CN109321496A - Citrobacter freundii ZJB-17010 and its application - Google Patents

Citrobacter freundii ZJB-17010 and its application Download PDF

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CN109321496A
CN109321496A CN201811153829.XA CN201811153829A CN109321496A CN 109321496 A CN109321496 A CN 109321496A CN 201811153829 A CN201811153829 A CN 201811153829A CN 109321496 A CN109321496 A CN 109321496A
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phenylacetyl
hydroxyl
phosphoryl
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柳志强
郑裕国
康雪梅
张晓健
金利群
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Zhejiang University of Technology ZJUT
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Abstract

The present invention relates to one plant of citrobacter freundii (Citrobacter freundii) ZJB-17010 and its applications; 99% is reached to catalysis N- phenylacetyl-DL- amino acids production l-amino acid enantio-selectivity value, 99% is reached to catalysis 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid preparation 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid enantio-selectivity value.

Description

Citrobacter freundii ZJB-17010 and its application
(1) technical field
The present invention relates to bacterial strain --- the citrobacter freundii (Citrobacter that one plant produces hydroamidase Freundii) ZJB-17010, and its chiral amino acid and catalytic amino acid derivative are prepared in catalysis N- phenylacetyl group amino acid N- phenylacetyl group substituent prepare the application in chiral amino acid derivative.
(2) background technique
L-glufosinate-ammonium chemical name are as follows: 4- [hydroxyl (methyl) phosphono]-L- high lactamine, is a kind of knot of Pidolidone Structure analog can inhibit glutamine synthelase (GS) active, accumulate ammonia in plant, and the accumulation of high concentration ammonia blocks plant Photorespiration, the destruction of chloroplast structure and the vesiculation of matrix, while Amino acid synthesis is obstructed, and leads to damaged membrane And cell death, to kill weeds.It, with broad spectrum activity, is second-biggest-in-the-world genetically modified crops herbicide-tolerant.
L-glufosinate-ammonium synthetic method has chemical synthesis and biological synthesis process at present.Chemical method mainly passes through following 4 kinds of sides The chiral centre of formula building L-glufosinate-ammonium: 1) utilizing chiral auxiliary reagent, and chiral induction constructs chiral centre；2) with native amino Acid is chiral source, and conversion obtains L-glufosinate-ammonium；3) asymmetric catalysis synthesis being catalyzed by chiral catalyst；4) disappear outside chiral resolution Revolve body.But chemical method synthesis L-glufosinate-ammonium process is more, yield is lower, and chiral reagent is with high costs, and the quantity of three wastes generated is big, It handles relatively difficult.Biological rule has stringent stereoselectivity, mild reaction condition, higher yield and is easily isolated The advantages such as purifying, therefore, bioanalysis produce L-glufosinate-ammonium technology and are worth with highly important industrialization development.
Bioanalysis production L-glufosinate-ammonium has protease hydrolytic bialaphos according to substrate specificity difference, passes through phosphodiesterase I, amidase I and glutaminase substep synergistic effect keep optical activity to hydrolyze L-3- acetylaminohydroxyphenylarsonic acid 4- (hydroxymethyl phosphono Base) butyramide, the fractionation bialaphos ethyl ester of alpha -chymotrypsin, deacetylase fractionation N- acetyl-glufosinate-ammonium, amidase Split 2- amino -4- (hydroxymethyl phosphono)-butyramide, ester hydrolase hydrolyzes L-glufosinate-ammonium-N- carboxylic acid anhydrides, nitrile hydratase water Solve glufosinate-ammonium substrate containing nitrile, transaminase-catalyzed 2- carbonyl -4- (hydroxymethyl phosphono) butyric acid etc..
The chemical method of synthesis L-glufosinate-ammonium mainly has at present: 1) chiral adjuvant revulsion, with (S) -2- hydroxyl -3- pinane Ketone is chiral adjuvant preparation, and yield 51%, e.e. value is 79%；Using D-Val methyl esters as chiral adjuvant, -78 are needed DEG C low-temp reaction, yield 51%, e.e. value are 93.5%.2) natural amino acid chiral source method, using L-Glu as chiral source, E.e. value is 99.4%；L-Methionine is chiral source, and total recovery 42.3%, e.e. value is 93.5%, needs to use hypertoxic iodine Methane.3) dissymmetric synthesis, asymmetric catalytic hydrogenation --- catalyst amount is big and third level natural division is expensive；It is asymmetric Strecker reaction --- use hypertoxic cyanide；Asymmetric Michael reaction --- catalyst amount is big, and product yield and E.e. it is worth low.4) racemic modification Split Method, the method highest yield 86%, e.e. value highest 99%, but D- glufosinate-ammonium after fractionation It is unconverted, it wastes raw material.
2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid system is split with hydroamidase selective catalysis Important effective component 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid of standby L-glufosinate-ammonium, to realize industrialized route L-glufosinate-ammonium synthesis provides foundation.
The effect of chiral amino acid and its derivative in pharmaceutical developments is increasing, and current pharmaceutical developments are chiral pure The requirement of degree is higher and higher.Chiral amino acid production process has the techniques such as chemical resolution and Enzymatic Resolution.Wherein Enzymatic Resolution because For its mild condition, high specificity, pollution is small, has a clear superiority compared with chemical resolution.Wherein hydroamidase selective catalysis is torn open N- phenylacetyl group amino acids production chiral amino acid is divided to have broad application prospects.
(3) summary of the invention
The object of the present invention is to provide bacterial strain --- the citrobacter freundiis that one kind can generate hydroamidase (Citrobacter freundii) ZJB-17010, and its in catalysis N- phenylacetyl-DL- amino acid derivativges (2-N- phenylacetyl Base -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid) prepare important effective component l-amino acid derivative (the 2- ammonia of L-glufosinate-ammonium Base -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid) or catalysis N- phenylacetyl-DL- amino acid preparation l-amino acid in application, L-glufosinate-ammonium effective component is produced for chemo-enzymatic process and l-amino acid provides new enzyme source, promotes Green Chemistry catalysis Development.
The technical solution adopted by the present invention is that:
In a first aspect, the present invention provides one plant of production hydroamidase bacterial strain --- citrobacter freundii (Citrobacter Freundii) ZJB-17010, is preserved in China typical culture collection center, and deposit number CCTCC No:M2017604 is protected October 23 2017 date is hidden, address: the Chinese Wuhan Wuhan University, 430072.The bacterial strain is by Zhejiang Polytechnical University's biology Graduate School of Engineering is separated from soil and is obtained, and has the ability for catalyzing and synthesizing L-glufosinate-ammonium through detecting.
Second aspect, the present invention provide a kind of citrobacter freundii ZJB-17010 and split N- in microorganism catalysis Phenylacetyl-DL- amino acid derivativges (preferably 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid) prepare L- grass In the important effective component l-amino acid derivative (preferably 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid) of ammonium phosphine Using the application are as follows: the wet thallus obtained using the fermented culture of citrobacter freundii ZJB-17010 is catalyst, with N- Phenylacetyl-DL- amino acid derivativges (preferably 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid) are substrate, It is the reaction system that reaction medium is constituted that pH value is 8.5 with buffer (preferably ammonium hydroxide), at 25-55 DEG C, 100-200rpm is (preferably 30-40 DEG C, 150rpm) under the conditions of carry out conversion reaction, after reaction, it is derivative that reaction solution is isolated and purified to obtain l-amino acid Object (preferably 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid).In the reaction system, catalyst amount is with wet thallus Weight is calculated as 10-200g/L, preferably 50g/L, and the substrate is initially added final concentration of 10-500mM, preferably 100mM.It is described anti- Answer system that can also be made of the fermentation liquid and substrate that the fermented culture of citrobacter freundii ZJB-17010 obtains, pH8.5； Wherein final concentration 10-200g/L of the wet thallus in entire reaction system in fermentation liquid, preferably 50g/L, substrate is in reaction system In final concentration 10-500mM, preferably 100mM.
The method that the reaction solution isolates and purifies are as follows: after reaction, reaction solution is extracted with dichloromethane, by water layer tune PH to 1.0-5.0 (preferably 2.5) carries out ion-exchange chromatography with the speed loading of 1-6.0Bv/h (preferably 4Bv/h), first spends Ion water washing, then eluted with the ammonium hydroxide of 0.2-4.5M (preferably 1M) with 0.5-3.0Bv/h (preferably 2Bv/h), with dip dyeing The filter paper of 0.2% ninhydrin solution detects eluent, and filter paper purpling shows that eluent contains 2- amino -4- [hydroxyl (methyl) Phosphoryl]-L- butyric acid, the eluent containing target components is collected, vacuum distillation takes crystal with methanol dissolving-recrystallization to paste It is dry, obtain l-amino acid derivative (preferably 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid).
The third aspect, the present invention also provides a kind of citrobacter freundii ZJB-17010 in catalysis N- phenylacetyl- DL- amino acid prepares the application of l-amino acid, and the application is obtained with the fermented culture of citrobacter freundii ZJB-17010 Wet thallus be catalyst, using N- phenylacetyl-DL- amino acid as substrate, with buffer (preferably ammonium hydroxide) be reaction medium constituted The transformation system of pH8.5 carries out conversion reaction under the conditions of 25-55 DEG C, 100-200rpm (preferably 30-40 DEG C, 150rpm), instead After answering, reaction solution is isolated and purified, and obtains l-amino acid.In the transformation system, catalyst amount is with wet thallus weight It is calculated as 20-300g/L (preferably 20-60g/L), the substrate is initially added final concentration of 50-500mM (preferably 100mM).It is described N- phenylacetyl-DL- amino acid is one of 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-proline, N- phenylacetyl- DL- aspartic acid, N- phenylacetyl-DL- methionine, N- phenylacetyl-DL-leucine, N- phenylacetyl-DL- isoleucine, N- benzene Acetyl-DL-phenylalanine.The method that the reaction solution isolates and purifies are as follows: after reaction, reaction solution methylene chloride is extracted It takes, by water layer tune pH to 1.0-5.0 (preferably 2.5), ion exchange is carried out with the speed loading of 1-6.0Bv/h (preferably 4Bv/h) Chromatography, is first washed with deionized, then carried out with the ammonium hydroxide of 0.2-4.5M (preferably 1M) with 0.5-3.0Bv/h (preferably 2Bv/h) Elution detects eluent with the filter paper of the ninhydrin solution of dip dyeing 0.2%, and filter paper purpling black shows that eluent contains L- amino Acid, collects the eluent containing target components, and vacuum distillation to paste is taken crystal dry, obtained L- ammonia with methanol dissolving-recrystallization Base acid.
N- phenylacetyl-DL- the amino acid is prepared as follows: amino acid and NaOH are added in distilled water, 4 DEG C of ice baths Under the conditions of to be stirred well to solution colorless and transparent, phenyllacetyl chloride is added dropwise, after being added dropwise to complete, continues 4 DEG C of condition of ice bath reaction 2h, then Room temperature (25-30 DEG C) is stirred to react 5h to solution in colorless and transparent, and HCl is added to adjust pH to 1.5-5.5 (preferably 2-4), is precipitated white Color solid filters drying, obtains, N- phenylacetyl-DL- amino acid；The amino acid and NaOH molar ratio be 1:1~7 (preferably 1: 1.5-2.5)；The amino acid and phenyllacetyl chloride molar ratio are 1:0.1~2 (preferably 1:0.5-1.0), and the distilled water volume is used Amount is calculated as 3-10ml/g with amino acid masses；The amino acid is one of following: alanine, serine, glutamic acid, tyrosine, Threonine, valine, aspartic acid, methionine, leucine, isoleucine, phenylalanine.
The wet thallus that the fermented culture of citrobacter freundii ZJB-17010 of the present invention obtains is made as follows It is standby:
(1) inclined-plane culture:
Citrobacter freundii ZJB-17010 is seeded to slant medium, in 30 DEG C of culture 48h, obtains inclined-plane thalline； The slant medium is final concentration of: 1~15g/L of mannitol, 1~15g/L of sodium glutamate, yeast extract 0.5~5g/L, K2HPO4 0~1.5g/L, KH2PO40~1.5g/L., MgSO40~1.0g/L, 0.2~1.8g/L of caprolactam, agar 20.0g/L, Solvent is deionized water, pH 7.0~7.5；It is preferred that slant medium final concentration forms are as follows: mannitol 10g/L, sodium glutamate 7g/ L, yeast extract 3g/L, K2HPO40.75g/L, KH2PO40.75g/L, MgSO40.5g/L, caprolactam 1g/L, agar are 20.0g/L, solvent are deionized water, pH 7.0~7.5.
(2) seed culture:
It is seeded to seed culture medium from one oese thallus of inclined-plane thalline picking, is cultivated at 30 DEG C for 24 hours, obtains seed liquor； The seed culture medium final concentration composition are as follows: 1~15g/L of mannitol, 1~15g/L of sodium glutamate, 0.5~5g/L of yeast extract, K2HPO40~1.5g/L, KH2PO40~1.5g/L., MgSO40~1.0g/L, 0.2~1.8g/L of caprolactam, solvent are to go Ionized water, pH 7.0~7.5；Preferred seed culture medium final concentration composition are as follows: mannitol 10g/L, sodium glutamate 7g/L, yeast extract 3g/L, K2HPO40.75g/L, KH2PO40.75g/L, MgSO40.5g/L, caprolactam 1g/L, solvent are deionized water, pH 7.0~7.5.
(3) fermented and cultured:
Seed liquor is seeded in fermentation medium with the inoculum concentration of volumetric concentration 1~10% (preferably 1%), 30 DEG C, After 150rpm shaken cultivation 60h, it is centrifuged 10min at 12000g, collects wet thallus；The fermentation medium final concentration composition Are as follows: 1~15g/L of mannitol, 1~15g/L of sodium glutamate, yeast extract 0.5~5g/L, K2HPO40~1.5g/L, KH2PO40~ 1.5g/L., MgSO40~1.0g/L, 0.2~1.8g/L of caprolactam, solvent are deionized water, pH 7.0~7.5；It is preferred that sending out Ferment culture medium final concentration composition are as follows: mannitol 10g/L, sodium glutamate 7g/L, yeast extract 3g/L, K2HPO40.75g/L, KH2PO4 0.75g/L, MgSO40.5g/L, caprolactam 1g/L, solvent are deionized water, pH 7.0~7.5.
The buffer that pH value of the present invention is 8.5 is constituted are as follows: citrate buffer solution (100mM, pH 4.0-6.0), phosphoric acid Buffer (100mM, pH 6.0-8.0), Tris-HCl (100mM, pH 7.0-9.0), borate buffer (100mM, pH 9.0- 10.5), ammonium hydroxide buffer (substrate solution pH is adjusted as 7.0-10.0 with ammonium hydroxide), sieve Er Shi buffer (100mM, pH 10.5- 12.0)；Preferred buffer composition are as follows: pH value of solution is adjusted as 8.5 with ammonium hydroxide.
The beneficial effects are mainly reflected as follows: the present invention provides the bacterial strains that one plant can generate hydroamidase -- Citrobacter freundii (Citrobacter freundii) ZJB-17010, the present invention also provides utilize Freund citric acid bar The fermented culture of bacterium (Citrobacter freundii) ZJB-17010 obtains wet thallus and is catalyzed 2-N- benzene as biocatalyst Acetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid prepares the important effective component 2- amino -4- [hydroxyl of L-glufosinate-ammonium (methyl) phosphoryl]-L- butyric acid method, 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl] butyric acid conversion ratio is reached 49.0%, product L-glufosinate-ammonium reaches e.e.%=99.9%, and the present invention also provides utilize citrobacter freundii ZJB- 17010 fermented cultures obtain wet thallus as the side of biocatalyst catalysis N- phenylacetyl-DL- amino acid preparation l-amino acid Method, conversion ratio can reach 49%, and product chiral amino acid can reach e.e.%=99-99.9%, split preparation process green Efficiently, bacterial strain is easy to cultivate collection application, and catalytic reaction condition is mild, with important application prospects.
(4) Detailed description of the invention:
Fig. 1 is the concentration standard curve of OPA/NAC- glufosinate-ammonium under high-throughput screening method.
Fig. 2 be pre-column derivatization reversed-phase high performance liquid chromatography under 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid and The liquid phase result map of 2- amino -4- [hydroxyl (methyl) phosphoryl]-D- butyric acid.
Fig. 3 is the Electronic Speculum for screening obtained strains citrobacter freundii (Citrobacter freundii) ZJB-17010 Observe result.
(5) specific embodiment:
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in This:
Ultrapure water of the present invention is UP water, refers to that resistivity reaches the water of 18M Ω * cm (25 DEG C).Ammonia of the present invention Water is the aqueous solution containing ammonia 25%~28%.Room temperature of the present invention is 25-30 DEG C.Ice bath described in the embodiment of the present invention is 4 ℃.2% ninhydrin solution preparation method of the invention are as follows: 2g ninhydrin and 0.08g stannous chloride are dissolved in 100mL ultrapure water, Then agitation and filtration takes filtrate to be kept in dark place.
Embodiment 1: the screening of citrobacter freundii (Citrobacter freundii) ZJB-17010
1, primary dcreening operation
The present invention is total to 80 parts of soil sampling from soil sampling in all parts of the country.The specific method of screening: it weighs 1g soil sample and is placed in 10mL In 0.85% physiological saline, is stood after rocking, take supernatant into enriched medium, in 30 DEG C, 150r/min shaken cultivation 2-3 days.It takes 1mL pregnant solution to be added in the fresh enriched medium of 50mL, is isolated and purified after being so repeated 3 times.
Bromothymol blue filter paper is selected to detect the bacterium colony for the substrate that can degrade as instruction filter paper.Its principle are as follows: contain mesh Enzyme bacterium colony using degradation substrate after, acidic by-products (phenylacetic acid, acetic acid, formic acid, benzoic acid) can be generated, to make to indicate The pH value of filter paper part changes.Indicator Bromothymol blue color change interval is (yellow-blue) 5.8-7.6, therefore can be utilized Yellow is presented in the periphery of bacterial colonies of chlorinated organics, and unavailable, and periphery of bacterial colonies plate color is still dark green.
Pregnant solution is diluted into 10 gradients with sterile 0.85% physiological saline step by step, chooses 10-4、10-5、10-6、10-7、10-8、10-9、10-10The dilution of five gradients respectively takes 0.1mL to be uniformly coated on plate screening culture medium, 30 DEG C of constant temperature incubations 48h.The aseptic filter paper for soaking the bromthymol blue containing 10% (instruction filter paper) is laid on plate, picking indicates on filter paper The corresponding colony inoculation of yellow is shown as into 96 orifice plates containing growth medium, is put into 30 DEG C, 180rpm shaking table constant temperature During which culture detects OD600Light absorption value, draw growth curve, when strain growth arrive logarithmic phase when, transfer 300 microlitres it is fresh Fermentation medium, be inoculated with 96 orifice plates after remaining bacterium solution as strain sterile preservation in 4 DEG C of refrigerators.Contain fermented and cultured after switching 96 orifice plate of base sufficiently grows it and producing enzyme in 30 DEG C, 180rpm shaking table constant temperature incubation 48h, obtains bacteria suspension.
2, OPA/NAC high flux screening
Substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid is dissolved in ammonium hydroxide, 1ml step 1 is added The bacteria suspension of preparation adjusts solution ph to 8.5 with ammonium hydroxide, constitutes 10mL reaction system, make Final substrate concentrations 50mM, in 30 DEG C, the isothermal reaction of 180rpm shaking table for 24 hours.For gained conversion fluid respectively with corresponding 10 times of the dilution of ultrapure water, 4 DEG C of ice baths use OPA/NAC High-throughput screening method is detected, and the higher bacterial strain of relative fluorescence is filtered out.
OPA/NAC high-throughput screening method: the 40 μ l of ice bath reaction solution after dilution is drawn to 96 hole fluorescence detections with the volley of rifle fire Plate, the A liquid after ice bath is added, vibrates 30s in microplate reader, adds 100 μ l ultrapure waters, 30s is vibrated again, in λex=340nm； λem=455nm measures fluorescence intensity level, gained fluorescence intensity level and 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid mark Directrix curve, which compares, can be obtained the concentration of contained 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid in sample, then can be with The yield for calculating 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid, obtains hydroamidase biocatalytic resolution 2-N- Phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid prepares the important effective component 2- amino -4- [hydroxyl of L-glufosinate-ammonium (methyl) phosphoryl]-L- butyric acid conversion ratio.
The A liquid: N-acetyl-L-cysteine (0.448g) and o-phthalaldehyde (0.185g) are dissolved in the anhydrous second of 10mL Borate buffer (140mM, pH 9.5) is added under condition of ice bath and is settled to 50mL, saves, can be reserved for 3 days under condition of ice bath for alcohol. N-acetyl-L-cysteine final concentration 0.313mol/L in the A liquid, o-phthalaldehyde final concentration 0.138mol/L.
2- amino -4- [hydroxyl (methyl) the phosphoryl]-L- butyric acid standard curve making method: it is prepared with ultrapure water 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid standard solution of 5g/L, is diluted to 3 concentration gradients: 0.01~ 0.1g/L (0.01,0.02,0.04,0.06,0.08,0.1g/L), 0.1~1.0g/L (0.1,0.2,0.4,0.6,0.8,1.0g/ L), (1.0,2.0,3.0,4.0,5.0g/L) 1.0~5.0g/L, the standard solution 40 of various concentration gradient is drawn with the volley of rifle fire respectively μ l is separately added into the A liquid that the prepared ice bath of 40 μ l saves, 30s is vibrated in microplate reader to 96 hole porous fluorescent detection plates, then 100 μ l ultrapure waters are separately added into, then vibrate 30s, in λex=340nm；λem=455nm measures fluorescence intensity level, with gained Fluorescence intensity level is ordinate, and corresponding mark product concentration is abscissa, makes standard curve.The standard of 3 concentration ranges is made altogether Curve, it is known that linear good (such as Fig. 1), R when sample concentration is 0.01~0.1g/L2=0.9978, show that this method has very High accuracy and applicability.
3, high performance liquid chromatography secondary screening
Step 2 is filtered out into strain inoculated to slant medium, after 30 DEG C of culture 48h, is saved as 4 DEG C of refrigerators.
By the strain inoculated being stored on inclined-plane into seed culture medium, cultivated for 24 hours at 30 DEG C.Seed liquor is dense with volume The inoculum concentration of degree 1% is seeded in fermentation medium, and 30 DEG C, 150rpm shaken cultivation 60h.It is centrifuged 5min at 12000g, receives Collect wet thallus.
Wet thallus is taken, substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid and ammonium hydroxide is added, uses ammonia Water adjusts pH value of solution 8.5, constitutes 10mL reaction system, wet thallus final concentration 50g/L, Final substrate concentrations 50mM are placed in 30 DEG C of water Bathe shaking table conversion for 24 hours.1mL conversion fluid is taken, supernatant is taken to carry out HPLC analysis after centrifuge separation.Finally obtain one plant of catalysis activity Higher wild strain, and this bacterial strain is denoted as ZJB-17010.
High performance liquid chromatography (HPLC) secondary screening method are as follows: by above-mentioned conversion fluid centrifuging and taking supernatant, dilute 10 times with ultrapure water Afterwards, it takes 200 μ l to clean 1.5mL EP to manage, 200 μ l of derivatization reagent is added, after 30 DEG C of reaction 5min, it is ultrapure that 600 μ l are added After water mixes, after (0.22 μm) of syringe filter membrane filtering, it is put into HPLC and is detected, obtain 2- amino -4- [hydroxyl in reaction solution (methyl) phosphoryl]-L- butyric acid and 2- amino -4- [hydroxyl (methyl) phosphoryl]-D- butyric acid concentration.
The derivatization reagent: 0.1g o-phthalaldehyde, 0.12g N-acetyl-L-cysteine, with 10mL dehydrated alcohol After dissolution, 50mL is settled to borate buffer (100mM, pH 9.8).
The HPLC condition: it wears peace U3000 high performance liquid chromatograph and is equipped with fluorescence detector in the U.S.；Wear peace C18Silicone hydroxyl Filled column (250mm × 4.6mm)；Mobile phase is ammonium acetate (50mM pH 4.7) solution containing 10% pure methanol, and column temperature control exists 35 DEG C, fluorescence exciting wavelength λex=350nm, emission wavelength lambdaem=450nm, sample volume are 10 μ l.With this condition, 2- amino- The appearance time of 4- [hydroxyl (methyl) phosphoryl]-L- butyric acid and 2- amino -4- [hydroxyl (methyl) phosphoryl]-D- butyric acid is 7.800min, 9.290min (such as Fig. 2).
Enriched medium composition: 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid 1g/L, glucose 5g/L, sodium chloride 1g/L, K2HPO4·3H2O 0.8g/L, KH2PO43.3g/L, MgSO4·7H2O 0.2g/L, solvent be go from Sub- water, pH 7.
Plate screening culture medium composition: 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid 1g/L, grape Sugared 5g/L, sodium chloride 1g/L, K2HPO4·3H2O 0.8g/L, KH2PO43.3g/L, MgSO4·7H2O 0.2g/L, agar 20g/ L, solvent are deionized water, pH 7.
Growth medium composition: sodium chloride 10g/L, peptone 10g/L, yeast powder 5g/L, solvent are deionized water.Inclined-plane Culture medium, seed culture medium, fermentation medium composition are the same as embodiment 3.
Embodiment 2: bacterial strain ZJB-17010 identification
1, Morphological Identification:
Embodiment 1 is screened obtained bacterial strain ZJB-17010 to be seeded on solid medium, 37 DEG C of shapes after culture 24 hours At round or subcircular, quality is soft, and surface is more smooth, flat, neat in edge, glossiness milky bacterium colony, diameter 2-4mm. Gram's staining observation: pink rod-short, no gemma.Solid medium composition: sodium chloride 10g/L, peptone 10g/L, ferment Female powder 5g/L, agar 20g/L, solvent are deionized water.
2, Physiology and biochemistry is identified:
94 kinds of phenotypes are carried out to bacterial strain ZJB-17010 using Biolog (GEN III) automatic microbe identification systems to test, Including 71 kinds of utilization of carbon source situation detections and 23 kinds of chemosensitivity detections: bacterial strain ZJB-17010 is inoculated in the training of BUG plate Support base (BIOLOG UNIVERSAL GROWTH AGAR), 33 DEG C constant temperature incubation 2 days, the thallus on plate is washed with aseptic cotton carrier Under, it is mixed with inoculation liquid (IF-A), bacteria suspension is made, adjusted with nephelometer to 91%T/IF-A.With 8 hole electric plus liquid devices by bacterium Suspension is added in respectively in each hole of III micropore identification plate of BiologGEN, every 100 μ L of hole.Micropore identification plate is placed on 33 DEG C of cultures In case, respectively culture 12h, for 24 hours, placed it in after 36h, 48h and read result on Biolog readout instrument.
Bacterial strain ZJB-17010 analyzes Metabolic Fingerprinting through Biolog readout instrument, and bacterial strain ZJB-17010 can utilize more by force 65 kinds of carbon Source cannot utilize other 11 kinds of carbon sources or Utilization ability is weaker；Bacterial strain ZJB-17010 is sensitive to 20 kinds of chemical substances. It is as shown in Table 1 and Table 2 that Biolog system provides 48h qualification result.
Utilization ability of the 1. bacterial strain ZJB-17010 of table to 71 kinds of carbon sources on III plate of BiologGEN
Chemosensitivity of the 2. bacterial strain ZJB-17010 of table to 23 kinds of chemical substances on III plate of BiologGEN
3, molecular biology identification:
Using the total DNA of bacterial strain ZJB-17010 as template, using primer P1:5'-AGAGTTTGATCCTGGCTCAG-3' and P2:5'-AAGGAGGTGATCCAGCCGCA-3' expands the 16S rDNA gene of bacterial strain, after gene product is connected with carrier T, It entrusts the raw work in Shanghai to expand and be sequenced the bacterium 16SrDNA, obtains the 16S rDNA sequence of the bacterial strain (shown in SEQ ID NO.1) Afterwards, the 16S rDNA gene order of related strain in GenBank is retrieved with BLAST on the website NCBI, and carries out homology ratio It is right.Microorganism of the present invention and 3246 homology highest of Citrobacter freundii bacterial strain LMG (homology, 99%, Based on 16S ribosomal RNA gene), according to Microbial Genetics identity principle, it is based on 16S rDNA homology Higher than 95%, identification bacterium substantially belongs to control bacterium.Therefore, this experimental identification bacterial strain ZJB-17010 is citrobacter freundii (Citrobacter freundii), it is quasi- to be named as citrobacter freundii (Citrobacter freundii) ZJB-17010, It is preserved in China typical culture collection center, deposit number CCTCC M 2017604, preservation date on October 23rd, 2017, Address: Wuhan, China Wuhan University, 430072.
It is any that the one or more nucleotide of the progress of nucleotide sequence shown in SEQ ID NO.1 in nucleotides sequence list are taken In generation, lacks or is inserted into resulting nucleotide sequence, as long as having 90% or more homology with the sequence, belongs to of the invention Protection scope.
Embodiment 3: the preparation of wet thallus
(1) inclined-plane culture:
Citrobacter freundii (Citrobacter freundii) ZJB-17010 is seeded to slant medium, 30 DEG C culture 48h, obtain inclined-plane thalline；
The slant medium is final concentration of: mannitol 10g/L, sodium glutamate 7g/L, yeast extract 3g/L, K2HPO4 0.75g/L, KH2PO40.75g/L, MgSO40.5g/L, caprolactam 1g/L, agar 20.0g/L, solvent are deionized water, PH 7.0~7.5.
(2) seed culture
It is seeded to seed culture medium from one oese thallus of inclined-plane thalline picking, is cultivated at 30 DEG C for 24 hours, obtains seed liquor；
The seed culture medium final concentration composition are as follows: mannitol 10g/L, sodium glutamate 7g/L, yeast extract 3g/L, K2HPO4 0.75g/L, KH2PO40.75g/L, MgSO40.5g/L, caprolactam 1g/L, solvent are deionized water, pH 7.0~7.5.
(3) fermented and cultured
Seed liquor is seeded in fermentation medium with the inoculum concentration of volumetric concentration 1%, 30 DEG C, 150rpm shaken cultivation After 60h, it is centrifuged 10min at 12000g, collects wet thallus；
The fermentation medium final concentration composition are as follows: mannitol 10g/L, sodium glutamate 7g/L, yeast extract 3g/L, K2HPO4 0.75g/L, KH2PO40.75g/L, MgSO40.5g/L, caprolactam 1g/L, solvent are deionized water, pH 7.0~7.5.
Embodiment 4: anti-as the bioconversion of substrate using 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid It answers
(1) substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid is dissolved in ammonium hydroxide, adds implementation The wet thallus 0.3g of 3 method of example preparation, and pH 8.5 is adjusted with ammonium hydroxide, 10mL reaction system is constituted, Final substrate concentrations are made 50mM, is placed in 30 DEG C of shaking baths, and 150rpm is converted for 24 hours, takes 1mL conversion fluid into ep pipe, taken after centrifuge separation supernatant by 1 method of embodiment carries out HPLC analysis detection.The result shows that ZJB-17010 can make substrate 2-N- phenylacetyl group -4- [hydroxyl (first Base) phosphoryl]-DL- butyric acid converts to obtain product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid, and conversion ratio is 49%, product optical purity is 99.9%.
(2) conversion fluid of the amino of 2- containing product -4- [hydroxyl (methyl) the phosphoryl]-L- butyric acid obtained enzymic catalytic reaction It is separated, extracts product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid.
1) it extracts:
Separatory funnel is added in obtained conversion fluid, while isometric methylene chloride is added, stands 3h after rocking, from point Liquid funnel lower end releases organic layer, pours out aqueous layer from upper end.Obtaining aqueous layer is containing product 2- amino -4- [hydroxyl (first Base) phosphoryl]-L- butyric acid aqueous solution.
2) product separation is carried out with anion exchange resin
1. resin pre-processes
Resin 201 × 7 is impregnated with 50 DEG C of warm water, is expanded resin sufficiently and is removed fine particle (inclination or flotation Method)；It with the NaOH aqueous solution soaking 3h of 1.0M, is washed with deionized water to neutrality, then is used after impregnating 3h with the HCL aqueous solution of 1.0M Deionized water is washed till neutrality, then uses the NaOH aqueous solution soaking 3h of 1.0M, is converted into OH-Form is finally washed with deionized water It is spare to neutrality.
2. filling column
Using wet method dress post (internal diameter 1.5cm, high 40cm), the deionization of 13cm height is first first added in ion exchange column Water, then 30mL wet resin 201 × 7 is taken to be packed into glass, 50mL deionized water is added, slowly stirs, the resin of suspension pours into In ion exchange column, its natural subsidence is allowed, keep it uniform in pillar endocrine, should not have apparent line of demarcation and bubble to generate Deng.
3. loading, elution
The resulting aqueous solution of step 1) is adjusted to pH 2.5, the above column flow rate 4.0Bv/h carries out loading, at regular intervals It takes out efflux progress liquid phase detection to be first washed with deionized when absorption reaches peak, then is carried out with the ammonium hydroxide of 1.0M Elution, elution speed 2.0Bv/h are detected with the filter paper of the ninhydrin solution of dip dyeing 0.2%, and filter paper purpling color table is bright to be washed De- liquid contains target substance 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid, and detect contained therein has at regular intervals 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid content, collect target components eluent.It is spent after elution Ion water washing ion exchange column, and convert the resin to OH-For separating next time.
4. purifying
Eluent is evaporated under reduced pressure to obtain clear yellow viscous substance, and methanol dissolution is added, and stirring is recrystallized to give white under ice bath Solid filters up to product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid.
With the conversion fluid 1L of step (1) reaction condition preparation after the above method isolates and purifies, 2- amino -4- [hydroxyl is obtained Base (methyl) phosphoryl]-L- butyric acid solid 4.7g, efficiency of pcr product 94.9%, optical purity 99.9%.
Embodiment 5: anti-as the bioconversion of substrate using 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid It answers
Substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid is dissolved in ammonium hydroxide, adds embodiment 3 The wet thallus 0.5g of method preparation adjusts pH 8.5 with ammonium hydroxide, constitutes 10mL reaction system, and wherein substrate is added final concentration of 100mM, is placed in 30 DEG C of shaking baths, and 150rpm is converted for 24 hours.Take 1mL conversion fluid into ep pipe, taken after centrifuge separation supernatant by 1 method of embodiment carries out HPLC analysis detection and obtains conversion ratio to be 49.2%.Conversion fluid 1L is prepared by implementation under same reaction condition 4 method of example isolates and purifies, and obtains product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid 9.63g, efficiency of pcr product 97.2%, optical purity 99.9%.
Embodiment 6: anti-as the bioconversion of substrate using 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid It answers
Substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid is dissolved in ammonium hydroxide, adds embodiment 3 The wet thallus 0.6g of method preparation adjusts pH 8.5 with ammonium hydroxide, constitutes 10mL reaction system, and wherein substrate is added final concentration of 200mM, is placed in 30 DEG C of shaking baths, and 150rpm is converted for 24 hours.Take 1mL conversion fluid into ep pipe, taken after centrifuge separation supernatant by 1 method of embodiment carries out HPLC analysis detection and obtains conversion ratio to be 49.1%.Conversion fluid 1L is prepared by implementation under same reaction condition 4 method of example isolates and purifies, and obtains product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid 18.7g, efficiency of pcr product 94.4%, optical purity 99.9%.
Embodiment 7: anti-as the bioconversion of substrate using 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid It answers
Substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid is dissolved in ammonium hydroxide, adds embodiment 3 The wet thallus 2g of method preparation adjusts pH 8.5 with ammonium hydroxide, constitutes 10mL reaction system, and wherein substrate is added final concentration of 200mM, is placed in 30 DEG C of shaking baths, and 150rpm is converted for 24 hours.Taken after taking 1mL conversion fluid to be centrifugated into ep pipe supernatant by 1 method of embodiment carries out HPLC analysis detection and obtains conversion ratio to be 49.7%.Conversion fluid 1L is prepared under same reaction condition, by implementation After 4 method of example isolates and purifies, product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid 19.52g, efficiency of pcr product are obtained 98.5%, optical purity 99.9%.
Embodiment 8: anti-as the bioconversion of substrate using 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid It answers
Substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid is dissolved in ammonium hydroxide, adds embodiment 3 The wet thallus 0.5g of method preparation adjusts pH 8.5 with ammonium hydroxide, constitutes 10mL reaction system, and wherein substrate is added final concentration of 50mM, is placed in 40 DEG C of shaking baths, and 150rpm is converted for 24 hours.Take 1mL conversion fluid into ep pipe, taken after centrifuge separation supernatant by 1 method of embodiment carries out HPLC analysis detection and obtains conversion ratio to be 49.9%.Conversion fluid 1L is prepared under same reaction condition, by implementation After 4 method of example isolates and purifies, product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid 4.9g, efficiency of pcr product are obtained 98.9%, optical purity 99.9%.
Embodiment 9: anti-as the bioconversion of substrate using 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid It answers
Substrate 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid is dissolved in ammonium hydroxide, adds embodiment 3 The wet thallus 2g of method preparation adjusts pH 8.5 with ammonium hydroxide, constitutes 10mL reaction system, and wherein substrate is added final concentration of 200mM, is placed in 50 DEG C of shaking baths, and 150rpm is converted for 24 hours.Take 1mL conversion fluid into ep pipe, taken after centrifuge separation supernatant by 1 method of embodiment carries out HPLC analysis detection and obtains conversion ratio to be 49.6%.Conversion fluid 1L is prepared under same reaction condition, by implementation After 4 method of example isolates and purifies, product 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid 19.2g, efficiency of pcr product are obtained 96.9%, optical purity 99.9%.
Embodiment 10: it is reacted by the bioconversion of substrate of N- phenylacetyl-DL-Alanine
6.0g alanine and 4.6g NaOH are added in 30ml distilled water, and it is colourless to be stirred well to solution under condition of ice bath It is bright, 5.0g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is in faint yellow, continuation condition of ice bath reaction 2h, then stirring at normal temperature reaction 5h in colorless and transparent, adds HCl to adjust pH to 2.0 or so to solution, and white solid is precipitated, and filters drying and obtains white solid and is N- phenylacetyl-DL-Alanine 13g.
Substrate N- phenylacetyl-DL-Alanine is dissolved in ammonium hydroxide, adds the wet thallus 0.6g of 3 method of embodiment preparation, is used Ammonium hydroxide adjusts pH 8.5, constitutes 10mL reaction system, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C of shaking baths, 150rpm is converted for 24 hours.It takes 1mL conversion fluid into ep pipe, supernatant is taken to carry out HPLC analysis by 1 method of embodiment after centrifuge separation Detect conversion ratio be 49.2%.Conversion fluid 1L is prepared under same reaction condition, by 4 method of embodiment (with the indenes of dip dyeing 0.2% The filter paper of triketone solution is detected, and filter paper purpling black shows that eluent contains target substance l-Alanine) isolate and purify after, Obtain product l-Alanine 4.2g, optical purity 99%.
Embodiment 11: it is reacted by the bioconversion of substrate of N- phenylacetyl-DL- threonine
6.0g threonine and 4.6g NaOH are added in 30ml distilled water, and it is colourless to be stirred well to solution under condition of ice bath It is bright, 5.0g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is in faint yellow, continuation condition of ice bath reaction 2h, then stirring at normal temperature reaction 5h in colorless and transparent, adds HCl to adjust pH to 2.0 or so to solution, and white solid is precipitated, and filters drying and obtains white solid and is N- phenylacetyl-DL- threonine 11g.
Substrate N- phenylacetyl-DL- threonine is dissolved in ammonium hydroxide, the wet thallus 3g of 3 method of embodiment preparation is added, uses ammonia Water tune pH 8.5 constitutes 100mL reaction system, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C of shaking baths, 150rpm is converted for 24 hours, takes 1mL conversion fluid into ep pipe, and supernatant is taken to carry out HPLC analysis by 1 method of embodiment after centrifuge separation Detect conversion ratio be 49.4%.Conversion fluid 1L is prepared under same reaction condition, by 4 method of embodiment (with the indenes of dip dyeing 0.2% The filter paper of triketone solution is detected, and filter paper purpling black shows that eluent contains target substance L-threonine) isolate and purify after, Obtain product L-threonine 5.72g, efficiency of pcr product 96.1%, optical purity 99.9%.
Embodiment 12: it is reacted by the bioconversion of substrate of N- phenylacetyl-DL- tyrosine
3.619g DL- tyrosine and 1.8g NaOH are added in 25ml distilled water, are stirred well to solution under condition of ice bath It is bright, 2.875g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is creamy white, and continues condition of ice bath and reacts 2h, then stirring at normal temperature After reacting 5h, HCl is added to adjust pH to 2.0 or so, a large amount of white solids are precipitated, filtering drying and obtaining white solid is N- benzene second Acyl-DL- tyrosinase 15 .0g.
Substrate N- phenylacetyl-DL- tyrosine is dissolved in ammonium hydroxide, adds the wet thallus 0.3g of 3 method of embodiment preparation, is used Ammonium hydroxide adjusts pH 8.5, constitutes 10mL reaction system, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C of shaking baths, 150rpm is converted for 24 hours, takes 1mL conversion fluid into ep pipe, and supernatant is taken to carry out HPLC analysis by 1 method of embodiment after centrifuge separation Detect conversion ratio be 49.2%.Conversion fluid 1L is prepared under same reaction condition, is isolated and purified by 4 method of embodiment (with dip dyeing The filter paper of 0.2% ninhydrin solution is detected, and filter paper purpling black shows that eluent contains target substance l-tyrosine) Afterwards, product l-tyrosine 8.62g, efficiency of pcr product 95.2%, optical purity 99.9% are obtained.
Embodiment 13: it is reacted by the bioconversion of substrate of N- phenylacetyl-DL-phenylalanine
3.3g DL-phenylalanine and 1.8g NaOH are added in 25ml distilled water, are sufficiently stirred under condition of ice bath, solution is saturating It is bright, 2.875g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is creamy white, and continuation condition of ice bath reaction 2h, then stirring at normal temperature are anti- After answering 5h, HCl is added to adjust pH to 4.0 or so, a large amount of white solids are precipitated, filtering drying and obtaining white solid is N- benzene second Acyl-DL-phenylalanine 5.2g.
Substrate N- phenylacetyl-DL-phenylalanine is dissolved in ammonium hydroxide, adds the wet thallus 0.4g of 3 method of embodiment preparation, PH 8.5 is adjusted with ammonium hydroxide, constitutes 10mL reaction system, wherein final concentration of 100mM is added in substrate, 30 DEG C of shaking baths are placed in, 150rpm is converted for 24 hours, takes 1mL conversion fluid into ep pipe, and supernatant is taken to carry out HPLC analysis by 1 method of embodiment after centrifuge separation Detect conversion ratio be 49.0%.Conversion fluid 1L is prepared under same reaction condition, by 4 method of embodiment (with the indenes of dip dyeing 0.2% The filter paper of triketone solution is detected, and filter paper purpling black shows that eluent contains target substance L-phenylalanine) it isolates and purifies Afterwards, product L-phenylalanine 7.94g, efficiency of pcr product 96.2%, optical purity 99.9% are obtained.
Embodiment 14: it is reacted by the bioconversion of substrate of N- phenylacetyl group DL-leucine
3.92g leucine and 3.2g NaOH are added in 30ml distilled water, and it is bright to be stirred well to solution under condition of ice bath, 6.2g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is in faint yellow, continuation condition of ice bath reaction 2h, then stirring at normal temperature reaction 5h Afterwards, add HCl to adjust pH to 2.0 or so, a large amount of white solids are precipitated, filtering drying and obtaining white solid is N- phenylacetyl-DL- Leucine 7.23g.
Substrate N- phenylacetyl-DL-leucine is dissolved in ammonium hydroxide, adds the wet thallus 0.6g of 3 method of embodiment preparation, is used Ammonium hydroxide adjusts pH 8.5, constitutes 10mL reaction system, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C of shaking baths, 150rpm is converted for 24 hours, takes 1mL conversion fluid into ep pipe, and supernatant is taken to carry out HPLC analysis by 1 method of embodiment after centrifuge separation Detect conversion ratio be 49.3%.Conversion fluid 1L is prepared under same reaction condition, by 4 method of embodiment (with the indenes of dip dyeing 0.2% The filter paper of triketone solution is detected, and filter paper purpling black shows that eluent contains target substance L-Leu) isolate and purify after, Obtain product L-Leu 6.02g, efficiency of pcr product 91.9%, optical purity 99.9%.
Embodiment 15: it is reacted by the bioconversion of substrate of N- phenylacetyl-DL- isoleucine
7.84g isoleucine and 6.4g NaOH are added in 60ml distilled water, are sufficiently stirred under condition of ice bath, and solution is bright, 5.78g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is in faint yellow, continuation condition of ice bath reaction 2h, then stirring at normal temperature reaction 5h Afterwards, HCl is added to adjust PH to 2.0 or so, solution becomes colorless transparent, is precipitated a small amount of white solid, and filtering drying, to obtain white solid Body is N- phenylacetyl-DL- isoleucine 14.92g.
Substrate N- phenylacetyl-DL- isoleucine is dissolved in ammonium hydroxide, adds the wet thallus 0.5g of 3 method of embodiment preparation, PH 8.5 is adjusted with ammonium hydroxide, constitutes 10mL reaction system, wherein final concentration of 100mM is added in substrate, 30 DEG C of shaking baths are placed in, 150rpm is converted for 24 hours, takes 1mL conversion fluid into ep pipe, and supernatant is taken to carry out HPLC analysis by 1 method of embodiment after centrifuge separation Detect conversion ratio be 49.5%.Conversion fluid 1L is prepared under same reaction condition, by 4 method of embodiment (with the indenes of dip dyeing 0.2% The filter paper of triketone solution is detected, and filter paper purpling black shows that eluent contains target substance l-Isoleucine) it isolates and purifies Afterwards, product l-Isoleucine 6.13g, efficiency of pcr product 93.5%, optical purity 99.9% are obtained.
Embodiment 16: it is reacted by the bioconversion of substrate of N- phenylacetyl-DL-serine
6.0g serine and 4.6g NaOH are added in 20ml distilled water, and it is colourless to be stirred well to solution under condition of ice bath It is bright, 5.2g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is in faint yellow, continuation condition of ice bath reaction 2h, then stirring at normal temperature reaction After 5h, solution adds HCl to adjust PH to 2.0 or so in colorless and transparent, and white solid is precipitated, and filters drying and obtains white solid i.e. For N- phenylacetyl-DL-serine 12.15g.
Substrate N- phenylacetyl-DL-serine is dissolved in ammonium hydroxide, the wet thallus 0.5g for adding the preparation of 3 method of embodiment is used It is 8.5 that ammonium hydroxide, which adjusts pH value of solution, constitutes 10mL reaction system, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C of water-baths and shakes Bed, 150rpm are converted for 24 hours, take 1mL conversion fluid into ep pipe, and supernatant is taken to carry out HPLC by 1 method of embodiment after centrifuge separation It is 49.4% that analysis detection, which obtains conversion ratio,.Conversion fluid 1L is prepared under same reaction condition, by 4 method of embodiment (with dip dyeing 0.2% The filter paper of ninhydrin solution detected, filter paper purpling black shows that eluent contains target substance Serine) separation is pure After change, product Serine 4.82g, efficiency of pcr product 91.8%, optical purity 99.9% are obtained.
Embodiment 17: it is reacted by the bioconversion of substrate of N- phenylacetyl-DL- methionine
8.94g methionine and 7.2g NaOH are added in 120ml distilled water, and it is saturating to be stirred well to solution under condition of ice bath It is bright, 10.3g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is transparent, continues condition of ice bath and reacts 2h, then stirring at normal temperature reaction After 5h, HCl is added to adjust PH to 2.0 or so, a large amount of white solids are precipitated, decompression filters drying and obtains N- phenylacetyl-DL- first sulphur ammonia Sour 15.32g.
Substrate N- phenylacetyl-DL- methionine is dissolved in ammonium hydroxide, adds the wet thallus of 3 method of embodiment preparation 0.45g, adjusting pH value of solution with ammonium hydroxide is 8.5 composition 10mL reaction systems, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C shaking bath, 150rpm are converted for 24 hours, are taken 1mL conversion fluid into ep pipe, take supernatant by 1 method of embodiment after centrifuge separation It carries out HPLC analysis detection and obtains conversion ratio to be 49.1%.Conversion fluid 1L is prepared under same reaction condition, (is used by 4 method of embodiment The filter paper of the ninhydrin solution of dip dyeing 0.2% is detected, and filter paper purpling black shows that eluent contains target substance L- first sulphur Propylhomoserin) isolate and purify after, obtain product L-methionine 7.08g, efficiency of pcr product 95.0%, optical purity 99.9%.
Embodiment 18: it is reacted by the bioconversion of substrate of N- phenylacetyl-DL-proline
9.36g valine and 7.2g NaOH are added in 60ml distilled water, and it is bright to be stirred well to solution under condition of ice bath, 14.24g phenyllacetyl chloride is added dropwise, after being added dropwise to complete, solution is in yellowish, slightly muddy, continuation condition of ice bath reaction 2h, then room temperature After being stirred to react 5h, solution bleach is transparent, and HCl is added to adjust PH to 2.0 or so, and a large amount of white solids are precipitated, and suction filtration is dried It is N- phenylacetyl-DL-proline 18.02g to white solid.
Substrate N- phenylacetyl-DL-proline is dissolved in ammonium hydroxide, adds the wet thallus 0.2g of 3 method of embodiment preparation, is used It is 8.5 that ammonium hydroxide, which adjusts pH value of solution, constitutes 10mL reaction system, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C of water-baths and shakes Bed, 150rpm are converted for 24 hours, take 1mL conversion fluid into ep pipe, and supernatant is taken to carry out HPLC by 1 method of embodiment after centrifuge separation It is 49.3% that analysis detection, which obtains conversion ratio,.Conversion fluid 1L is prepared under same reaction condition, by 4 method of embodiment (with dip dyeing 0.2% The filter paper of ninhydrin solution detected, filter paper purpling black shows that eluent contains target substance Valine) separation is pure After change, product Valine 5.42g, efficiency of pcr product 92.6%, optical purity 99.9% are obtained.
Embodiment 19: it is reacted by the bioconversion of substrate of N- phenylacetyl-DL- glutamic acid
5g glutamic acid and 4.6g NaOH are added in 30ml distilled water, and it is bright to be stirred well to solution under condition of ice bath, are added dropwise After 5.735g phenyllacetyl chloride, after being added dropwise to complete, solution is transparent, continues condition of ice bath and reacts 2h, then stirring at normal temperature reacts 5h, add HCl adjusts PH to 2.0 or so, and solution becomes cloudy, and a large amount of white solids are precipitated in 4 DEG C of refrigerator cold-storages after overnight, and decompression filters drying Obtain N- phenylacetyl-DL- glutamic acid 8.45g.
Substrate N- phenylacetyl-DL- glutamic acid is dissolved in ammonium hydroxide, adds the wet thallus 0.4g of 3 method of embodiment preparation, is used It is 8.5 that ammonium hydroxide, which adjusts pH value of solution, constitutes 10mL reaction system, and wherein final concentration of 100mM is added in substrate, is placed in 30 DEG C of water-baths and shakes Bed, 150rpm are converted for 24 hours, take 1mL conversion fluid into ep pipe, and supernatant is taken to carry out HPLC by 1 method of embodiment after centrifuge separation It is 49.4% that analysis detection, which obtains conversion ratio,.Conversion fluid 1L is prepared under same reaction condition, by 4 method of embodiment (with dip dyeing 0.2% The filter paper of ninhydrin solution detected, filter paper purpling black shows that eluent contains target substance Pidolidone) separation is pure After change, product Pidolidone 6.92g, efficiency of pcr product 94.1%, optical purity 99.9% are obtained.
Sequence table
<110>Zhejiang Polytechnical University
<120>citrobacter freundii ZJB-17010 and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1384
<212> DNA
<213>citrobacter freundii (Citrobacter freundii)
<400> 1
cgccctcccg aaggttaagc tacctacttc ttttgcaccc actcccatgg tgtgacgggc 60
ggtgtgtaca aggcccggga acgtattcac cgtggcattc tgatccacga ttactagcga 120
ttccgacttc atggagtcga gttgcagact ccaatccgga ctacgacata ctttatgagg 180
tccgcttgct ctcgcgaggt cgcttctctt tgtatatgcc attgtagcac gtgtgtagcc 240
ctactcgtaa gggccatgat gacttgacgt catccccacc ttcctccagt ttatcactgg 300
cagtctcctt tgagttcccg accgaatcgc tggcaacaaa ggataagggt tgcgctcgtt 360
gcgggactta acccaacatt tcacaacacg agctgacgac agccatgcag cacctgtctc 420
agagttcccg aaggcaccaa agcatctctg ctaagttctc tggatgtcaa gagtaggtaa 480
ggttcttcgc gttgcatcga attaaaccac atgctccacc gcttgtgcgg gcccccgtca 540
attcatttga gttttaacct tgcggccgta ctccccaggc ggtcgactta acgcgttagc 600
tccggaagcc acgcctcaag ggcacaacct ccaagtcgac atcgtttacg gcgtggacta 660
ccagggtatc taatcctgtt tgctccccac gctttcgcac ctgagcgtca gtctttgtcc 720
agggggccgc cttcgccacc ggtattcctc cagatctcta cgcatttcac cgctacacct 780
ggaattctac ccccctctac aagactctag cctgccagtt tcggatgcag ttcccaggtt 840
gagccccggg gatttcacat ccgacttgac agaccgcctg cgtgcgcttt acgcccagta 900
attccgatta acgcttgcac cctccgtatt accgcggctg ctggcacgga gttagccggt 960
gcttcttctg cgagtaacgt caatcgctgc ggttattaac cacaacgcct tcctcctcgc 1020
tgaaagtact ttacaacccg aaggccttct tcatacacgc ggcatggctg catcaggctt 1080
gcgcccattg tgcaatattc cccactgctg cctcccgtag gagtctggac cgtgtctcag 1140
ttccagtgtg gctggtcatc ctctcagacc agctagggat cgtcgcctag gtgagccgtt 1200
accccaccta ctagctaatc ccatctgggc acatccgatg gcaagaggcc cgaaggtccc 1260
cctctttggt cttgcgacgt tatgcggtat tagctaccgt ttccagtagt tatccccctc 1320
catcgggcag tttcccagac attactcacc cgtccgccac tcgtcaccca aggagcaagc 1380
tcct 1384

Claims (10)

1. citrobacter freundii (Citrobacter freundii) ZJB-17010, is preserved in China typical culture collection Center, deposit number CCTCC M 2017604, preservation date on October 23rd, 2017, address: Wuhan, China Wuhan is big It learns, 430072.
2. citrobacter freundii ZJB-17010 described in a kind of claim 1 is in catalysis 2-N- phenylacetyl group -4- [hydroxyl (first Base) phosphoryl]-DL- butyric acid prepares the application in 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid.
3. application as claimed in claim 2, it is characterised in that the application is fermented with citrobacter freundii ZJB-17010 It is catalyst that culture, which obtains wet thallus, using 2-N- phenylacetyl group -4- [hydroxyl (methyl) phosphoryl]-DL- butyric acid as substrate, with slow Fliud flushing is the reaction system that reaction medium constitutes pH8.5, and conversion reaction is carried out under the conditions of 25-55 DEG C, 100-200rpm, is reacted After, reaction solution is isolated and purified to obtain 2- amino -4- [hydroxyl (methyl) phosphoryl]-L- butyric acid.
4. application as claimed in claim 3, it is characterised in that in the reaction system, catalyst amount is in terms of wet thallus weight For 10-200g/L, substrate is initially added final concentration of 10-500mM.
5. application as claimed in claim 3, it is characterised in that the method that the reaction solution isolates and purifies are as follows: after reaction, Reaction solution is extracted with dichloromethane, by water layer tune pH to 1.0-5.0, ion exchange is carried out with the speed loading of 1-6.0Bv/h Chromatography, is first washed with deionized, then eluted with the ammonium hydroxide of 0.2-4.5M with 0.5-3.0Bv/h, collects and contains target components Eluent, vacuum distillation with methanol dissolving-recrystallization, taken crystal dry, obtains 2- amino -4- [hydroxyl (methyl) to paste Phosphoryl]-L- butyric acid.
6. citrobacter freundii ZJB-17010 described in a kind of claim 1 prepares L- in catalysis N- phenylacetyl-DL- amino acid The application of amino acid.
7. application as claimed in claim 6, it is characterised in that the application is with the fermented training of Xiangfang enterobacteria ZJB-17001 It supports the wet thallus obtained and is constituted pH8.5 by reaction medium of buffer using N- phenylacetyl-DL- amino acid as substrate for catalyst Transformation system, conversion reaction is carried out under the conditions of 25-55 DEG C, 100-200rpm, after reaction, reaction solution through separate it is pure Change, obtains N- phenylacetyl-l-amino acid.
8. the use as claimed in claim 7, it is characterised in that in the transformation system, catalyst amount is in terms of wet thallus weight For 20-300g/L, the substrate is initially added final concentration of 50-500mM.
9. application as claimed in claim 7, it is characterised in that the N- phenylacetyl-DL- amino acid is one of following: N- benzene second Acyl-DL-Alanine, N- phenylacetyl-DL-serine, N- phenylacetyl-DL- glutamic acid, N- phenylacetyl-DL- tyrosine, N- benzene second Acyl-DL- threonine, N- phenylacetyl-DL-proline, N- phenylacetyl-DL- aspartic acid, N- phenylacetyl-DL- methionine, N- Phenylacetyl-DL-leucine, N- phenylacetyl-DL- isoleucine, N- phenylacetyl-DL-phenylalanine.
10. the use as claimed in claim 7, it is characterised in that the wet thallus is prepared as follows:
(1) inclined-plane culture: being seeded to slant medium for citrobacter freundii ZJB-17010, in 30 DEG C of culture 48h, obtains Inclined-plane thalline；The slant medium is final concentration of: 1~15g/L of mannitol, 1~15g/L of sodium glutamate, and yeast extract 0.5~ 5g/L, K2HPO40~1.5g/L, KH2PO40~1.5g/L., MgSO40~1.0g/L, 0.2~1.8g/L of caprolactam, fine jade Rouge is 20.0g/L, and solvent is deionized water, pH 7.0~7.5；
(2) seed culture: being seeded to seed culture medium from one oese thallus of inclined-plane thalline picking, cultivates for 24 hours, obtains at 30 DEG C Seed liquor；The seed culture medium final concentration composition are as follows: 1~15g/L of mannitol, 1~15g/L of sodium glutamate, yeast extract 0.5~ 5g/L, K2HPO40~1.5g/L, KH2PO40~1.5g/L., MgSO40~1.0g/L, caprolactam 0.2~1.8g/L, it is molten Agent is deionized water, pH 7.0~7.5；
(3) fermented and cultured: seed liquor being seeded in fermentation medium with the inoculum concentration of volumetric concentration 1~10%, 30 DEG C, After 150rpm shaken cultivation 60h, it is centrifuged 10min at 12000g, collects wet thallus；1~15g/L of mannitol, sodium glutamate 1~ 15g/L, yeast extract 0.5~5g/L, K2HPO40~1.5g/L, KH2PO40~1.5g/L., MgSO40~1.0g/L, acyl in oneself 0.2~1.8g/L of amine, solvent are deionized water, pH 7.0~7.5.
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