is put into an EP tube and centrifuged at 12000r/min for 2min, and the supernatant is taken for chiral liquid phase detection. 1L of the converted solution was prepared under the same reaction conditions, and after separation and purification by the method of example 3, 6g of L-2-amino-4 (hydroxyethylmethylphosphoryl) -butyric acid was obtained as a product with an optical purity of 99.9%.

SEQUENCE LISTING

<110> Zhejiang industrial university

Serratia marcescens and application thereof

<130>

<160>1

<170>PatentIn version 3.5

<210>1

<211>1389

<212>DNA

<213>Serratia marcescens

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ggcaccaaag catctctgct aagttctctg gatgtcaaga gtaggtaagg ttcttcgcgt 480

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atcctgtttg ctccccacgc tttcgcacct gagcgtcagt cttcgtccag ggggccgcct 720

tcgccaccgg tattcctcca gatctctacg catttcaccg ctacacctgg aattctaccc 780

ccctctacga gactctagct tgccagtttc aaatgcagtt cccaggttga gcccggggat 840

ttcacatctg acttaacaaa ccgcctgcgt gcgctttacg cccagtaatt ccgattaacg 900

cttgcaccct ccgtattacc gcggctgctg gcacggagtt agccggtgct tcttctgcga 960

gtaacgtcaa ttgatgaacg tattaagttc accaccttcc tcctcgctga aagtgcttta 1020

caacccgaag gccttcttca cacacgcggc atggctgcat caggcttgcg cccattgtgc 1080

aatattcccc actgctgcct cccgtaggag tctggaccgt gtctcagttc cagtgtggct 1140

ggtcatcctc tcagaccagc tagggatcgt cgcctaggtg agccattacc ccacctacta 1200

gctaatccca tctgggcaca tctgatggca agaggcccga aggtccccct ctttggtctt 1260

gcgacgttat gcggtattag ctaccgtttc cagtagttat ccccctccat caggcagttt 1320

cccagacatt actcacccgt ccgccgctcg tcacccaggg agcaagctcc cctgtgctac 1380

cgctcgact 1389

Claims

1. Serratia marcescens (Serratia marcocens) ZJB-16006, preserved in China center for type culture Collection with the preservation number of CCTCC No: m2017033, date of deposit 2017, month 01, day 13, address: china, wuhan university, 430072.

2. The use of serratia marcescens ZJB-16006 in claim 1 for catalyzing 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile to prepare L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid, wherein the structural formula of the 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile is as follows:

the structural formula of the L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid is as follows:

3. the use according to claim 2, characterized in that said use is: taking wet thalli obtained by fermentation culture of serratia marcescens ZJB-16006 as a catalyst, taking 2-amino-4 (hydroxyethyl methyl phosphoryl) -butyronitrile as a substrate, taking distilled water or 100mM phosphate buffer solution with pH of 7.0 as a reaction medium, carrying out conversion reaction at 30-50 ℃ and 200rpm under the condition of 100 plus materials, separating and purifying the reaction solution after the reaction is finished, and obtaining L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid.

4. The use according to claim 3, wherein the amount of catalyst is 20-100g/L of reaction medium based on the weight of wet cells, and the final concentration of substrate initially added is 2-10g/L of reaction medium.

5. The use according to claim 3, wherein the catalyst is prepared by the following process: (1) slant culture: inoculating serratia marcescens ZJB-16006 to a slant culture medium, and culturing at 30 deg.C for 48 hr to obtain slant thallus; the final concentration of the slant culture medium is as follows: 1-20.0 g/L of mannitol, 1-10.0 g/L of sodium glutamate, 1-5.0 g/L of yeast extract and K₂HPO₄0.1～1.0g/L，KH₂PO₄0.1～1.0g/L，MgSO₄0.1-1.0 g/L, 0.1-2.0 g/L caprolactam, 20.0g/L agar, deionized water as solvent and pH 7.0-7.5;

(2) seed culture: selecting slant thallus, inoculating to seed culture medium, and culturing at 30 deg.C for 24 hr to obtain seed solution; the final concentration of the seed culture medium is as follows: 1-20.0 g/L of mannitol, 1-10.0 g/L of sodium glutamate, 1-5.0 g/L of yeast extract and K₂HPO₄0.1～1.0g/L，KH₂PO₄0.1～1.0g/L，MgSO₄0.1-1.0 g/L, 0.1-2.0 g/L caprolactam, deionized water as solvent and pH 7.0-7.5;

(3) fermentation culture: inoculating the seed solution into a fermentation culture medium in an inoculation amount of 1% of volume concentration, carrying out shaking culture at 30 ℃ and 150rpm for 48h, centrifuging at 12000g for 10min, and collecting wet thalli; the final concentration of the fermentation medium is as follows: 1-20.0 g/L of mannitol, 1-10.0 g/L of sodium glutamate, 1-5.0 g/L of yeast extract and K₂HPO₄0.1～1.0g/L，KH₂PO₄0.1～1.0g/L，MgSO₄0.1-1.0 g/L, 0.1-2.0 g/L caprolactam, deionized water as solvent and pH 7.0-7.5.

6. The use of claim 3, wherein the reaction solution is separated and purified by the following steps: adjusting the pH value of the reaction liquid to 2.5, performing column chromatography by adopting anion exchange resin 201 multiplied by 7, loading the sample at the flow rate of 1-6.0 BV/h, washing with deionized water, then eluting with 0.5-2.0M ammonia water at the elution speed of 1.0-4.0 BV/h, and collecting the eluent containing the target substance; distilling the eluent under reduced pressure until no solvent flows out, adding methanol for dissolving, stirring in ice bath, recrystallizing, filtering, and drying the filter cake to obtain the product L-2-amino-4 (hydroxyethyl methyl phosphoryl) -butyric acid.