CN109321490B - Enterobacter aerogenes ZJB-17003 and application thereof - Google Patents
Enterobacter aerogenes ZJB-17003 and application thereof Download PDFInfo
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Abstract
The invention relates to Enterobacter aerogenes (Enterobacter aeogensa) ZJB-17003 and application thereof, wherein the enantioselectivity value of catalyzing N-phenylacetyl-DL-amino acid to prepare L-amino acid reaches 99 percent, and the enantioselectivity value of catalyzing 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] butyric acid to prepare 2-amino-4- [ hydroxy (methyl) phosphoryl ] butyric acid reaches 99 percent.
Description
(I) technical field
The invention relates to a strain for producing amidohydrolase, namely Enterobacter aerogenes (Enterobacter aerogenes) ZJB-17003, and application thereof in catalyzing N-phenylacetyl amino acid to prepare chiral amino acid and catalyzing N-phenylacetyl-L-amino acid derivatives of the N-phenylacetyl-DL-amino acid derivatives.
(II) background of the invention
The chemical name of the L-glufosinate-ammonium is as follows: 4- [ hydroxyl (methyl) phosphonyl ] -L-homoalanine is a structural analogue of L-glutamic acid, can inhibit the activity of Glutamine Synthetase (GS), enables ammonia accumulation in plants, blocks the light respiration of the plants due to high-concentration ammonia accumulation, destroys chloroplast structures and cysts of matrixes, and simultaneously inhibits amino acid synthesis to damage cell membranes and kill cells, thereby killing weeds. It has broad spectrum and is herbicide tolerant to the second major transgenic crop in the world.
The existing L-glufosinate-ammonium synthesis method comprises a chemical synthesis method and a biological synthesis method. The chemical method mainly constructs the chiral center of L-glufosinate-ammonium by the following 4 ways: 1) constructing a chiral center by chiral induction by using a chiral auxiliary reagent; 2) taking natural amino acid as a chiral source, and converting to obtain L-glufosinate-ammonium; 3) asymmetric synthesis reaction catalyzed by chiral catalyst; 4) chiral resolution of the racemate. However, the chemical method for synthesizing L-glufosinate-ammonium has the disadvantages of multiple working procedures, low yield, high chiral reagent cost, large amount of three wastes and difficult treatment. The biological method has the advantages of strict stereoselectivity, mild reaction conditions, high yield, easy separation and purification and the like, so that the technology for producing the L-glufosinate-ammonium by the biological method has very important industrial development value.
Biological production of L-glufosinate-ammonium according to the substrate, there are protease hydrolysis bialaphos, hydrolysis of L-3-acetamido-4- (hydroxymethylphosphono) butanamide by phosphodiesterase I, amidase I and glutaminase stepwise synergy maintaining optical activity, resolving bialaphos ethyl ester by alpha-chymotrypsin, deacetylase resolution of N-acetyl-glufosinate, amidase resolution of 2-amino-4- (hydroxymethylphosphono) -butanamide, ester hydrolase hydrolysis of L-glufosinate-N-carboxylic acid anhydride, nitrile hydratase hydrolysis of glufosinate nitrile-containing substrate, transaminase catalysis of 2-carbonyl-4- (hydroxymethylphosphono) butyric acid and the like.
The existing chemical method for synthesizing the L-glufosinate-ammonium mainly comprises the following steps: 1) the chiral auxiliary inducing method is characterized in that (S) -2-hydroxy-3-pinone is used as the chiral auxiliary for preparation, the yield is 51 percent, and the e.e. value is 79 percent; the D-valine methyl ester is used as a chiral auxiliary agent, the reaction is carried out at a low temperature of-78 ℃, the yield is 51 percent, and the e.e. value is 93.5 percent. 2) The natural amino acid chiral source method takes L glutamic acid as a chiral source, and the e.e. value is 99.4 percent; l-methionine is chiral source, total yield is 42.3%, e.e. value is 93.5%, and virulent iodomethane is needed. 3) Asymmetric synthesis method, asymmetric catalytic hydrogenation-large catalyst dosage and expensive trimethyl silicon cyanide price; asymmetric Strecker reaction-use highly toxic cyanide; asymmetric Michael addition-the catalyst consumption is large, and the product yield and the e.e. value are low. 4) The racemate resolution method has the highest yield of 86 percent and the highest e.e. value of 99 percent, but the D-glufosinate-ammonium is not converted after the resolution, so that the raw materials are wasted.
The important effective component 2-amino-4- [ hydroxyl (methyl) phosphoryl ] -L-butyric acid of the L-glufosinate-ammonium is prepared by selectively catalyzing and resolving 2-N-phenylacetyl-4- [ hydroxyl (methyl) phosphoryl ] -DL-butyric acid by using amidohydrolase, and a basis is provided for realizing the synthesis of the L-glufosinate-ammonium in an industrial route.
Chiral amino acids and derivatives thereof have an increasing role in pharmaceutical development, and the current pharmaceutical development has an increasing demand for chiral purity. The chiral amino acid production process includes chemical resolution, enzyme resolution and other processes. The enzymatic resolution has the advantages of mild condition, strong specificity, less pollution and obvious advantages compared with the chemical resolution. The amide hydrolase selectively catalyzes and resolves the N-phenylacetyl amino acid to produce the chiral amino acid, so that the chiral amino acid has wide application prospect.
Disclosure of the invention
The invention aims to provide a strain capable of producing amidohydrolase, namely Enterobacter aerogenes ZJB-17003, and application thereof in catalyzing N-phenylacetyl-DL-amino acid derivatives (2-N-phenylacetyl-4- [ hydroxyl (methyl) phosphoryl ] butyric acid) to prepare L-amino acid derivatives (2-amino-4- [ hydroxyl (methyl) phosphoryl ] -L-butyric acid) which are important effective components of L-glufosinate-ammonium, and application thereof in catalyzing N-phenylacetyl-DL-amino acid to prepare L-amino acid, thereby providing a new enzyme source for producing the effective components of the L-glufosinate-ammonium and the L-amino acid by a chemical-enzymatic method and promoting the development of green chemical catalysis.
The technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides an amide hydrolase producing strain, namely Enterobacter aerogenes (Enterobacteriaceae) ZJB-17003, which is preserved in China center for type culture Collection with the preservation number of CCTCC No: m2017599, date of deposit 2017, 10 month, 23 day, address: wuhan university, 430072. The strain is separated from soil by the institute of bioengineering of Zhejiang industrial university, and has the capability of catalyzing and synthesizing L-glufosinate-ammonium through detection.
In a second aspect, the present invention provides an application of said enterobacter aerogenes ZJB-17003 in the microbial catalytic resolution of N-phenylacetyl-DL-amino acid derivatives (preferably 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] butyric acid) to prepare L-amino acid derivatives (preferably 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid) which are important active ingredients of L-glufosinate-ammonium, said application being: the wet thallus catalyst of Enterobacter aerogenes ZJB-17003 after fermentation culture, a reaction system with the pH value of 8.5 is formed by taking N-phenylacetyl-DL-amino acid derivative (preferably 2-N-phenylacetyl-4- [ hydroxyl (methyl) phosphoryl ] -DL-butyric acid) as a substrate and a buffer solution (preferably ammonia water) as a reaction medium, the conversion reaction is carried out at the temperature of 25-55 ℃, the temperature of 100 and 200rpm (preferably 30-40 ℃, 150rpm), and after the reaction is finished, the L-amino acid derivative (preferably 2-amino-4- [ hydroxyl (methyl) phosphoryl ] -L-butyric acid) is obtained by separating and purifying the reaction solution. In the reaction system, the dosage of the catalyst is 10-200g/L, preferably 50g/L, and the final concentration of the substrate added initially is 10-500mM, preferably 100mM, calculated by the weight of wet bacteria. The reaction system can also be composed of fermentation liquor and substrate obtained by fermenting and culturing Enterobacter aerogenes ZJB-17003, and the pH value is 8.5; wherein the final concentration of wet thallus in the fermentation liquor in the whole reaction system is 10-200g/L, preferably 50g/L, and the final concentration of the substrate in the reaction system is 10-500mM, preferably 100 mM.
The method for separating and purifying the reaction liquid comprises the following steps: after completion of the reaction, the reaction mixture is extracted with methylene chloride, the pH of the aqueous layer is adjusted to 1.0 to 5.0 (preferably 2.5), loading the sample at a rate of 1-6.0Bv/h (preferably 4Bv/h) for ion exchange chromatography, washing with deionized water, eluting with 0.5-3.0Bv/h (preferably 2Bv/h) with 0.2-4.5M (preferably 1M) ammonia water, detecting the eluate with 0.2% ninhydrin solution-impregnated filter paper, collecting eluate containing target components, distilling under reduced pressure to paste, dissolving in methanol for recrystallization, and drying to obtain L-amino acid derivative (preferably 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid).
In a third aspect, the invention also provides an application of the enterobacter aerogenes ZJB-17003 in catalyzing N-phenylacetyl-DL-amino acid to prepare L-amino acid, wherein wet thalli obtained by fermenting and culturing the enterobacter aerogenes ZJB-17003 is used as a catalyst, N-phenylacetyl-DL-amino acid is used as a substrate, a buffer solution (preferably ammonia water) is used as a reaction medium to form a conversion system with the pH value of 8.5, the conversion reaction is carried out under the conditions of 25-55 ℃, 100-200rpm (preferably 30-40 ℃ and 150rpm), and after the reaction is finished, the reaction solution is separated and purified to obtain the L-amino acid. In the transformation system, the dosage of the catalyst is 20-300g/L (preferably 20-60g/L) based on the weight of wet bacteria, and the final concentration of the substrate added is 50-500mM (preferably 100 mM). The N-phenylacetyl-DL-amino acid is one of the following: N-phenylacetyl-DL-alanine, N-phenylacetyl-DL-serine, N-phenylacetyl-DL-glutamic acid, N-phenylacetyl-DL-tyrosine, N-phenylacetyl-DL-threonine, N-phenylacetyl-DL-valine, N-phenylacetyl-DL-aspartic acid, N-phenylacetyl-DL-methionine, N-phenylacetyl-DL-leucine, N-phenylacetyl-DL-isoleucine, N-phenylacetyl-DL-phenylalanine. The method for separating and purifying the reaction liquid comprises the following steps: after the reaction is finished, extracting the reaction solution by using dichloromethane, adjusting the pH of an aqueous layer to 1.0-5.0 (preferably 2.5), loading the aqueous layer to perform ion exchange chromatography at the speed of 1-6.0Bv/h (preferably 4Bv/h), firstly washing by using deionized water, then eluting by using 0.2-4.5M (preferably 1M) of ammonia water at the speed of 0.5-3.0Bv/h (preferably 2Bv/h), detecting the eluent by using filter paper impregnated with 0.2% ninhydrin solution, changing the filter paper to be purple black to indicate that the eluent contains L-amino acid, collecting the eluent containing the target component, distilling under reduced pressure to be pasty, dissolving and recrystallizing by using methanol, taking crystals and drying to obtain the L-amino acid.
The N-phenylacetyl-DL-amino acid is prepared by the following method: adding amino acid and NaOH into distilled water, fully stirring under an ice bath condition of 4 ℃ until the solution is colorless and transparent, dropwise adding phenylacetyl chloride, continuing to react for 2 hours under the ice bath condition of 4 ℃ after dropwise adding is finished, stirring at normal temperature (25-30 ℃) for 5 hours until the solution is colorless and transparent, adding HCl to adjust the pH value to 1.5-5.5 (preferably 2-4), separating out a solid, and performing suction filtration and drying to obtain N-phenylacetyl-DL-amino acid; the molar ratio of the amino acid to NaOH is 1:1 to 7 (preferably 1: 1.5-2.5); the molar ratio of the amino acid to the phenylacetyl chloride is 1: 0.1-2 (preferably 1:0.5-1.0), wherein the volume consumption of the distilled water is 3-10ml/g in terms of the mass of the amino acid; the amino acid is one of the following: alanine, serine, glutamic acid, tyrosine, threonine, valine, aspartic acid, methionine, leucine, isoleucine, phenylalanine.
The wet thallus obtained by fermenting and culturing the enterobacter aerogenes ZJB-17003 is prepared by the following method:
(1) slant culture:
inoculating Enterobacter aerogenes ZJB-17003 to a slant culture medium, and culturing at 30 ℃ for 48h to obtain slant thallus; the final concentration of the slant culture medium is as follows: 1-15 g/L of mannitol, 1-15 g/L of sodium glutamate, 0.5-5 g/L of yeast extract and K2HPO40～1.5g/L，KH2PO40～1.5g/L.，MgSO40-1.0 g/L, 0.2-1.8 g/L caprolactam, 20.0g/L agar, deionized water as solvent and pH of 7.0-7.5; preferably, the final concentration composition of the slant culture medium is as follows: 10g/L of mannitol, 7g/L of sodium glutamate, 3g/L of yeast extract and K2HPO40.75g/L，KH2PO40.75g/L，MgSO40.5g/L, 1g/L caprolactam, 20.0g/L agar, deionized water as a solvent, and pH 7.0-7.5.
(2) Seed culture
Selecting one strain of the thallus on the inclined plane, inoculating the strain to a seed culture medium, and culturing at 30 ℃ for 24 hours to obtain a seed solution; the final concentration of the seed culture medium is as follows: 1-15 g/L of mannitol, 1-15 g/L of sodium glutamate, 0.5-5 g/L of yeast extract and K2HPO40～1.5g/L，KH2PO40～1.5g/L，MgSO40-1.0 g/L of caprolactam, 0.2-1.8 g/L of caprolactam, deionized water as a solvent, and 7.0-7.5 of pH; preferably, the final concentration composition of the seed culture medium is as follows: 10g/L of mannitol, 7g/L of sodium glutamate, 3g/L of yeast extract and K2HPO40.75g/L，KH2PO40.75g/L，MgSO40.5g/L, 1g/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 1-10% (preferably 1%) by volume concentration, carrying out shaking culture at 30 ℃ and 150rpm for 60h, centrifuging at 12000g for 10min, and collecting wet thalli; the final concentration of the fermentation medium is as follows: 1-15 g/L of mannitol, 1-15 g/L of sodium glutamate, 0.5-5 g/L of yeast extract and K2HPO40～1.5g/L，KH2PO40～1.5g/L，MgSO40-1.0 g/L of caprolactam, 0.2-1.8 g/L of caprolactam, deionized water as a solvent, and 7.0-7.5 of pH; preferably, the final concentration of the fermentation medium consists of: 10g/L of mannitol, 7g/L of sodium glutamate, 3g/L of yeast extract and K2HPO40.75g/L，KH2PO40.75g/L，MgSO40.5g/L, 1g/L caprolactam, deionized water as solvent and pH 7.0-7.5.
Further, the buffer solution having a pH of 8.5 is composed of: a citric acid buffer (100mM, pH 4.0-6.0), a phosphoric acid buffer (100mM, pH 6.0-8.0), Tris-HCl (100mM, pH 7.0-9.0), a boric acid buffer (100mM, pH 9.0-10.5), an aqueous ammonia buffer (pH 7.0-10.0 of a substrate solution adjusted with aqueous ammonia), a Roche buffer (100mM, pH 10.5-12.0); preferably the buffer composition is: the pH of the substrate solution was adjusted to 8.5 with ammonia.
The invention has the following beneficial effects: the invention provides a strain capable of producing an amidohydrolase, namely Enterobacter aerogenes ZJB-17003, and also provides a method for preparing L-glufosinate-L by using wet thalli obtained by fermentation culture of the Enterobacter aerogenes ZJB-17003 as a biocatalyst to catalyze 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid to prepare an important effective component 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, wherein the conversion rate of the 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] butyric acid reaches 49.5%, and the product L-glufosinate-L reaches e.e.% 99.9% The conversion rate of the amino acid method can reach 49%, the product chiral amino acid can reach e.e% of 99-99.9%, the resolution preparation process is green and efficient, the strain is easy to culture, collect and apply, the catalytic reaction condition is mild, and the method has an important application prospect.
(IV) description of the drawings:
FIG. 1 is a standard curve of OPA/NAC-glufosinate concentration under a high throughput screening method.
FIG. 2 is a liquid phase results diagram of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid and 2-amino-4- [ hydroxy (methyl) phosphoryl ] -D-butyric acid under pre-column derivatization reversed phase high performance liquid chromatography.
FIG. 3 shows the result of electron microscope observation of the strain selected for Enterobacter aerogenes ZJB-17003.
(V) specific embodiment:
the invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
the ultrapure water is UP water, and is water with the resistivity of 18M omega cm (25 ℃). The ammonia water is an aqueous solution containing 25-28% of ammonia. The normal temperature of the invention is 25-30 ℃. The ice bath was 4 ℃ in the examples of the invention. The preparation method of the 2% ninhydrin solution comprises the following steps: dissolving 2g of ninhydrin and 0.08g of stannous chloride in 100mL of ultrapure water, stirring and filtering, and taking the filtrate and storing away from light.
Example 1: screening of Enterobacter aerogenes (Enterobacter aerogenesa) ZJB-17003
1. Preliminary screening
The invention takes soil samples from all parts of the country, and takes 80 parts of soil samples. The screening method comprises the following specific steps: weighing 1g of soil sample, placing the soil sample in 10 mL0.85% of physiological saline, shaking, standing, taking supernatant into an enrichment medium, and carrying out shaking culture at 30 ℃ and 150r/min for 2-3 days. Adding 1mL of enrichment solution into 50mL of fresh enrichment medium, repeating the process for 3 times, and then carrying out separation and purification.
Bromothymol blue filter paper is selected as indicating filter paper to detect colonies capable of degrading substrates. The principle is as follows: the colonies containing the enzyme of interest, after degradation of the substrate, produce acidic by-products (phenylacetic acid, acetic acid, formic acid, benzoic acid) which cause a change in the pH locally on the indicator filter. The indicator bromothymol blue has the color change range of 5.8-7.6 (yellow-blue), so that the color of the periphery of the colony utilizing chlorine-containing organic matters is yellow, and the color of the plate around the colony which cannot be utilized is green.
Gradually diluting the enriched solution with sterile 0.85% physiological saline for 10 gradients, and selecting 10-4、10-5、10-6、10-7、10-8、10-9、10-100.1mL of each of the five dilutions was spread on the plate screening medium and incubated at 30 ℃ for 48 h. Spreading sterile filter paper (indicator filter paper) soaked with 10% bromothymol blue on the plate, picking out corresponding colony showing yellow on the indicator filter paper, inoculating into 96-well plate containing growth medium, placing into shaker at 30 deg.C and 180rpm for constant temperature culture, during which OD is detected600And drawing a growth curve, transferring 300 mu l of fresh fermentation medium when the strain grows to the logarithmic phase, inoculating the residual strain liquid after a 96-well plate is inoculated, sealing and storing in a refrigerator at 4 ℃ for later use. And culturing the transferred 96-well plate containing the fermentation medium for 48 hours at 30 ℃ and 180rpm by a shaking table at constant temperature to ensure that the plate can fully grow and produce enzyme.
Preparing a high-concentration substrate-ammonia buffer solution, adding a small amount of the substrate-ammonia buffer solution into a bacterial suspension containing a fermentation culture medium to enable the final concentration of the substrate to be 50mM, and placing the mixture in a shaker at 30 ℃ and 180rpm for constant-temperature reaction for 24 hours to obtain the bacterial suspension.
2. OPA/NAC high throughput screening
Dissolving a substrate 2-N-phenylacetyl-4- [ hydroxyl (methyl) phosphoryl ] -DL-butyric acid in ammonia water, adding 1mL of the bacterial suspension prepared in the step 1, adjusting the pH value of the solution to 8.5 by using the ammonia water to form a 10mL reaction system, enabling the final concentration of the substrate to be 50mM, and carrying out constant temperature reaction on a shaker at 30 ℃ and 180rpm for 24 hours. The obtained conversion solution is correspondingly diluted by 10 times by ultrapure water respectively, ice bath is carried out at 4 ℃, an OPA/NAC high-throughput screening method is used for detecting, and the strains with higher relative fluorescence values are screened out.
OPA/NAC high throughput screening method: absorbing 40 mul of diluted ice-bath reaction liquid to a 96-hole fluorescence detection plate by using a line gun, adding the A liquid after ice-bath, oscillating for 30s in an enzyme-labeling instrument, adding 100 mul of ultrapure water, oscillating for 30s again, and carrying out gamma-fluorescence detection on the solution at the lambda positionex＝340nm；λem(455 nm) and the fluorescence intensity value was measured, and the obtained value was compared with 2-amino-4- [ hydroxy (methyl) phosphoryl group]Comparing the standard curve of the (E) -L-butyric acid to obtain the 2-amino-4- [ hydroxyl (methyl) phosphoryl group contained in the sample]The concentration of L-butyric acid, from which 2-amino-4- [ hydroxy (methyl) phosphoryl can be calculated]The yield of the (E) -L-butyric acid is obtained, and the strain obtained is used for biocatalytic resolution of the 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl]2-amino-4- [ hydroxy (methyl) phosphoryl group as important effective component for preparing L-glufosinate-ammonium from-DL-butyric acid]-conversion of L-butyric acid.
The solution A comprises: dissolving N-acetyl-L-cysteine (0.448g) and o-phthalaldehyde (0.185g) in 10mL of absolute ethyl alcohol, adding a boric acid buffer solution (140mM, pH 9.5) under an ice bath condition to a constant volume of 50mL, and storing under an ice bath condition for 3 days. The final concentration of N-acetyl-L-cysteine in the solution A is 0.313mol/L, and the final concentration of o-phthalaldehyde is 0.138 mol/L.
The 2-amino-4- [ hydroxy (methyl) phosphoryl group]-L-butyric acid standard curve preparation method: 5g/L of 2-amino-4- [ hydroxy (methyl) phosphoryl group was prepared with ultrapure water]-standard solution of L-butyric acid, diluted in 3 concentration gradients: 0.01 to 0.1g/L (0.01, 0.02, 0.04, 0.06, 0.08, 0.1g/L), 0.1 to 1.0g/L (0.1, 0.2, 0.4, 0.6, 0.8, 1.0g/L), 1.0 to 5.0g/L (1.0, 2.0, 3.0, 4.0, 5.0g/L),respectively sucking 40 μ l of standard solution with different concentration gradients to 96-well microporous fluorescent detection plate with a row gun, respectively adding 40 μ l of prepared solution A stored in ice bath, oscillating in enzyme labeling instrument for 30s, respectively adding 100 μ l of ultrapure water, oscillating for 30s, and measuring at lambdaex＝340nm；λemThe fluorescence intensity value was measured at 455nm, and the obtained fluorescence intensity value was plotted on the ordinate and the concentration of the standard on the abscissa to prepare a calibration curve. The standard curves of 3 concentration ranges are prepared, and the good linearity (shown in figure 1) can be known when the sample concentration is 0.01-0.1 g/L, R20.9978, showing that the method has high accuracy and applicability.
3. High performance liquid chromatography rescreening
Inoculating the strain screened in the step 2 into a slant culture medium, culturing at 30 ℃ for 48h, and storing in a refrigerator at 4 ℃. The strain preserved on the slant was inoculated into a seed medium and cultured at 30 ℃ for 24 hours. Inoculating the seed liquid into a fermentation medium at an inoculation amount of 1% by volume concentration, and performing shaking culture at 30 ℃ and 150rpm for 60 h. The cells were centrifuged at 12000g for 5min to collect wet cells. Adding a substrate 2-N-phenylacetyl-4- [ hydroxyl (methyl) phosphoryl ] -DL-butyric acid and ammonia water into wet thalli, adjusting the pH of the solution to 8.5 by using the ammonia water to form a 10mL reaction system, adjusting the final concentration of the wet thalli to 50g/L and the final concentration of the substrate to 50mM, placing the wet thalli in a water bath shaker at 30 ℃ for conversion for 24h, taking 1mL of conversion solution, performing centrifugal separation, taking a supernatant for HPLC analysis, and finally obtaining a wild strain with high catalytic activity, wherein the strain is marked as a strain ZJB-17003.
High Performance Liquid Chromatography (HPLC) rescreening method: centrifuging the conversion solution to obtain a supernatant, diluting the supernatant by 10 times with ultrapure water, taking 200 mu L of clean 1.5mL of EP tube, adding 200 mu L of derivatization reagent, reacting at 30 ℃ for 5min, adding 600 mu L of ultrapure water, mixing uniformly, filtering by using a syringe filter membrane (0.22 mu m), and detecting by HPLC to obtain the concentrations of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid and 2-amino-4- [ hydroxy (methyl) phosphoryl ] -D-butyric acid in the conversion solution.
The derivatization reagent is: 0.1g of o-phthalaldehyde and 0.12g of 0.12g N-acetyl-L-cysteine were dissolved in 10mL of absolute ethanol, and the resulting solution was diluted to 50mL with a boric acid buffer (100mM, pH 9.8).
The HPLC conditions are as follows: a U3000 high performance liquid chromatograph of Daian American and equipped with a fluorescence detector; daian C18A silicon hydroxyl packed column (250mM × 4.6.6 mM), a mobile phase of ammonium acetate (50mM pH 4.7) solution containing 10% pure methanol, a column temperature controlled at 35 deg.C, and a fluorescence excitation wavelength lambdaex350nm, emission wavelength λemThe sample size was 10. mu.l at 450 nm. Under these conditions, 2-amino-4- [ hydroxy (methyl) phosphoryl group]-L-butyric acid and 2-amino-4- [ hydroxy (methyl) phosphoryl]The peak time of the-D-butyric acid is 7.800min and 9.290min (as shown in figure 2).
The enrichment medium comprises the following components: 2-N-Phenylacetyl-4- [ hydroxy (methyl) phosphoryl]1g/L of DL-butyric acid, 5g/L of glucose, 1g/L of sodium chloride, K2HPO4·3H2O 0.8g/L，KH2PO43.3g/L，MgSO4·7H2O0.2 g/L, deionized water as solvent, and pH7.
Plate screening medium composition: 2-N-Phenylacetyl-4- [ hydroxy (methyl) phosphoryl]1g/L of DL-butyric acid, 5g/L of glucose, 1g/L of sodium chloride, K2HPO4·3H2O 0.8g/L，KH2PO43.3g/L，MgSO4·7H20.2g/L of O, 20g/L of agar, deionized water as a solvent and pH7.
Growth medium composition: 10g/L of sodium chloride, 10g/L of peptone, 5g/L of yeast powder and deionized water as a solvent. The composition of the slant medium, seed medium and fermentation medium was the same as in example 3.
Example 2: identification of Strain ZJB-17003
1. Morphological identification:
the strain ZJB-17003 obtained by screening in the embodiment 1 of the invention is inoculated on a solid culture medium, and forms a round or nearly round, soft texture, smooth and flat surface, regular edge and glossy milky colony with the diameter of 2-4mm after being cultured for 24 hours at 37 ℃. And (3) gram staining observation: pink short rod shape, no spores. Solid medium composition: 10g/L of sodium chloride, 10g/L of peptone, 5g/L of yeast powder, 20g/L of agar and deionized water as a solvent.
2. Physiological and biochemical identification:
94 phenotypic tests were performed on strain ZJB-17003 using the Biolog (GEN III) automated microorganism identification system, including 71 carbon source utilization assays and 23 chemosensitivity assays: inoculating ZJB-17003 into BUG plate culture medium (BIOLOG UNIVERSAL GROWTH AGAR), culturing at 33 deg.C for 2 days, washing thallus on the plate with sterile cotton swab, mixing with inoculating liquid (IF-A), making into bacterial suspension, and adjusting to 91% T/IF-A with turbidimeter. The bacterial suspensions were added to each well of the BiologGEN iii microwell assay plate using an 8-well electric applicator, 100 μ L per well. The plate was placed in a 33 ℃ incubator and read on a Biolog reader after 12h, 24h, 36h, 48h incubation, respectively.
The strain ZJB-17003 analyzes the metabolic fingerprint through a Biolog reader, and the strain ZJB-17003 can strongly utilize 65 carbon sources and cannot or has weak utilization capacity on other 6 carbon sources; the strain ZJB-17003 is sensitive to 22 chemical substances. The 48h identification results given by the Biolog system are shown in tables 1 and 2.
TABLE 1 ability of the Strain ZJB-17003 to utilize 71 carbon sources on the BiologGEN III plate
TABLE 2 chemosensitivity of Strain ZJB-17003 to 23 chemicals on the BiologGEN III plate
3. Molecular biological identification:
the total DNA of the strain ZJB-17003 is used as a template, primers P1:5'-AGAGTTTGATCCTGGCTCAG-3' and P2:5'-AAGGAGGTGATCCAGCCGCA-3' are used for amplifying the 16S rDNA gene of the strain, a gene product is connected with a T vector, Shanghai workers are entrusted to amplify and sequence the 16S rDNA of the strain to obtain a 16S rDNA sequence (shown in SEQ ID NO. 1) of the strain, BLAST is used for searching the 16S rDNA gene sequence of related strains in GenBank on an NCBI website, and homology comparison is carried out. The strain ZJB-17003 has the highest homology with Enterobacter aerogenes strain NBRC 13534 (homology, 99%, based on 16S ribosomal RNA gene), and the 16S rDNA homology is higher than 95% according to the principle of microbial genetic identification, so that the identified strain basically belongs to a control strain. Therefore, the strain ZJB-17003 identified by the experiment is Enterobacter aerogenes (Enterobacter aerogenes), is supposed to be named as Enterobacter aerogenes (Enterobacter aerogenes) ZJB-17003 and is deposited in China center for type culture Collection with the preservation number of CCTCC No: m2017599, date of deposit 2017, 10 month 23 day, address: wuhan university, 430072.
Any nucleotide sequence obtained by substituting, deleting or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.1 in the nucleotide sequence table is within the protection scope of the present invention as long as the nucleotide sequence has homology of more than 90%.
Example 3: preparation of Wet cells
(1) Slant culture:
inoculating Enterobacter aerogenes (Enterobacter aerogenesa) ZJB-17003 to a slant culture medium, and culturing at 30 ℃ for 48h to obtain slant thallus;
the final concentration of the slant culture medium is as follows: 10g/L of mannitol, 7g/L of sodium glutamate, 3g/L of yeast extract and K2HPO40.75g/L，KH2PO40.75g/L，MgSO40.5g/L, 1g/L caprolactam, 20.0g/L agar, deionized water as a solvent, and pH 7.0-7.5.
(2) Seed culture
Selecting one strain of the thallus on the inclined plane, inoculating the strain to a seed culture medium, and culturing at 30 ℃ for 24 hours to obtain a seed solution;
the final concentration of the seed culture medium is as follows: 10g/L of mannitol, 7g/L of sodium glutamate, 3g/L of yeast extract and K2HPO40.75g/L，KH2PO40.75g/L，MgSO40.5g/L, 1g/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 60h, centrifuging at 12000g for 10min, and collecting wet thalli;
the final concentration of the fermentation medium is as follows: 10g/L of mannitol, 7g/L of sodium glutamate, 3g/L of yeast extract and K2HPO40.75g/L，KH2PO40.75g/L，MgSO40.5g/L, 1g/L caprolactam, deionized water as solvent and pH 7.0-7.5.
Example 4 bioconversion reactions Using 2-N-Phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid as substrate
(1) The substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid was dissolved in aqueous ammonia, 0.3g of the wet cell prepared in example 3 was added thereto, and the pH was adjusted to 8.5 with aqueous ammonia to prepare 10mL of a reaction system, the final substrate concentration was adjusted to 50mM, the reaction system was placed in a 30 ℃ water bath shaker, and the resultant was transformed at 150rpm for 24 hours, and 1mL of the transformed solution was taken out into an ep tube, and after centrifugation, the supernatant was subjected to HPLC analysis as described in example 1. The results show that ZJB-17003 can convert the substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid into the product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, the conversion rate is 49.5%, and the optical purity of the product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid is 99.9%.
(2) Separating the conversion mixed liquor containing the product 2-amino-4- [ hydroxyl (methyl) phosphoryl ] -L-butyric acid obtained by the enzyme catalysis reaction to extract the product 2-amino-4- [ hydroxyl (methyl) phosphoryl ] -L-butyric acid.
1) And (3) extraction:
the resulting conversion solution was added to a separatory funnel, and simultaneously, dichloromethane of equal volume was added, and after shaking, it was allowed to stand for 3 hours, the organic layer was discharged from the lower end of the separatory funnel, and the aqueous layer was poured out from the upper end. The aqueous layer obtained is an aqueous solution containing the product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid.
2) Product separation using anion exchange resins
(ii) resin pretreatment
Soaking ion exchange resin 201 × 7 in 50 deg.C warm water to fully expand the resin and remove fine particles (inclined or floating method), and soaking in 1.0Soaking in NaOH aqueous solution of M for 3h, washing with deionized water to neutrality, soaking in HCl aqueous solution of 1.0M for 3h, washing with deionized water to neutrality, soaking in NaOH aqueous solution of 1.0M for 3h, and converting into OH-And finally washing the mixture to be neutral by deionized water for later use.
② mounting columns
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 with the height of 13cm into an ion exchange column, then 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, naturally settling, and enabling the resin to be uniformly secreted in the column without obvious boundary lines, bubbles and the like.
③ sample loading and elution
Adjusting the pH of the aqueous solution obtained in the step 1) to 2.5, loading the aqueous solution at the column flow rate of 4.0Bv/h, taking out effluent liquid at intervals for liquid phase detection, when the adsorption reaches the maximum value, washing the effluent liquid with deionized water, then eluting the effluent liquid with 1.0M ammonia water at the elution speed of 2.0Bv/h, detecting the effluent liquid with filter paper impregnated with 0.2% ninhydrin solution, wherein the filter paper changes purple to show that the eluent contains the target substance 2-amino-4- [ hydroxy (methyl) phosphoryl group]L-butyric acid, collecting the eluate containing the target component, and detecting the 2-amino-4- [ hydroxy (methyl) phosphoryl group contained therein at intervals]-the content of L-butyric acid. Washing the ion exchange column with deionized water after the elution is finished, and converting the resin into OH-For the next separation.
Purification
Distilling the eluent under reduced pressure to obtain a yellow viscous substance, adding methanol to dissolve the yellow viscous substance, stirring the yellow viscous substance under ice bath to recrystallize the yellow viscous substance to obtain a white solid, and filtering the white solid to obtain the product 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid.
After 1L of the conversion solution prepared under the reaction condition of the step (1) is separated and purified by the method, 4.85g of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid solid is obtained, the yield of the product is 97.9%, and the optical purity is 99.9%.
Example 5: biotransformation reaction using 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid as substrate
The substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid was dissolved in aqueous ammonia, 0.5g of the wet cell prepared in example 3 was added thereto, and the pH was adjusted to 8.5 with aqueous ammonia to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 100mM, and the reaction system was placed in a 30 ℃ water bath shaker and inverted at 150rpm for 24 hours. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.5%. 1L of the converted solution was prepared under the same reaction conditions and separated and purified by the method in example 4 to obtain 9.3g of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, the yield of which was 93.9% and the optical purity was 99.9%.
Example 6: biotransformation reaction using 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid as substrate
The substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid was dissolved in aqueous ammonia, 0.6g of the wet cell prepared in example 3 was added thereto, and the pH was adjusted to 8.5 with aqueous ammonia to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 200mM, and the reaction system was placed in a 30 ℃ water bath shaker and inverted at 150rpm for 24 hours. 1mL of the conversion solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.2%. 1L of the converted solution was prepared under the same reaction conditions and separated and purified by the method in example 4 to obtain 18.5g of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, the yield of which was 93.4% and the optical purity was 99.9%.
Example 7: biotransformation reaction using 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid as substrate
The substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid was dissolved in aqueous ammonia, 2g of the wet cell prepared in example 3 was added thereto, and the pH was adjusted to 8.5 with aqueous ammonia to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 200mM, and the reaction system was placed in a 30 ℃ water bath shaker and inverted at 150rpm for 24 hours. 1mL of the conversion solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.9%. 1L of the converted solution was prepared under the same reaction conditions and separated and purified by the method in example 4 to obtain 19.4g of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, the yield of which was 97.9% and the optical purity was 99.9%.
Example 8: biotransformation reaction using 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid as substrate
The substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid was dissolved in aqueous ammonia, 0.5g of the wet cell prepared in example 3 was added thereto, and the pH was adjusted to 8.5 with aqueous ammonia to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 50mM, and the reaction system was placed in a 40 ℃ water bath shaker and inverted at 150rpm for 24 hours. 1mL of the conversion solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.7%. 1L of the converted solution was prepared under the same reaction conditions and separated and purified by the method in example 4 to obtain 4.58g of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, the yield of which was 92.5% and the optical purity was 99.9%.
Example 9: biotransformation reaction using 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid as substrate
The substrate 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid was dissolved in aqueous ammonia, 2g of the wet cell prepared in example 3 was added thereto, and the pH was adjusted to 8.5 with aqueous ammonia to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 200mM, and the reaction system was placed in a 50 ℃ water bath shaker and inverted at 150rpm for 24 hours. 1mL of the conversion solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.9%. 1L of the converted solution was prepared under the same reaction conditions and separated and purified by the method in example 4 to obtain 19.6g of 2-amino-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid, the yield of which was 98.9% and the optical purity of which was 99.9%.
Example 10: biotransformation reaction using N-phenylacetyl-DL-alanine as substrate
Adding 6.0g of alanine and 4.6g of NaOH into 30ml of distilled water, fully stirring under an ice bath condition until the solution is colorless and transparent, dropwise adding 5.0g of phenylacetyl chloride, after the dropwise adding is completed, the solution is light yellow, continuing to react for 2 hours under the ice bath condition, stirring at normal temperature for reaction for 5 hours until the solution is colorless and transparent, adding HCl to adjust the pH value to about 2.0, separating out a white solid, and performing suction filtration and drying to obtain 13g of white solid namely the N-phenylacetyl-DL-alanine.
Substrate N-phenylacetyl-DL-alanine was dissolved in aqueous ammonia, 0.6g of wet cell prepared in example 3 was added thereto, pH8.5 was adjusted with aqueous ammonia to prepare 10mL of a reaction system, and the reaction system was stirred in a 30 ℃ water bath shaker at 150rpm for 24 hours with the substrate added to a final concentration of 100 mM. 1mL of the conversion solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.9%. 1L of the conversion solution was prepared under the same reaction conditions, and after separation and purification by the method in example 4 (detection was performed with filter paper impregnated with 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance L-alanine), the product L-alanine was obtained in an amount of 4.2g, with a yield of 94.4% and an optical purity of 99.9%.
Example 11: biotransformation reaction using N-phenylacetyl-DL-threonine as substrate
Adding 6.0g of threonine and 4.6g of NaOH into 30ml of distilled water, fully stirring under an ice bath condition until the solution is colorless and transparent, dropwise adding 5.0g of phenylacetyl chloride, after dropwise adding is completed, keeping the solution in light yellow, reacting for 2 hours under the ice bath condition, stirring at normal temperature for reaction for 5 hours until the solution is colorless and transparent, adding HCl to adjust the pH value to about 2.0, separating out a white solid, and performing suction filtration and drying to obtain the white solid, namely 10.35g of N-phenylacetyl-DL-threonine.
Substrate N-phenylacetyl-DL-threonine was dissolved in aqueous ammonia, 3g of the wet cell prepared in example 3 was added thereto, pH8.5 was adjusted with aqueous ammonia to prepare 100mL of a reaction system, and the reaction system was stirred in a 30 ℃ water bath shaker at 150rpm for 24 hours with the substrate added to a final concentration of 100 mM. 1mL of the conversion solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.7%. 1L of the conversion solution was prepared under the same reaction conditions, and after separation and purification by the method in example 4 (detection was carried out with a filter paper impregnated with a 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance L-threonine), the product L-threonine was obtained in an amount of 5.58g, with a yield of 93.7% and an optical purity of 99.9%.
Example 12: biotransformation reaction using N-phenylacetyl-DL-tyrosine as substrate
3.619g of DL-tyrosine and 1.8g of NaOH are added into 25ml of distilled water, the mixture is fully stirred under the ice bath condition until the solution is transparent, 2.875g of phenylacetyl chloride is dropwise added, the solution is milky after the dropwise addition, the ice bath condition is continued for reaction for 2h, the mixture is stirred at normal temperature for reaction for 5h, HCl is added for regulating the pH value to about 2.0, a large amount of white solid is separated out, and the white solid is 5.5g of N-phenylacetyl-DL-tyrosine after suction filtration and drying.
Substrate N-phenylacetyl-DL-tyrosine was dissolved in ammonia water, 0.3g of wet cell prepared in example 3 was added thereto, pH8.5 was adjusted with ammonia water to prepare a 10mL reaction system, and the reaction system was stirred in a 30 ℃ water bath shaker at 150rpm for 24 hours with the substrate added to a final concentration of 100 mM. 1mL of the conversion solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.8%. After 1L of the conversion solution was prepared under the same reaction conditions and separated and purified by the method in example 4 (detection was carried out using filter paper impregnated with 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance L-tyrosine), the product L-tyrosine was obtained in an amount of 8.82g, with a yield of 97.4% and an optical purity of 99.9%.
Example 13: biotransformation reaction using N-phenylacetyl-DL-phenylalanine as substrate
Adding 3.3g of DL-phenylalanine and 1.8g of NaOH into 25ml of distilled water, fully stirring under an ice bath condition, thoroughly brightening the solution, dropwise adding 2.875g of phenylacetyl chloride, reacting for 2 hours under the ice bath condition after dropwise adding is finished, adding HCl to adjust the pH value to about 4.0 after stirring and reacting for 5 hours at normal temperature, separating out a large amount of white solid, and performing suction filtration and drying to obtain 5.2g of white solid, namely N-phenylacetyl-DL-phenylalanine.
Dissolving a substrate N-phenylacetyl-DL-phenylalanine in ammonia water, adding 0.4g of the wet thallus prepared by the method in example 3, adjusting the pH to 8.5 with ammonia water to form a 10mL reaction system, adding the substrate to the reaction system to a final concentration of 100mM, placing the reaction system in a water bath shaker at 30 ℃, converting the substrate for 24h at 150rpm, taking 1mL of conversion solution to an EP tube, centrifuging, taking the supernatant, and performing HPLC analysis according to the method in example 1 to detect that the conversion rate is 49.0%. 1L of the conversion solution was prepared under the same reaction conditions, and after separation and purification by the method in example 4 (detection was carried out with filter paper impregnated with 0.2% ninhydrin solution, and the purple-black color of the filter paper showed that the eluate contained the target substance L-phenylalanine), the product L-phenylalanine 7.92g was obtained, the product yield was 96%, and the optical purity was 99.9%.
Example 14: biotransformation reaction using N-phenylacetyl-DL-leucine as substrate
Adding 3.92g of leucine and 3.2g of NaOH into 30ml of distilled water, fully stirring under an ice bath condition until the solution is bright, dropwise adding 6.2g of phenylacetyl chloride, reacting for 2 hours under the ice bath condition continuously, stirring at normal temperature for 5 hours, adding HCl to adjust the pH value to about 2.0, precipitating a large amount of white solid, and performing suction filtration and drying to obtain 7.43g of white solid, namely N-phenylacetyl-DL-leucine.
Substrate N-phenylacetyl-DL-leucine was dissolved in ammonia water, 0.6g of wet cell prepared in example 3 was added thereto, pH8.5 was adjusted with ammonia water to prepare 10mL of a reaction system, and the reaction system was stirred in a 30 ℃ water bath shaker at 150rpm for 24 hours with the substrate added to a final concentration of 100 mM. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.5%. After 1L of the conversion solution was prepared under the same reaction conditions and separated and purified by the method in example 4 (detection was performed with filter paper impregnated with 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance), the product L-leucine (6.2 g) was obtained, the yield of the product was 94.6%, and the optical purity was 99.9%.
Example 15: biotransformation reaction using N-phenylacetyl-DL-isoleucine as substrate
Adding 7.84g of isoleucine and 6.4g of NaOH into 60ml of distilled water, fully stirring under an ice bath condition, dripping 5.78g of phenylacetyl chloride into the solution to obtain a light yellow solution, continuing to react for 2 hours under the ice bath condition, stirring at normal temperature for 5 hours, adding HCl to adjust the pH value to about 2.0, enabling the solution to become colorless and transparent, separating out a small amount of white solid, and performing suction filtration and drying to obtain 7.28g of white solid, namely N-phenylacetyl-DL-isoleucine.
Substrate N-phenylacetyl-DL-isoleucine was dissolved in ammonia, 0.5g of wet cell prepared in example 3 was added thereto, pH8.5 was adjusted with ammonia to prepare a 10mL reaction system, and the reaction system was stirred in a 30 ℃ water bath with a final concentration of 100mM and then transferred at 150rpm for 24 hours. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.5%. After 1L of the conversion solution was prepared under the same reaction conditions and separated and purified by the method in example 4 (detection was carried out using filter paper impregnated with 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance), the product L-isoleucine was obtained in an amount of 6.3g, the yield of the product was 96.2%, and the optical purity was 99.9%.
Example 16: biotransformation reaction using N-phenylacetyl-DL-serine as substrate
Adding 6.0g of serine and 4.6g of NaOH into 20ml of distilled water, fully stirring under an ice bath condition until the solution is colorless and transparent, dropwise adding 5.2g of phenylacetyl chloride, after dropwise adding is completed, keeping the solution in light yellow, continuing to react for 2 hours under the ice bath condition, stirring at normal temperature for 5 hours until the solution is colorless and transparent, adding HCl to adjust the pH value to about 2.0, separating out a white solid, and performing suction filtration and drying to obtain 11g of white solid, namely N-phenylacetyl-DL-serine.
Dissolving a substrate N-phenylacetyl-DL-serine in ammonia water, adding 0.5g of wet thallus prepared in the method of example 3, adjusting the pH of the solution to 8.5 with the ammonia water to form 10mL of a reaction system, adding the substrate to the reaction system to a final concentration of 100mM,
the mixture was placed on a 30 ℃ water bath shaker and transformed at 150rpm for 24 h. 1mL of the transformation solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.5%. After 1L of the conversion solution was prepared under the same reaction conditions and separated and purified by the method in example 4 (detection was carried out using filter paper impregnated with 0.2% ninhydrin solution, and the purplish black color of the filter paper showed that the eluate contained the target substance L-serine), 4.85g of the product L-serine was obtained, the yield of the product was 92.3%, and the optical purity was 99.9%.
Example 17: biotransformation reaction using N-phenylacetyl-DL-methionine as substrate
Adding 8.94g of methionine and 7.2g of NaOH into 120ml of distilled water, fully stirring under an ice bath condition until the solution is transparent, dropwise adding 10.3g of phenylacetyl chloride, after dropwise adding is completed, the solution is transparent, continuing to react for 2 hours under the ice bath condition, stirring at normal temperature for 5 hours, adding HCl to adjust the pH value to about 2.0, separating out a large amount of white solid, and performing reduced pressure suction filtration and drying to obtain 14.8g of N-phenylacetyl-DL-methionine.
Substrate N-phenylacetyl-DL-methionine was dissolved in ammonia, 0.45g of wet cell prepared in example 3 was added thereto, and the pH of the solution was adjusted to 8.5 with ammonia to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 100mM, and the reaction system was placed in a 30 ℃ water bath shaker and inverted at 150rpm for 24 hours. 1mL of the conversion solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.2%. 1L of the conversion solution was prepared under the same reaction conditions, and after separation and purification by the method in example 4 (detection was carried out using a filter paper impregnated with a 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance L-methionine), the product L-methionine was obtained in an amount of 6.92g, with a yield of 92.9% and an optical purity of 99.9%.
Example 18: biotransformation reaction using N-phenylacetyl-DL-valine as substrate
9.36g of valine and 7.2g of NaOH are added into 60ml of distilled water, the mixture is fully stirred under the ice bath condition until the solution is transparent, 14.24g of phenylacetyl chloride is dropwise added, the solution is yellowish and slightly turbid after the dropwise addition, the mixture is continuously reacted for 2 hours under the ice bath condition, the solution is colorless and transparent after being stirred at normal temperature for 5 hours, HCl is added to adjust the pH value to about 2.0, a large amount of white solid is separated out, and the white solid, namely 17.3g of N-phenylacetyl-DL-valine, is obtained after suction filtration and drying.
Substrate N-phenylacetyl-DL-valine was dissolved in aqueous ammonia, 0.2g of the wet cell prepared in example 3 was added thereto, and the pH of the solution was adjusted to 8.5 with aqueous ammonia to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 100mM, and the reaction system was placed in a 30 ℃ water bath shaker and inverted at 150rpm for 24 hours. 1mL of the conversion solution was transferred to an EP tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.3%. 1L of the converted solution was prepared under the same reaction conditions, and after separation and purification by the method in example 4 (detection was carried out using a filter paper impregnated with a 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance L-valine), the product L-alanine was obtained in an amount of 5.28g, and the product yield was 90.2% and the optical purity was 99.9%.
Example 19: biotransformation reaction using N-phenylacetyl-DL-glutamic acid as substrate
Adding 5g of glutamic acid and 4.6g of NaOH into 30ml of distilled water, fully stirring under an ice bath condition until the solution is transparent, dripping 5.735g of phenylacetyl chloride, after the dripping is finished, the solution is transparent, continuing to react for 2 hours under the ice bath condition, stirring at normal temperature for reaction for 5 hours, adding HCl to adjust the pH value to about 2.0, enabling the solution to become turbid, refrigerating overnight at 4 ℃ in a refrigerator, precipitating a large amount of white solid, and performing reduced pressure suction filtration and drying to obtain 8.1g of N-phenylacetyl-DL-glutamic acid.
Substrate N-phenylacetyl-DL-glutamic acid was dissolved in ammonia water, 0.4g of wet cell prepared in example 3 was added thereto, and the pH of the solution was adjusted to 8.5 with ammonia water to prepare 10mL of a reaction system, wherein the substrate was added to a final concentration of 100mM, and the reaction system was placed in a 30 ℃ water bath shaker and inverted at 150rpm for 24 hours. 1mL of the transformation solution was added to an ep tube, centrifuged, and the supernatant was analyzed by HPLC as described in example 1 to determine a conversion of 49.4%. After 1L of the transformant was prepared under the same reaction conditions and separated and purified by the method in example 4 (detection was carried out using filter paper impregnated with 0.2% ninhydrin solution, and the purplish black color of the filter paper indicated that the eluate contained the target substance L-glutamic acid), the product L-glutamic acid (6.93 g) was obtained, the yield of the product was 94.2%, and the optical purity was 99.9%.
Sequence listing
<110> Zhejiang industrial university
<120> Enterobacter aerogenes ZJB-17003 and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>1453
<212>DNA
<213> Enterobacter aerogenes (Enterobacter aerogenesa)
<400>1
tgaatccaaa gtggtaagcg ccctcccgaa ggttaagcta cctacttctt ttgcaaccca 60
ctcccatggt gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gtagcattct 120
gatctacgat tactagcgat tccgacttca tggagtcgag ttgcagactc caatccggac 180
tacgacatac tttatgaggt ccgcttgctc tcgcgaggtc gcttctcttt gtatatgcca 240
ttgtagcacg tgtgtagccc tactcgtaag ggccatgatg acttgacgtc atccccacct 300
tcctccagtt tatcactggc agtctccttt gagttcccga ccgaatcgct ggcaacaaag 360
gataagggtt gcgctcgttg cgggacttaa cccaacattt cacaacacga gctgacgaca 420
gccatgcagc acctgtctca gagttcccga aggcaccaaa gcatctctgc taagttctct 480
ggatgtcaag agtaggtaag gttcttcgcg ttgcatcgaa ttaaaccaca tgctccaccg 540
cttgtgcggg cccccgtcaa ttcatttgag ttttaacctt gcggccgtac tccccaggcg 600
gtcgacttaa cgcgttagct ccggaagcca ctcctcaagg gaacaacctc caagtcgaca 660
tcgtttacag cgtggactac cagggtatct aatcctgttt gctccccacg ctttcgcacc 720
tgagcgtcag tctttgtcca gggggccgcc ttcgccaccg gtattcctcc agatctctac 780
gcatttcacc gctacacctg gaattctacc cccctctaca agactctagc ctgccagttt 840
cgaatgcagt tcccaggttg agcccgggga tttcacatcc gacttgacag accgcctgcg 900
tgcgctttac gcccagtaat tccgattaac gcttgcaccc tccgtattac cgcggctgct 960
ggcacggagt tagccggtgc ttcttctgcg agtaacgtca atcgctaagg ttattaacct 1020
taacgccttc ctcctcgctg aaagtacttt acaacccgaa ggccttcttc atacacgcgg 1080
catggctgca tcaggcttgc gcccattgtg caatattccc cactgctgcc tcccgtagga 1140
gtctggaccg tgtctcagtt ccagtgtggc tggtcatcct ctcagaccag ctagggatcg 1200
tcgcctaggt gagccattac cccacctact agctaatccc atctgggcac atctgatggc 1260
atgaggcccg aaggtccccc actttggtct tgcgacgtta tgcggtatta gctaccgttt 1320
ccagtagtta tccccctcca tcaggcagtt tcccagacat tactcacccg tccgccgctc 1380
gtcacccgag agcaagctct ctgtgctacc gctcgacttg catgtgttat gcctgccgcc 1440
agcgttcatc tga 1453
Claims (10)
1. Enterobacter aerogenes (Enterobacter aerogene) ZJB-17003 preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2017599, date of deposit 2017, 10 month 23 day, address: university of Wuhan, China 430072.
2. Use of enterobacter aerogenes ZJB-17003 of claim 1 for catalyzing 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid to prepare 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -L-butyric acid.
3. The method as claimed in claim 2, wherein the method comprises the steps of using wet thalli obtained by fermentation culture of enterobacter aerogenes ZJB-17003 as a catalyst, using 2-N-phenylacetyl-4- [ hydroxy (methyl) phosphoryl ] -DL-butyric acid as a substrate, using a buffer solution as a reaction medium to form a reaction system with a pH value of 8.5, carrying out conversion reaction at 25-55 ℃ and 200rpm under 100-.
4. The use according to claim 3, wherein in the reaction system, the amount of the catalyst is 10 to 200g/L based on the weight of wet cells, and the final concentration of the substrate is 10 to 500mM when initially added.
5. The use of claim 3, wherein the reaction solution is separated and purified by the following steps: after the reaction is finished, extracting the reaction liquid by using dichloromethane, adjusting the pH value of a water layer to 1.0-5.0, loading the water layer to perform ion exchange chromatography at the speed of 1-6.0Bv/h, washing the water layer by using deionized water, eluting the water layer by using 0.2-4.5M ammonia water at the speed of 0.5-3.0Bv/h, collecting eluent containing the target component, distilling the eluent under reduced pressure to paste, dissolving the paste by using methanol for recrystallization, taking crystals and drying the crystals to obtain the 2-amino-4- [ hydroxyl (methyl) phosphoryl ] -L-butyric acid.
6. Use of enterobacter aerogenes ZJB-17003 of claim 1 for catalyzing N-phenylacetyl-DL-amino acid to prepare N-phenylacetyl-L-amino acid.
7. The method as claimed in claim 6, wherein the method comprises using wet cells obtained by fermentation and culture of Enterobacter aerogenes ZJB-17003 as catalyst, using N-phenylacetyl-DL-amino acid as substrate, using buffer solution as reaction medium to form a conversion system with pH8.5, performing conversion reaction at 25-55 deg.C and 100-200rpm, and separating and purifying the reaction solution after the reaction is finished to obtain N-phenylacetyl-L-amino acid.
8. The use according to claim 7, wherein in the conversion system, the amount of catalyst is 20-300g/L based on the weight of wet cells, and the final concentration of the substrate is 50-500mM when initially added.
9. Use according to claim 7, characterized in that the N-phenylacetyl-DL-amino acid is one of the following: N-phenylacetyl-DL-alanine, N-phenylacetyl-DL-serine, N-phenylacetyl-DL-glutamic acid, N-phenylacetyl-DL-tyrosine, N-phenylacetyl-DL-threonine, N-phenylacetyl-DL-valine, N-phenylacetyl-DL-aspartic acid, N-phenylacetyl-DL-methionine, N-phenylacetyl-DL-leucine, N-phenylacetyl-DL-isoleucine, N-phenylacetyl-DL-phenylalanine.
10. The use according to claim 7, wherein the catalyst is prepared by the following method:
(1) slant culture: inoculating Enterobacter aerogenes ZJB-17003 to a slant culture medium, and culturing at 30 ℃ for 48h to obtain slant thallus; the final concentration of the slant culture medium is as follows: 1-15 g/L of mannitol, 1-15 g/L of sodium glutamate, 0.5-5 g/L of yeast extract and K2HPO40～1.5g/L，KH2PO40～1.5g/L.，MgSO40 to 1.0g/L, 0.2 to 1.8g/L of caprolactam, 20.0g/L of agar, and deionized water as a solvent，pH 7.0～7.5；
(2) Seed culture: selecting one strain of the thallus on the inclined plane, inoculating the strain to a seed culture medium, and culturing at 30 ℃ for 24 hours to obtain a seed solution; the final concentration of the seed culture medium is as follows: 1-15 g/L of mannitol, 1-15 g/L of sodium glutamate, 0.5-5 g/L of yeast extract and K2HPO40～1.5g/L，KH2PO40～1.5g/L.，MgSO40-1.0 g/L of caprolactam, 0.2-1.8 g/L of caprolactam, deionized water as a solvent, and 7.0-7.5 of pH;
(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 60h, centrifuging at 12000g for 10min, and collecting wet thalli; the final concentration composition of the fermentation medium is as follows: 1-15 g/L of mannitol, 1-15 g/L of sodium glutamate, 0.5-5 g/L of yeast extract and K2HPO40～1.5g/L，KH2PO40～1.5g/L.，MgSO40 to 1.0g/L, 0.2 to 1.8g/L caprolactam, deionized water as a solvent, and pH7.0 to 7.5.
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