CN107142227A - Klebsiella oxytoca and its application - Google Patents
Klebsiella oxytoca and its application Download PDFInfo
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- CN107142227A CN107142227A CN201710207706.9A CN201710207706A CN107142227A CN 107142227 A CN107142227 A CN 107142227A CN 201710207706 A CN201710207706 A CN 201710207706A CN 107142227 A CN107142227 A CN 107142227A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/22—Klebsiella
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
Abstract
Prepared the invention discloses a kind of Klebsiella oxytoca and its catalysis α amino nitriles (2 amino 4 (hydroxyethyl methyl phosphoryl) butyronitrile) and be used as the application in L 2 amino 4 (hydroxyethyl methyl phosphoryl) butyric acid of the important chiral precursor of L glufosinate-ammoniums, the invention provides bacterial strain Klebsiella oxytoca (Klebsiella oxytoca) ZJB 16005 that one plant can produce nitrilase, present invention also offers the method that the wet thallus obtained by the use of the fermented cultures of Klebsiella oxytoca ZJB 16005 is biological obtained L 2 amino 4 (hydroxyethyl methyl phosphoryl) butyric acid as the important chiral precursor of L glufosinate-ammoniums of catalyst 2 amino 4 (hydroxyethyl methyl phosphoryl) butyronitrile, with important application prospect.
Description
(1) technical field
The present invention relates to the bacterial strain of one plant of production nitrilase, and its in living things catalysis alpha-aminonitriles the substrate ((hydroxyl of 2- amino -4
Ethyl-methyl phosphoryl)-butyronitrile) prepare as the important chiral precursor of L-glufosinate-ammonium (the hydroxyethyl methyl phosphinylidyne of L-2- amino -4
Base)-butyric acid application.
(2) background technology
Cyano group in nitrile compounds can be converted into carboxyl system by nitrilase (Nitrilase, EC 3.5.5.1)
Standby carboxylic acid, it has unique chemo-selective, stereoselectivity and regioselectivity to nitrile compound, can be used for synthesis many
Kind of chemical method combined coefficient is low or is difficult to the carboxylic acid that synthesizes.Its good, high conversion rate of selectivity, meanwhile, the approach and traditional work
Skill, which is compared, has reaction condition gentle, of reduced contamination without HTHP, strong acid and strong base, has chemical method unrivaled
Superiority, meets the developing direction of atom economy type and Green Chemistry, and huge application potential, nothing are shown in chemical synthesis
There is large development by number of applications or species, in medicine and its intermediate, agricultural chemicals and its intermediate, food and feed
The fields such as additive are widely applied：Nitrilase catalysis is prepared in the middle of the key of Rosuvastatin (Rosuvastatin)
Body (R) -3- hydroxyl pentanedioic acid diethyl esters；Prepare the key intermediate R of medicament for resisting platelet aggregation clopidogrel (Clopidogrel)
Type o-chloromandelic acid；Prepare key intermediate iminodiacetic acid of IDA routes production herbicide glyphosate etc..
L-2- amino -4 (hydroxyethyl methyl phosphoryl)-butyric acid is the important chiral precursor for preparing L-glufosinate-ammonium.DL- grass ammoniums
Phosphine, active ingredient is phosphinothricin (abbreviation PPT), and its chemical name is：4- [hydroxymethyl phosphono]-DL- is high
Alanine (alias：D, L-glufosinate-ammonium amine salt), it is wide spectrum, contact killing type, natural disposition of going out, non-residual herbicide, is by German Hirst
The new steriland herbicide that company's (being related companies of Beyer Co., Ltd at present) develops in 1980s.Except with weeding
Outside active also there is bactericidal and insecticidal activity can be reached with agrochemical mixture while the effect prevented and treated.The herbicide has height
Effect, low toxicity, it is degradable the features such as.Glufosinate-ammonium is raceme mixture, and only L-type has phytotoxicity, and its activity of weeding is
2 times of racemic mixture.At present, commercially available glufosinate-ammonium is typically all racemic mixture, if can be with the glufosinate-ammonium of L-type
To be used instead of it, use cost can be not only reduced, moreover it is possible to mitigate environmental pressure.The synthetic method of glufosinate-ammonium has a lot, bag
Include chemical method synthesis racemic glufosinate-ammonium：Drop cloth riel (Gabriel)-diethyl malonate, A Buzuofu
(Arbuzov) synthetic method, high-pressure catalytic synthetic method, haler-Bei Gesi (Bucherer-Bergs) method, haler-Bei Gesi
(Bucherer-Bergs) method, (Neber) rearrangement method, Michael free radical additions method, the careless ammoniums of (Srecker) method synthesis DL-
Phosphine；Chemical method synthesizes L-glufosinate-ammonium：D-Val methyl esters and (S) -2- hydroxyl -3- pinones induce for the chiral adjuvant of adjuvant
Method, Pidolidone and L-Methionine for chiral source natural amino acid chiral source method, asymmetric Strecker reaction and it is asymmetric
Michael addition processes, utilize quinine carry out racemate resolution method；Enzymatic clarification L-glufosinate-ammonium mainly have proteinase hydrolization method,
Phosphodiesterase I Hydrolyze methods, acyltransferase I catalysis methods, three kinds of enzyme synergetic hydrolysis methods of glutaminase；Transaminase method；α-pancreas
Galactase Split Method, amidase Split Method, deacetylase Split Method, D-AAO Split Method etc. (modern,
2009,8 (3)：1-5).
The method applied to industrialized production glufosinate-ammonium can be attributed to following three routes at present：Beyer Co., Ltd utilizes
Michael free radical additions method prepares D, L-glufosinate-ammonium；Srecker methods synthesize D, L-glufosinate-ammonium；Meiji Seika Kaisba asymmetry catalysis
Hydrogenation synthesis L-glufosinate-ammonium.Wherein Srecker methods synthesis D, L-glufosinate-ammonium route reaction condition is gentle, is the big life of current industrialization
The used route of production.The route using triethyl phosphite and phosphorus trichloride as initiation material, through disproportionation, grignard, methylate, plus
D, L-glufosinate-ammonium is made into, nitrile ammonium, acidolysis, ammoniumization reaction.Srecker is replaced with nitrilase selective catalysis hydrolysis
Alpha-aminonitriles substrate (2- amino -4 (hydroxyethyl methyl phosphoryl)-butyronitrile) system in hydrolysis step in route, catalysis route
The important chiral precursor of standby synthesis L-glufosinate-ammonium：L-2- amino -4 (hydroxyethyl methyl phosphoryl)-butyric acid, to realize industrialization road
The Srecker methods synthesis L-glufosinate-ammonium of line provides foundation.
(3) content of the invention
It is an object of the present invention to provide bacterial strain -- the Klebsiella oxytoca (Klebsiella of one plant of production nitrilase
Oxytoca) ZJB-16005, and its living things catalysis alpha-aminonitriles substrate (2- amino -4 (hydroxyethyl methyl phosphoryl)-butyronitrile) system
The application of standby (hydroxyethyl methyl phosphoryl)-butyric acid of L-2- amino -4.
The technical solution adopted by the present invention is：
The present invention provides one plant of new strains -- Klebsiella oxytoca (Klebsiella with production nitrilase performance
Oxytoca) ZJB-16005, is preserved in China typical culture collection center, deposit number CCTCC No：M2017032, preservation
Date 2017 year 01 month 13, address：Chinese Wuhan Wuhan Universitys, postcode 430072.
The invention further relates to described Klebsiella oxytoca (Klebsiella oxytoca) ZJB-16005 catalysis alpha-amidos
Nitrile (2- amino -4 (hydroxyethyl methyl phosphoryl)-butyronitrile) prepares the L-2- amino -4 as the important chiral precursor of L-glufosinate-ammonium
Application in (hydroxyethyl methyl phosphoryl)-butyric acid, described application is：With the fermented trainings of Klebsiella oxytoca ZJB-16005
It is catalyst to support the wet thallus obtained, using 2- amino -4 (hydroxyethyl methyl phosphoryl)-butyronitrile as substrate, with distilled water or
The phosphate buffer of 100mM, pH 7.0 is reaction medium, and conversion reaction is carried out under the conditions of 30-50 DEG C, 100-200rpm, is reacted
Terminate, reaction solution is isolated and purified, obtain (hydroxyethyl methyl the phosphoryl)-butyric acid of L-2- amino -4.
Nitrilase living things catalysis alpha-aminonitriles (2- amino -4 (hydroxyethyl methyl phosphoryl)-butyronitrile) generate L-2- ammonia
Base -4 (hydroxyethyl methyl phosphoryl)-butyric acid process is as follows：
Further, the catalyst amount is calculated as 10-100g/L reaction mediums, preferably 35g/L with wet thallus weight, described
Substrate is initially added final concentration of 2-100g/L reaction mediums, preferably 5g/L.
Further, the catalyst is prepared as follows：(1) inclined-plane culture：Klebsiella oxytoca ZJB-16005 is connect
Plant to slant medium, 48h are cultivated at 30 DEG C, obtain inclined-plane thalline；The slant medium final concentration is constituted：Mannitol 1
~20.0g/L, 1~10.0g/L of sodium glutamate, yeast extract 1~5.0g/L, K2HPO40.1~1.0g/L, KH2PO40.1~
1.0g/L, MgSO40.1~1.0g/L, 0.1~2.0g/L of caprolactam, agar 20.0g/L, solvent are deionized water, pH
7.0-7.5；It is preferred that the slant medium final concentration composition is：Mannitol 10.0g/L, sodium glutamate 7.0g/L, yeast extract
3.0g/L, K2HPO40.75g/L, KH2PO40.75g/L, MgSO40.5g/L, caprolactam 1.0g/L, agar 20.0g/L are molten
Agent is deionized water, pH 7.0-7.5；
(2) seed culture：Picking inclined-plane thalline is seeded to seed culture medium, and 24h is cultivated at 30 DEG C, obtains seed liquor；Institute
Stating seed culture medium final concentration composition is：1~20.0g/L of mannitol, 1~10.0g/L of sodium glutamate, 1~5.0g/L of yeast extract,
K2HPO40.1~1.0g/L, KH2PO40.1~1.0g/L, MgSO40.1~1.0g/L, caprolactam 0.1~2.0g/L, it is molten
Agent is deionized water, pH 7.0-7.5；It is preferred that the seed culture medium final concentration composition is：Mannitol 10.0g/L, sodium glutamate
7.0g/L, yeast extract 3.0g/L, K2HPO40.75g/L, KH2PO40.75g/L, MgSO40.5g/L, caprolactam 1.0g/L,
Solvent is 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%, 30 DEG C,
After 150rpm shaken cultivations 48h, 10min is centrifuged under 12000g, wet thallus is collected；The fermentation medium final concentration composition
For：1~20.0g/L of mannitol, 1~10.0g/L of sodium glutamate, yeast extract 1~5.0g/L, K2HPO40.1~1.0g/L,
KH2PO40.1~1.0g/L, MgSO40.1~1.0g/L, 0.1~2.0g/L of caprolactam, solvent are deionized water, pH
7.0-7.5, preferably described fermentation medium final concentration, which is constituted, is：Mannitol 10.0g/L, sodium glutamate 7.0g/L, yeast extract
3.0g/L, K2HPO40.75g/L, KH2PO40.75g/L, MgSO40.5g/L, caprolactam 1.0g/L, solvent is deionization
Water, pH 7.0-7.5.
The method that reaction solution of the present invention is isolated and purified is：Reaction solution is adjusted to pH 2.5, using anion exchange tree
Fat 201 × 7 carries out column chromatography, and above column flow rate is that 1~6.0BV/h (preferably 4.0BV/h) carries out loading, is first washed with deionized water
Wash, then eluted with 0.5~2.0M (preferably 1M) ammoniacal liquor, elution speed is 1.0~4.0BV/h (preferably 2BV/h), is collected
Eluent containing object；By eluent vacuum distillation to solvent-free outflow, methanol dissolving is added, is stirred under ice bath, weight
Crystallization, filtering, filtration cakes torrefaction produces product L-2- amino -4 (hydroxyethyl methyl phosphoryl)-butyric acid.
It is made in the present invention using (hydroxyethyl methyl the phosphoryl)-butyronitrile of nitrilase selective catalysis hydrolysis 2- amino -4
As L-2- amino -4 (hydroxyethyl methyl phosphoryl)-butyric acid of the important chiral precursor of L-glufosinate-ammonium, to realize industrialized route
Ripe Srecker methods synthesis L-glufosinate-ammonium provides foundation.The acid in domestic industry Strecker routes is instead of simultaneously
Solution reaction, reduce acidolysis reaction generation acid water pollution and acidolysis reaction to the corrosion-damaged of equipment.
The beneficial effects are mainly as follows：The invention provides the bacterial strain that one plant can produce nitrilase -- production acid
Klebsiella (Klebsiella oxytoca) ZJB-16005, present invention also offers utilize Klebsiella oxytoca ZJB-16005
Conduct is made for (hydroxyethyl methyl the phosphoryl)-butyronitrile of biology catalyst 2- amino -4 in the wet thallus that fermented culture is obtained
The method of L-2- amino -4 (hydroxyethyl methyl phosphoryl)-butyric acid of the important chiral precursor of L-glufosinate-ammonium, before important application
Scape.
(4) illustrate
Fig. 1 is D-2- amino -4 (hydroxyethyl methyl phosphoryl)-fourth under the conditions of pre-column derivatization RPLC
The liquid phase result spectrogram of acid and L-2- amino -4 (hydroxyethyl methyl phosphoryl)-butyric acid.
Fig. 2 is ammonium ion concentration standard curve.
(5) embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1：Produce the screening and identification of nitrilase bacterial strain
1st, the screening of nitrilase bacterial strain is produced
(1) present invention respectively weighs 1g and suspended with 0.85% physiological saline from soil sampling in all parts of the country, stands, plus 1ml supernatants
In 50mL the richness of sole carbon source is used as added with 1g/L alpha-aminonitriles substrate (2- amino -4 (hydroxyethyl methyl phosphoryl)-butyronitrile)
Collect in culture medium, cultivated 3 days on 30 DEG C, 150rpm shaking table；After culture medium is muddy, 1ml nutrient solutions are taken to be transferred to addition
In the fresh enriched medium for having 1g/L2- amino -4 (hydroxyethyl methyl phosphoryl)-butyronitrile, it is further cultured for 3 days.So circulation
3~4 circulations of enrichment；
(2) last time enrichment culture liquid sterilized water is diluted into 10 gradients step by step, chooses 10-4、10-5、10-6、10-7、
10-8The dilution of five gradients respectively takes 0.1mL to be uniformly coated on added with 1g/L2- amino -4 (hydroxyethyl methyl phosphoryl)-fourth
On the plating medium of nitrile, cultivated 2-3 days in 30 DEG C of constant incubators, by the single bacterium colony picking on flat board, be inoculated in inclined-plane
On culture medium, cultivated 2-3 days in 30 DEG C of constant incubators, in 4 DEG C of preservations；
(3) by the inoculation being stored on inclined-plane into seed culture medium, 24h is cultivated at 30 DEG C.By seed liquor with body
The inoculum concentration of product concentration 1% is seeded in fermentation medium, 30 DEG C, 150rpm shaken cultivations 48h.Centrifuged under 12000g
10min, collects wet thallus, takes a certain amount of thalline and substrate (2- amino -4 (hydroxyethyl methyl phosphoryl)-butyronitrile) to be suspended in
In distilled water, 0.2g/10mL bacteria concentrations are made, the reaction system of 10g/L concentration of substrate is converted, are placed in 30 DEG C of shaking baths
Convert 24h.Sampling, carries out phenol-hypochlorite method primary dcreening operation, nitrilase vigor is relatively good, then carries out high performance liquid chromatography
The concentration and optical purity of (hydroxyethyl methyl the phosphoryl)-butyric acid of product L-2- amino -4 are detected, final screening obtains bacterial strain
ZJB-16005。
The enriched medium final concentration composition is (g/L)：Glucose 5.0, sodium chloride 1.0, K2HPO4·3H2O 0.8,
KH2PO43.3, MgSO4·7H2O 0.2, solvent is deionized water, and pH is 7.0, and 1g/L 2- ammonia is added in this culture medium
Base -4 (hydroxyethyl methyl phosphoryl)-butyronitrile is used as unique carbon source.
The plate screening culture medium is (g/L)：Glucose 5.0, sodium chloride 1.0, K2HPO4·3H2O 0.8, KH2PO4
3.3, MgSO4·7H2O 0.2, agar 20.0, solvent is deionized water, and pH is 7.0.115 DEG C of 30min sterilizings.It is cooled to room temperature
(hydroxymethyl the phosphoryl)-butyronitrile 1.0g/L of 2- amino -4 is added afterwards.
The slant medium final concentration composition is (g/L)：Mannitol 10.0, sodium glutamate 7.0, yeast extract 3.0,
K2HPO40.75, KH2PO40.75, MgSO40.5, caprolactam 1.0, agar 20.0, solvent is deionized water, pH 7.0-
7.5。
The fermentation medium final concentration composition is (g/L)：Mannitol 10.0, sodium glutamate 7.0, yeast extract 3.0,
K2HPO40.75, KH2PO40.75, MgSO40.5, caprolactam 1.0, solvent is deionized water, pH 7.0-7.5.
(4) phenol-hypochlorite method primary dcreening operation
Take 2ml A liquid to be placed in 10ml centrifuge tube with pipette, add 4 μ L prepare liquids, close the lid, firmly mix.
Add 2ml B liquid with pipette again afterwards, close the lid, mix again.It is placed in after 37 DEG C of heating water bath 5min, to centrifuge tube
Interior color no longer changes, and takes 200 μ L to be placed in 96 orifice plates, is obtained at 620nm after measurement absorbance with standard curve contrast
The concentration of ammonium root in prepare liquid, ammonium root concentration is equal to 2- amino -4 (hydroxyethyl methyl the phosphoryl)-butyric acid obtained after conversion
Concentration, the yield of 2- amino -4 in conversion process (hydroxyethyl methyl phosphoryl)-butyric acid can be detected with this.
The A liquid：5g phenol and 25mg sodium nitroprussides are weighed, is placed in 500mL volumetric flasks, ultra-pure water is added and determines
Rong Hou, is stored in brown bottle, is stored in 4 DEG C of refrigerators.
The B liquid：The NaOH for weighing 2.5g is placed in 500mL volumetric flasks, and adding 4.4mL with pipette contains 5.2% effective chlorine
Aqueous sodium hypochlorite solution after, use ultra-pure water constant volume, be stored in brown bottle, in 4 DEG C of refrigerators store.
The preparation of standard curve：Phenol-hypochlorite method at room temperature with cuvette and ultraviolet-visible spectrophotometer or
Person can measure under 96 orifice plates and general microplate spectrophotometer to the concentration of ammonium root, under 620nm wavelength,
The concentration of OD620 values and ammonium ion has good linear relationship (such as Fig. 2), R2=0.9982, show that this method has very high
Feasibility and accuracy.
(5) pre-column derivatization reversed-phased high performace liquid chromatographic secondary screening
Learn from else's experience the preferable μ L of reaction solution 500 of result obtained after primary dcreening operation, adds isometric derivatization reagent, mixes, enters
Row derivative reaction 5min, then the μ L of sample introduction 15, carry out HPLC analyses.
Described derivatization reagent：10mg OPAs (OPA) and 12mg N-acetyl-L-cysteines are weighed respectively
(NAC) after, being dissolved with l mL absolute ethyl alcohols, 5mL is diluted to 0.2mol/L borate buffers (pH value 9.8), -4 DEG C of refrigerators are protected
Deposit standby (resting period is no more than 3d).
Efficient liquid phase chromatographic analysis condition is：The gas chromatograph used wears peace U3000 liquid chromatograph (configurations for the U.S.
Fluorescence detector)；Chromatographic column：Wear peace C18 (4.6mm × 250mm, silicone hydroxyl filler)；Mobile phase：Methanol：0.05mol/L acetic acid
Ammonium salt solution (volume ratio 10:90, pH value 5.7)；Flow velocity：1mL/min；Column temperature：35℃；Sample size：15μL；Fluoroscopic examination wavelength：
Excitation wavelength Ex 350nm, launch wavelength Em 450nm.
Under this condition, (hydroxyethyl methyl the phosphoryl)-butyric acid of L-2- amino -4 and (the hydroxyethyl methyl phosphorus of D-2- amino -4
Acyl group) appearance time of-butyric acid is respectively 9.2min and 11.1min, sees Fig. 1.
2nd, bacterial strain ZJB-16005 Molecular Identification
Step 1 is screened into obtained bacterial strain ZJB-16005 and extracts its STb gene with molecular agents box, with bacterial strain ZJB-
16005 STb gene is template, utilizes primer:P1:5'-AGAGTTTGATCCTGGCTCAG-3' and P2:5'-
AAGGAGGTGATCCAGCCGCA-3' expands 16S rDNA genes (the SEQ ID NO of bacterial strain：Shown in 1), by the same T of gene outcome
After carrier connection, commission Shanghai life work is expanded and is sequenced to the bacterium 16S rDNA, after the 16S rDNA sequences for obtaining the bacterial strain,
The 16S rDNA gene orders of related strain in GenBank are retrieved on NCBI websites with BLAST, and carry out sequence analysis.This
Invention microorganism and Klebsiella oxytoca bacterial strain JKo3 homologys highests (homology, 99%, based on 16S
RDNA), according to microbial molecules science of heredity identity principle, the homology based on 16S rDNA sequences is higher than 95%, identifies bacterium base
Originally control bacterium is belonged to.Therefore, the bacterial strain ZJB-16005 of this experimental identification is Klebsiella oxytoca (Klebsiella
Oxytoca), intend being named as Klebsiella oxytoca (Klebsiella oxytoca) ZJB-16005.
It is any to SEQ ID NO：Nucleotide sequence shown in 1 carries out substitution, missing or the insertion of one or more nucleotides
The nucleotide sequence obtained is handled, as long as it has more than 90% homology with the nucleotides, the protection of the present invention is belonged to
Scope.
Embodiment 2：The preparation of wet thallus
(1) inclined-plane culture
Klebsiella oxytoca (Klebsiella oxytoca) ZJB-16005 is seeded to slant medium, in 30 DEG C of trainings
48h is supported, inclined-plane thalline is obtained.
The slant medium final concentration composition is (g/L)：Mannitol 10.0, sodium glutamate 7.0, yeast extract 3.0,
K2HPO40.75, KH2PO40.75, MgSO40.5, caprolactam 1.0, agar 20.0, solvent is deionized water, pH 7.0-
7.5。
(2) seed culture
Seed culture medium is seeded to from inclined-plane thalline picking thalline, 24h is cultivated at 30 DEG C, seed liquor is obtained；The seed
Culture medium final concentration composition is (g/L)：Mannitol 10.0, sodium glutamate 7.0, yeast extract 3.0, K2HPO40.75, KH2PO4
0.75, MgSO40.5, caprolactam 1.0, solvent is 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 cultivations
After 48h, 10min is centrifuged under 12000g, wet thallus is collected.
The fermentation medium final concentration composition is (g/L)：Mannitol 10.0, sodium glutamate 7.0, yeast extract 3.0,
K2HPO40.75, KH2PO40.75, MgSO40.5, caprolactam 1.0, solvent is deionized water, pH 7.0-7.5.
Embodiment 3：It is anti-for the bioconversion of substrate with alpha-aminonitriles (2- amino -4 (hydroxyethyl methyl phosphoryl)-butyronitrile)
Should
(1) wet thallus 0.2g prepared by the method for embodiment 2, bottom are added in 10mL phosphate buffers (100mM, pH 7.0)
Thing 2- amino -4 (hydroxyethyl methyl phosphoryl) final concentration of 2g/L of-butyronitrile, is put into 30 DEG C of shaking bath, turns under the conditions of 150rpm
Change 24h.1ml conversion fluids are taken in EP pipes, 12000r/min centrifugation 2min take supernatant to carry out chiral liquid phase detection.As a result show
Conversions of the bacterial strain ZJB-16005 to 2- amino -4 (hydroxyethyl methyl phosphoryl)-butyronitrile obtains the (ethoxy of product L-2- amino -4
Methyl phosphoryl)-butyric acid optical purity be 99.9%.
(2) enzyme reaction obtains the conversion fluid of the amino of L-2- containing product -4 (hydroxyethyl methyl phosphoryl)-butyric acid in ion friendship
Change on post and the separation of product is carried out using anion exchange resin 201 × 7.
1) resin is pre-processed
Soaked with 50 DEG C of warm water, it is fully expanded and remove fine particle (tilting or floatation)；Use successively l.0M
NaOH aqueous solution soakings 3h is washed with deionized water to neutrality, then with l.0M HCl/water solution soak 3h after be washed with deionized water to
Neutrality, then with NaOH aqueous solution soaking 3h l.0M, is converted into OH-Form, is finally washed with deionized water standby to neutrality.
2) post is filled
Using wet method dress post (internal diameter 1.5cm, high 40cm), the deionization of certain altitude is first first added in ion exchange column
Water (generally the 1/3 of column length), is taking 30mL wet resins 201 × 7 to load glass, is adding 50mL deionized waters, slowly stir,
The resin of suspension is poured into ion exchange column, allows its natural subsidence, and resin must be evenly distributed in post, should not there is obvious
Line of demarcation, should not there is bubble generation.
3) loading, elution
Step (1) conversion fluid is adjusted to pH 2.5, above column flow rate is that 4.0BV/h carries out loading, is taken at regular intervals
The liquid of outflow carries out liquid phase detection, and when absorption has reached peak, pillar is first washed with deionized, then with 1.0M ammonia
Water is eluted, and elution speed is 2.0BV/h, collects eluent, and detect at regular intervals L-2- amino contained therein-
4 (hydroxyethyl methyl phosphoryl)-butyric acid.Elution finishes rear ion exchange column and is washed with deionized, and by 201 × 7 turns of resin
Turn to OH-For separation and Extraction next time.
4) purify
Eluent vacuum distillation obtains clear yellow viscous material, adds methanol dissolving, is stirred under ice bath, recrystallize white solid
Body, filtering produces product L-2- amino -4 (hydroxyethyl methyl phosphoryl)-butyric acid.
Conversion fluid 1L is prepared with step (1) reaction condition, after above-mentioned isolate and purify, (the hydroxyl of L-2- amino -4 is finally obtained
Ethyl-methyl phosphoryl)-butyric acid solid 0.7g, optical purity is 99.9%.
Embodiment 4：
Wet thallus 0.2g prepared by the method for embodiment 2, substrate are added in 10mL phosphate buffers (100mM, pH 7.0)
2- amino -4 (hydroxyethyl methyl phosphoryl) final concentration of 10g/L of-butyronitrile, is put into 30 DEG C of shaking bath, turns under the conditions of 150rpm
Change 24h.1ml conversion fluids are taken in EP pipes, 12000r/min centrifugation 2min take supernatant to carry out chiral liquid phase detection.Same is anti-
Conversion fluid 1L is prepared under the conditions of answering, after being isolated and purified through the method for embodiment 3, (the ethoxy of product L-2- amino -4 is obtained
Methyl phosphoryl)-butyric acid 3g, optical purity is 99.9%.
Embodiment 5：
Wet thallus 1g, substrate 2- prepared by the method for embodiment 2 are added in 10mL phosphate buffers (100mM, pH 7.0)
Amino -4 (hydroxyethyl methyl phosphoryl) final concentration of 10g/L of-butyronitrile, is put into 30 DEG C of shaking bath, is converted under the conditions of 150rpm
24h.1ml conversion fluids are taken in EP pipes, 12000r/min centrifugation 2min take supernatant to carry out chiral liquid phase detection.Same reaction
Under the conditions of prepare conversion fluid 1L, after being isolated and purified through the method for embodiment 3, obtain (the ethoxy first of product L-2- amino -4
Base phosphoryl)-butyric acid 4.2g, optical purity is 99.9%.
Embodiment 6：
Wet thallus 0.2g prepared by the method for embodiment 2, substrate are added in 10mL phosphate buffers (100mM, pH 7.0)
2- amino -4 (hydroxyethyl methyl phosphoryl) final concentration of 10g/L of-butyronitrile, is put into 40 DEG C of shaking bath, turns under the conditions of 150rpm
Change 24h.1ml conversion fluids are taken in EP pipes, 12000r/min centrifugation 2min take supernatant to carry out chiral liquid phase detection.Same is anti-
Conversion fluid 1L is prepared under the conditions of answering, after being isolated and purified through the method for embodiment 3, (the ethoxy of product L-2- amino -4 is obtained
Methyl phosphoryl)-butyric acid 3.2g, optical purity is 99.9%.
Embodiment 7：
Wet thallus 5g, substrate 2- prepared by the method for embodiment 2 are added in 100mL phosphate buffers (100mM, pH 7.0)
Amino -4 (hydroxyethyl methyl phosphoryl) final concentration of 10g/L of-butyronitrile, is put into 30 DEG C of shaking bath, is converted under the conditions of 150rpm
48h.1ml conversion fluids are taken in EP pipes, 12000r/min centrifugation 2min take supernatant to carry out chiral liquid phase detection.Same reaction
Under the conditions of prepare conversion fluid 1L, after being isolated and purified through the method for embodiment 3, obtain (the ethoxy first of product L-2- amino -4
Base phosphoryl)-butyric acid 5.1g, optical purity is 99.9%.
SEQUENCE LISTING
<110>Zhejiang Polytechnical University
<120>Klebsiella oxytoca and its application
<130>
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1458
<212> DNA
<213> Klebsiella oxytoca
<400> 1
cgcattgcgg cagctaccat gcaagtcgaa cggtagcaca gagagcttgc tctcgggtga 60
cgagtggcgg acgggtgagt aatgtctggg aaactgcctg atggaggggg ataactactg 120
gaaacggtag ctaataccgc ataacgtcgc aagaccaaag agggggacct tcgggcctct 180
tgccatcaga tgtgcccaga tgggattagc tagtaggtgg ggtaacggct cacctaggcg 240
acgatcccta gctggtctga gaggatgacc agccacactg gaactgagac acggtccaga 300
ctcctacggg aggcagcagt ggggaatatt gcacaatggg cgcaagcctg atgcagccat 360
gccgcgtgta tgaagaaggc cttcgggttg taaagtactt tcagcgggga ggaaggcgat 420
aaggttaata accttgtcga ttgacgttac ccgcagaaga agcaccggct aactccgtgc 480
cagcagccgc ggtaatacgg agggtgcaag cgttaatcgg aattactggg cgtaaagcgc 540
acgcaggcgg tctgtcaagt cggatgtgaa atccccgggc tcaacctggg aactgcattc 600
gaaactggca ggctggagtc ttgtagaggg gggtagaatt ccaggtgtag cggtgaaatg 660
cgtagagatc tggaggaata ccggtggcga aggcggcccc ctggacaaag actgacgctc 720
aggtgcgaaa gcgtggggag caaacaggat tagataccct ggtagtccac gctgtaaacg 780
atgtcgactt ggaggttgtt cccttgagga gtggcttccg gagctaacgc gttaagtcga 840
ccgcctgggg agtacggccg caaggttaaa actcaaatga attgacgggg gcccgcacaa 900
gcggtggagc atgtggttta attcgatgca acgcgaagaa ccttacctac tcttgacatc 960
cagagaactt agcagagatg ctttggtgcc ttcgggaact gtgagacagg tgctgcatgg 1020
ctgtcgtcag ctcgtgttgt gaaatgttgg gttaagtccc gcaacgagcg caacccttat 1080
cctttgttgc cagcggtccg gccgggaact caaaggagac tgccagtgat aaactggagg 1140
aaggtgggga tgacgtcaag tcatcatggc ccttacgagt agggctacac acgtgctaca 1200
atggcatata caaagagaag cgacctcgcg agagcaagcg gacctcataa agtatgtcgt 1260
agtccggatt ggagtctgca actcgactcc atgaagtcgg aatcgctagt aatcgtggat 1320
cagaatgcca cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccatggga 1380
gtgggttgca aaagaagtag gtagcttaac cttcgggagg gcgcttacca ctttgtgatt 1440
catgactggg ggaagtcg 1458
Claims (6)
- Klebsiella oxytoca 1. (Klebsiella oxytoca) ZJB-16005, is preserved in China typical culture collection center, Deposit number CCTCC No：M 2017032, preservation date 2017 year 01 month 13 days, address：China, Wuhan, Wuhan University, postal Compile 430072.
- 2. Klebsiella oxytoca ZJB-16005 described in a kind of claim 1 is in catalysis alpha-aminonitriles ((the ethoxy first of 2- amino -4 Base phosphoryl)-butyronitrile) prepare application in L-2- amino -4 (hydroxyethyl methyl phosphoryl)-butyric acid.
- 3. application as claimed in claim 2, it is characterised in that described application is：Passed through with Klebsiella oxytoca ZJB-16005 The wet thallus that fermented and cultured is obtained is catalyst, and using 2- amino -4 (hydroxyethyl methyl phosphoryl),-butyronitrile is substrate, with distilled water Or the phosphate buffer of 100mM, pH 7.0 is reaction medium, conversion reaction is carried out under the conditions of 30-50 DEG C, 100-200rpm, instead It should terminate, reaction solution is isolated and purified, obtain (hydroxyethyl methyl the phosphoryl)-butyric acid of L-2- amino -4.
- 4. application as claimed in claim 3, it is characterised in that the catalyst amount is calculated as 20-100g/L with wet thallus weight Reaction medium, the substrate is initially added final concentration of 2-10g/L reaction mediums.
- 5. application as claimed in claim 3, it is characterised in that the catalyst is prepared as follows：(1) inclined-plane culture：Will Klebsiella oxytoca ZJB-16005 is seeded to slant medium, and 48h is cultivated at 30 DEG C, obtains inclined-plane thalline；The inclined-plane culture Base final concentration is constituted：1~20.0g/L of mannitol, 1~10.0g/L of sodium glutamate, yeast extract 1~5.0g/L, K2HPO4 0.1 ~1.0g/L, KH2PO40.1~1.0g/L, MgSO40.1~1.0g/L, 0.1~2.0g/L of caprolactam, agar 20.0g/L, Solvent is deionized water, pH 7.0-7.5；(2) seed culture：Picking inclined-plane thalline is seeded to seed culture medium, and 24h is cultivated at 30 DEG C, obtains seed liquor；The kind Sub- culture medium final concentration is constituted：1~20.0g/L of mannitol, 1~10.0g/L of sodium glutamate, 1~5.0g/L of yeast extract, K2HPO40.1~1.0g/L, KH2PO40.1~1.0g/L, MgSO40.1~1.0g/L, caprolactam 0.1~2.0g/L, it is molten Agent is 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 shakes Swing after culture 48h, 10min is centrifuged under 12000g, collect wet thallus；The fermentation medium final concentration is constituted：Mannitol 1 ~20.0g/L, 1~10.0g/L of sodium glutamate, yeast extract 1~5.0g/L, K2HPO40.1~1.0g/L, KH2PO40.1~ 1.0g/L, MgSO40.1~1.0g/L, 0.1~2.0g/L of caprolactam, solvent are deionized water, pH 7.0-7.5.
- 6. application as claimed in claim 3, it is characterised in that the method that the reaction solution is isolated and purified is：Reaction solution is adjusted to PH 2.5, is firmly chromatographed using anion exchange resin 201 × 7, and above column flow rate is that 1~6.0BV/h carries out loading, is first used Deionized water is washed, then is eluted with 0.5~2.0M ammoniacal liquor, and elution speed is 1.0~4.0BV/h, and collection contains target The eluent of thing；By eluent vacuum distillation to solvent-free outflow, methanol dissolving is added, is stirred under ice bath, recrystallization, mistake Filter, filtration cakes torrefaction produces product L-2- amino -4 (hydroxyethyl methyl phosphoryl)-butyric acid.
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| CN111235132A (en) * | 2019-12-23 | 2020-06-05 | 浙江工业大学 | β -galactosidase, gene, engineering bacterium and application thereof |
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| CN102559558A (en) * | 2012-02-17 | 2012-07-11 | 湖南省植物保护研究所 | Klebsiella oxytoca strain and application thereof, and triazophos pesticide residue degrading bacterium and preparation method thereof |
| CN104404011A (en) * | 2014-11-05 | 2015-03-11 | 浙江大学 | Amidase, coding gene and applications thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559558A (en) * | 2012-02-17 | 2012-07-11 | 湖南省植物保护研究所 | Klebsiella oxytoca strain and application thereof, and triazophos pesticide residue degrading bacterium and preparation method thereof |
| CN104404011A (en) * | 2014-11-05 | 2015-03-11 | 浙江大学 | Amidase, coding gene and applications thereof |
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| YU-GUO ZHENG ET AL.: "recent advances in biotechnological applications of alcohol dehydrogenases", 《APPL MICROBIOL BIOTECHNOL》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111235132A (en) * | 2019-12-23 | 2020-06-05 | 浙江工业大学 | β -galactosidase, gene, engineering bacterium and application thereof |
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