CN108642130A - A kind of high-throughput screening method of tyrosine phenol lyase high dynamic strain - Google Patents
A kind of high-throughput screening method of tyrosine phenol lyase high dynamic strain Download PDFInfo
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Abstract
The invention discloses a kind of high-throughput screening method of tyrosine phenol lyase high dynamic strain, the method is：The wet thallus that the fermented culture of strain to be tested obtains is added as strain to be tested sample in substrate reactions liquid, the centrifugation of shaking table reaction solution obtains supernatant；Supernatant is taken to be added in chromogenic reaction liquid, it is stored at room temperature colour developing, light absorption value is surveyed at 465nm, according to Sodium Pyruvate standard curve, obtain the content of Sodium Pyruvate in supernatant, and then the vigor of tyrosine phenol lyase in strain to be tested sample is obtained, to filter out tyrosine phenol lyase high dynamic strain；The method of the present invention, directly the tyrosine phenol lyase and its mutant improved to levodopa synthesis capability can be obtained by high flux screening, time needs 20min from one sample of analysis, and shortening to nearly 60 samples of analysis only needs 1min, and measured enzyme activity control errors are within 3%.
Description
(1) technical field
Tyrosine phenol lyase that the present invention relates to a kind of high flux screenings to improve levodopa synthesis capability and its prominent
The method of variant.
(2) background technology
Tyrosine phenol lyase (tyrosine phenol-lyase, TPL) also known as beta-Tyrosinase, it is a kind of phosphoric acid
Pyridoxal (pyridoxal-5 '-phosphate, PLP) dependent form lyases.TPL can be catalyzed l-tyrosine and α occurs, and β-disappears
Dereaction generates pyruvic acid, phenol and ammonia.This reaction is reversible reaction, if substituting phenol with catechol, which can be catalyzed life
At levodopa, catalytic mechanism can be indicated with following formula：
Levodopa is active material important in biologic artifact, is the key agents for treating Parkinson's disease.It is controlled
Treat Parkinson's disease principle be：The derivative of levodopa-dopamine is a kind of important nerve for treating Parkinson's disease
Mediator, but it can not be by blood-brain barrier, therefore Parkinson's disease cannot be treated by external supplement dopamine, and it is left-handed
DOPA can reach central nervous system, and be changed into dopamine under the action of decarboxylase in vivo by blood-brain barrier, from
And DOPAMINE CONTENT IN RABBIT in brain tissue is made to increase, and then play the effect for the treatment of parkinsonism.
It is unstable in view of levodopa plant extraction process production capacity, step is complicated, chemical synthesis process is more, of high cost, ring
Border is seriously polluted, and microbial method synthesis levodopa increasingly attracts attention.Tyrosine phenol lyase is catalyzed catechol, acetone
Acid and ammonia synthesis levodopa have the significant advantages such as reaction condition is mild, Atom economy is high.
As the fast development of technique for gene engineering can be carried effectively by technologies such as enzyme high flux screening, molecular modifications
High tyrosine phenol lyase catalyzes and synthesizes the efficiency of levodopa.Since tyrosine phenol lyase type present in nature is numerous
More, the mutated library mutant enormous amount that enzyme molecule house of correction obtains (usually contains 104~106Mutant), pass through traditional color
The detection means such as spectrum are difficult that efficiently screening obtains the tyrosine phenol lyase that catalytic performance improves.Therefore, it establishes quickly, accurately
Tyrosine phenol lyase high-throughput screening method be of great significance.
(3) invention content
The present invention provides one to overcome the problems such as the high vigor tyrosine phenol lyase period length of screening, heavy workload
Micromation culture of the kind based on deep-well plates and the high-throughput tyrosine phenol lyase screening technique based on the detection of microplate reader milligram ammonia,
Its mechanism is as follows：The substrate pyruvate sodium for participating in levodopa synthesis generates the 1 of a kind of colour developing in strong base solution with salicylide,
Bis- (the 2- hydroxyphenyls) -1 of 5-, 4- pentadienone products, have absorption value in 465nm wavelength.With the reduction of Sodium Pyruvate concentration, face
Color becomes faint yellow from Chinese red.If the vigor of tyrosine phenol lyase is lower, illustrate that the ability of its synthesis levodopa is weaker,
The residual concentration of Sodium Pyruvate is bigger, then chromogenic reaction liquid color is deeper, if the vigor of tyrosine phenol lyase is higher, illustrates it
The ability for synthesizing levodopa is stronger, and the residual concentration of Sodium Pyruvate is smaller, and chromogenic reaction liquid color is more shallow.
The technical solution adopted by the present invention is：
The present invention provides a kind of high-throughput screening method of tyrosine phenol lyase high dynamic strain, and the method is：(1)
Conversion reaction：The wet thallus that the fermented culture of strain to be tested (the preferably strain to be tested of the gene containing tyrosine phenol lyase) is obtained
It is added in substrate reactions liquid as strain to be tested sample, in 10-30 DEG C, 100-300rpm shaking tables reaction 20-120min (preferably 1M
HCl places 5-10min terminations reaction at 95 DEG C), reaction solution is centrifuged, supernatant is obtained；The substrate reactions liquid is dense eventually
Degree group becomes：Catechol 2-15g/L, Sodium Pyruvate 2-20g/L, ammonium acetate 5-50g/L, sodium sulfite 0.1-2g/L, EDTA-
2Na 0.1-2g/L, phosphopyridoxal pyridoxal phosphate (PLP) 0.2-2mM, pH 7.0-8.0, solvent is ultra-pure water；(2) chromogenic reaction：Take step
Suddenly (1) supernatant is added in chromogenic reaction liquid, is stored at room temperature colour developing, and light absorption value is surveyed at 465nm, bent according to Sodium Pyruvate standard
Line obtains the content of Sodium Pyruvate in supernatant, and then obtains the vigor of tyrosine phenol lyase in strain to be tested sample, to
Filter out the tyrosine phenol lyase high dynamic strain improved to levodopa synthesis capability；The chromogenic reaction liquid is by 10-
250g/L sodium hydrate aqueous solutions are with salicylide and ultra-pure water with volume ratio 3:0.1~3:5-10 is formed；The standard curve is
In the case where strain to be tested detects the same terms, the recombination bacillus coli of substrate reactions liquid and the gene containing tyrosine phenol lyase is converted
Supernatant after reaction carries out chromogenic reaction, with a concentration of abscissa of Sodium Pyruvate, is drawn by ordinate of light absorption value at 465nm
It forms.
Further, the substrate reactions liquid final concentration group becomes：Catechol 4-8g/L, Sodium Pyruvate 4-10g/L, acetic acid
Ammonium 30-50g/L, sodium sulfite 0.5-1g/L, EDTA-2Na 1-2g/L, phosphopyridoxal pyridoxal phosphate 0.5-1mM, pH 7.0-8.0, solvent
For ultra-pure water, more preferable substrate reactions liquid final concentration group becomes：Catechol 5g/L, Sodium Pyruvate 5g/L, ammonium acetate 50g/L,
Sodium sulfite 1g/L, EDTA-2Na 2g/L, PLP 1mM, solvent are ultra-pure water.
Further, the chromogenic reaction liquid by 250g/L sodium hydrate aqueous solutions and salicylide and ultra-pure water with volume ratio 3:
0.1:6.7 composition.
Further, step (2) described chromogenic reaction carries out as follows：Successively it is separately added into 1ml 250g/L NaOH
Aqueous solution, 200 μ L steps (1) supernatants, 6.7ml ultra-pure waters, 100 μ L salicylides, 2ml 250g/L NaOH aqueous solutions, mixing
Uniformly, 10ml coloring reaction systems are constituted, colour developing is stored at room temperature, light absorption value is surveyed at 465nm.
Further, the Sodium Pyruvate standard curve is prepared as follows：(1) conversion reaction：Tyrosine phenol will be contained to split
The recombination bacillus coli wet thallus for solving enzyme gene is added in substrate reactions liquid, 30 DEG C, 150rpm shaking tables reaction 5min-5h, timing
Sampling (respectively 10,20,30,40,50,60,80,100,120,140,160,180,200,220,240,260,280,
Sampled when 300min) it is added after 1M HCl terminate reaction, 10min is centrifuged in 3000rpm, obtains the supernatant of differential responses time
Liquid；The substrate reactions liquid final concentration group becomes：Catechol 5g/L, Sodium Pyruvate 5g/L, ammonium acetate 50g/L, sodium sulfite
1g/L, EDTA-2Na 2g/L, phosphopyridoxal pyridoxal phosphate 1mM, solvent are ultra-pure water, pH 7.0-8.0；Final concentration is added in the wet thallus
For 4g/L；
(2) chromogenic reaction：1mL 250g/L NaOH aqueous solutions are sequentially added, 200 μ L steps (1) differential responses times were obtained
The supernatant obtained, 6.7mL ultra-pure waters, 100 μ L salicylides, 2mL 250g/L NaOH aqueous solutions are uniformly mixed, and it is anti-to constitute colour developing
System 10mL is answered, places 2h at room temperature, absorbance is measured in 465nm with microplate reader, using absorbance as ordinate, with Sodium Pyruvate
Conversion ratio is abscissa, obtains Sodium Pyruvate standard curve.
Further, the recombination bacillus coli of the gene containing tyrosine phenol lyase is by nucleosides shown in SEQ ID NO.1
What the channel genes Escherichia coli of acid sequence obtained.
Further, strain to be tested fermentation process is：Strain to be tested is seeded to fermentation medium, at 20-37 DEG C, 100-
300rpm shaking table culture 6-12h obtain wet thallus；The fermentation medium group becomes：Peptone 5-20g/L, yeast powder 2-
10g/L, sodium chloride 2-10g/L, IPTG 10-30g/L, solvent are water, and pH value is natural.
Further, the strain to be tested first carries out seed activation culture before fermentation, then by seed liquor with volumetric concentration 2-
5% inoculum concentration is seeded to fermentation medium, and the seed activation cultural method is：Strain to be tested is seeded to seed culture
Base obtains seed liquor at 30-37 DEG C, 100-300rpm shaking table culture 10-14h；The seed culture medium group becomes：Peptone
5-20g/L, yeast powder 2-10g/L, sodium chloride 2-10g/L block that enzyme element 50-100 μ g/mL, and solvent is water, and pH value is natural.
Further, the method is reacted in 96 orifice plates, and 96 orifice plate is deep-well plates, the silica gel hole of deep-well plates
Hole is corresponding with the deep hole of deep-well plates on pad, ensure each micropore independently with extraneous exchange of air.Culture medium liquid amount is deep-well plates
The 30%-50% of pore volume, inoculum concentration are the 20%-50% of fermentation medium volume.
Further, the method is：(1) strain to be tested is inoculated in the deep-well plates I equipped with seed culture medium, in 30-
37 DEG C, 100-300rpm shaking table culture 10-14h, obtain seed liquor；(2) seed liquor in deep-well plates I is accordingly inoculated into dress
Have in the deep-well plates II of fermentation medium, in 20-37 DEG C, 100-300rpm shaking table cultures 6-12h；Deep-well plates II is positioned over hole
Plate centrifuge, 1000-3000 × g centrifuge 10-30min, discard supernatant liquid；(3) 100-400 μ L substrates are added in deep-well plates II
Reaction solution, 10-30 DEG C, 100-300rpm shaking tables react 20min-2h, and 100-400 μ L 1M HCl are added and terminate reaction；By deep hole
Plate II is positioned over orifice plate centrifuge, and 1000-3000 × g centrifuges 10-30min, obtains supernatant；(4) successively divide to deep-well plates III
Not Jia Ru 1ml 250g/L NaOH aqueous solutions, 200 μ L, 6.7ml ultra-pure waters of step (3) supernatant, 100 μ L salicylides, 2ml
250g/L NaOH aqueous solutions are uniformly mixed, and constitute 10ml coloring reaction systems, are placed 20min-2h at room temperature and develop the color instead
It answers, with light absorption value at microplate reader detection 465nm, the content of Sodium Pyruvate in supernatant is obtained according to Sodium Pyruvate standard curve, into
And conversion ratio and vigor of the recombinant bacterium to substrate are obtained, when vigor is higher than 20% or more control strain, screening, which obtains, contains tyrosine
The bacterial strain that phenols cracking enzyme activity improves.
Since tyrosine phenol lyase vigor is higher, the content for reacting remaining Sodium Pyruvate is lower, dense according to Sodium Pyruvate
Degree obtains the content of Sodium Pyruvate in supernatant with the correspondence of light absorption value, and it is left-handed to obtain the synthesis of tyrosine phenol lyase recombinant bacterium
The ability of DOPA is determined as the bacterial strain of vigor raising when the vigor is higher than 20% or more of starting strain, into traveling one
Efficient liquid phase chromatographic analysis is walked to determine.
The strain to be tested includes the recombination bacillus coli of gene containing tyrosine phenol lyase and its mutator, the junket
Propylhomoserin phenols cracking enzyme gene sequence source is unlimited.
The present invention provides a kind of high pass for the bacterial strain containing tyrosine phenol lyase for screening and being improved to levodopa synthesis capability
Amount method, this method generate a kind of colour developing in strong base solution based on the substrate pyruvate sodium that levodopa synthesizes with salicylide
1,5- bis- (2- hydroxyphenyls) -1,4- pentadienone products carry out, and Sodium Pyruvate concentration is different, and colour developing degree is different, according to acetone
The correspondence of sour na concn and light absorption value at 465nm, quickly screening obtain the tyrosine phenol that levodopa synthesis capability improves
Lyases and its mutant.
Compared with prior art, advantageous effect of the present invention is mainly reflected in：Screening technique according to the present invention, Ke Yizhi
It connects high flux screening and obtains the tyrosine phenol lyase and its mutant that are improved to levodopa synthesis capability, and it is traditional efficient
Liquid phase detection method is compared, and the time needs 20min from one sample of analysis, and shortening to nearly 60 samples of analysis only needs 1min, and this
Invention comprehensive utilization porous plate carries out the fast culture of strain, fermentation, is quickly measured using microplate reader, has operation letter
Just the advantages that quick and economical and practical, automatic and mechanical operation easy to implement, to efficiently synthesize the junket ammonia of levodopa, are detected
The quick screening of sour phenols cracking enzyme brings great convenience, and the high-throughput screening method is sensitive effectively, with high-efficient liquid phase technique phase
Compare, measured enzyme activity control errors can be sieved directly within 3%, and compared to the bacterial strain of control strain enzyme activity raising 20%
Choosing obtains.
(4) it illustrates
Fig. 1 is the high flux screening flow of tyrosine phenol lyase and its high vigor bacterium.
Fig. 2 is the correspondence of Sodium Pyruvate concentration and light absorption value during high flux screening.
Fig. 3 is that light absorption value of the different concentration of substrate in different developing times changes over time song to high flux screening in the process
Line；Sodium Pyruvate concentration：0mM (■), 2.5mM (●), 5mM (▲), 10mM (◆), 20mM (▼), 40mM (), 60mM
(zero), 80mM (△), 100mM (◇).
Fig. 4 is the correspondence of Sodium Pyruvate concentration and light absorption value in simulation reaction process when being not added with thalline.
Fig. 5 is the correspondence of light absorption value and conversion ratio in reaction process when thalline is added.
(5) specific implementation mode
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Ultra-pure water ultra-pure water described in the embodiment of the present invention (Ultrapure water) is also known as UP water, refers to that resistivity reaches
The water of 18M Ω * cm (25 DEG C).
The correspondence of embodiment 1 Sodium Pyruvate concentration and light absorption value
With the third of ultra-pure water configuration 0-100mM (0mM, 2.5mM, 5mM, 10mM, 20mM, 40mM, 60mM, 80mM, 100mM)
Ketone acid sodium solution.Reaction system 10mL is successively separately added into 1mL 250g/L NaOH aqueous solutions, the acetone of 200 μ L various concentrations
Acid sodium solution, 6.7mL ultra-pure waters, 100 μ L salicylides, 2mL 250g/L NaOH aqueous solutions, wherein being shaken up after salicylide is added
Make itself and the abundant reaction solution of Sodium Pyruvate.2h is placed at room temperature, and absorbance is measured in 465nm with microplate reader.It is with absorbance
Ordinate, a concentration of abscissa of Sodium Pyruvate draw light absorption curve, as a result such as Fig. 2.The concentration of Sodium Pyruvate is higher, light absorption value
It is bigger, and the two is in a linear relationship：Y=0.0434X+0.3046, R2=0.9998.
The determination of 2 chromogenic reaction time of embodiment
With the third of ultra-pure water configuration 0-100mM (0mM, 2.5mM, 5mM, 10mM, 20mM, 40mM, 60mM, 80mM, 100mM)
Ketone acid sodium solution.Reaction system 10mL is successively separately added into 1mL 250g/L NaOH aqueous solutions, the acetone of 200 μ L various concentrations
Acid sodium solution, 6.7mL ultra-pure waters, 100 μ L salicylides, 2mL 250g/L NaOH aqueous solutions are uniformly mixed.It places at room temperature, often
Every 10min samplings microplate reader absorbance, reaction time 2h are measured in 465nm.Using absorbance as ordinate, the chromogenic reaction time
For abscissa, light absorption curve is drawn, as a result such as Fig. 3.Show more than chromogenic reaction 1h, light absorption value tends towards stability substantially.
Embodiment 3 is not added under thalline reaction system, the correspondence of Sodium Pyruvate concentration and light absorption value
Substrate reactions liquid (pH 7.0-8.0) final concentration group becomes：Catechol 5g/L, Sodium Pyruvate (be respectively 0,1,
2.5,5,7.5,10,15,20,30,40g/L), ammonium acetate 50g/L, sodium sulfite 1g/L, EDTA-2Na2g/L, phosphopyridoxal pyridoxal phosphate
(PLP) 1mM, solvent are ultra-pure water.Various concentration Sodium Pyruvate reacts 30min successively in 30 DEG C, 150rpm shaking tables, is separately added into
400 μ L 1M HCl terminate reaction, obtain the reaction solution of various concentration Sodium Pyruvate.
Chromogenic reaction step is：Coloring reaction system 10mL, is successively separately added into 1mL 250g/L NaOH aqueous solutions, and 200
The reaction solution of μ L difference Sodium Pyruvate concentration, 6.7mL ultra-pure waters, 100 μ L salicylides, 2mL 250g/L NaOH aqueous solutions, mixing
Uniformly.2h is placed at room temperature, and absorbance is measured in 465nm with microplate reader.Respectively using absorbance as ordinate, Sodium Pyruvate concentration
For abscissa, curve is drawn, as a result such as Fig. 4.Show under the conditions of being not added with thalline, the light absorption value under different Sodium Pyruvate concentration and
Sodium Pyruvate concentration is linear：Y=0.0282X+0.1630, R2=0.9973.
Embodiment 4 is added under thalline reaction system, the correspondence of Sodium Pyruvate concentration and light absorption value
(1) structure of the tyrosine phenol lyase gene recombined escherichia coli containing Fusobacterium nucleatum source：It is described to contain tool
Core Fusobacterium source tyrosine phenol lyase gene recombined escherichia coli is by tyrosine phenol lyase encoding gene (SEQ ID
Shown in NO.1) it imports Escherichia coli structure and obtains, specific construction method bibliography (Enzyme Microb Tech, 2018,
266:20-26)。
The tyrosine phenol lyase recombination bacillus coli of source containing Fusobacterium nucleatum cultivates acquisition in the following manner：It will
Recombinant bacterium is cultivated to OD for 37 DEG C in LB culture mediums600After 0.6~0.8, the IPTG of 1mmol/L is added, 10- is induced at 28 DEG C
12h.After culture, wet thallus is collected by centrifugation, with 0.85% brine 2 times.
(2) substrate reactions liquid (pH 7.0-8.0) final concentration group becomes：Catechol 5g/L, Sodium Pyruvate 5g/L, acetic acid
Ammonium 50g/L, sodium sulfite 1g/L, EDTA-2Na 2g/L, PLP 1mM, solvent is ultra-pure water.
Specifically conversion reaction step is：400 μ L of substrate reactions liquid are drawn, is added and contains Fusobacterium nucleatum source tyrosine phenol
Lyases recombinates large intestine wet thallus (final concentration of 4g/L is added in wet thallus), and thalline and substrate reactions liquid is made to be uniformly mixed, 30 DEG C,
150rpm shaking tables react 5min-5h (be respectively 10,20,30,40,50,60,80,100,120,140,160,180,200,220,
240,260,280,300min), it is added after 400 μ L 1M HCl terminate reaction, centrifuges 10min in 3000rpm, take differential responses
The supernatant of time carries out chromogenic reaction.
(4) chromogenic reaction
Coloring reaction system 10mL is successively separately added into 1mL 250g/L NaOH aqueous solutions, 200 μ L differential responses times
The supernatant of acquisition, 6.7mL ultra-pure waters, 100 μ L salicylides, 2mL 250g/L NaOH aqueous solutions are uniformly mixed.It places at room temperature
2h measures absorbance with microplate reader in 465nm, and high performance liquid chromatography surveys Sodium Pyruvate conversion ratio.It is respectively vertical with absorbance
Coordinate, Sodium Pyruvate conversion ratio are abscissa, curve are drawn, as a result such as Fig. 5.Show under the conditions of thalline is added, Sodium Pyruvate
Conversion ratio is linear with light absorption value：Y=-0.01076X+1.2423, R2=1.
Liquid phase testing conditions are as follows：Liquid chromatogram is Shimadzu LC-16 (Japan), and chromatographic column is：C18 columns (Welch,
5μm×250×4.6mm)；Column temperature：34℃；Flow velocity：1mL/min；Sample size：10mL；Detection wavelength：UV210nm；Mobile phase：
20mM KH2PO4(HCl tune pH to 2.6)：Methanol=9:1.
The comparison of 5 high-throughput screening method of the present invention of embodiment and traditional HPLC analytical method
(1) recombination bacillus coli for the gene containing tyrosine phenol lyase that 4 method of Example obtains is as strain to be tested
The LB agar mediums of that resistance of card containing 100ug/ml are coated on, 37 DEG C of culture 12h, the single bacterium colony of acquisition is as junket ammonia to be screened
Sour phenols cracking enzyme high dynamic strain；
(2) it under aseptic condition, takes the single bacterium colony of tyrosine phenol lyase high dynamic strain to be screened to be inoculated in and is trained containing seed
Support base in 96 orifice plates, it is for use after activation；
(3) bacterium solution after activation is inoculated in by volumetric concentration 2% containing fermentation medium in 96 orifice plates, 28 DEG C of cultures
10h, 1000-3000 × g centrifuge 30min, obtain thalline；
(4) be added substrate reactions liquid 400 μ L in 96 orifice plates that step (3) obtains thalline, 30 DEG C, 150rpm shaking tables it is anti-
It answers, separately sampled after reaction 10,20,30,40,50,60min, samples taken places 5-10min at 95 DEG C and terminates reaction,
3000 × g centrifuges 10min.Above-mentioned substrate reactions liquid (pH 8.0) final concentration group becomes：Catechol 5g/L, Sodium Pyruvate 8g/
L, ammonium acetate 50g/L, sodium sulfite 2g/L, EDTA-2Na 1g/L, PLP 1mM, solvent is ultra-pure water；
(5) supernatant after taking step (4) to centrifuge carries out chromogenic reaction 1h, and absorbance is measured in 465nm with microplate reader, on
It is 10ml to state coloring reaction system, is successively separately added into 1ml 250g/L NaOH aqueous solutions, 200 μ L are by step (4) differential responses
The supernatant after sample centrifugation acquired by time, 6.7ml ultra-pure waters, 100 μ L salicylides, 2ml 250g/L NaOH aqueous solutions,
It is uniformly mixed.The Sodium Pyruvate concentration and light absorption value correspondence curve Y=-0.01076X+ obtained according to embodiment 4
1.2423, the content for obtaining Sodium Pyruvate in supernatant is calculated, conversion ratio of the recombinant bacterium to substrate is further obtained, screening obtains
The higher bacterial strain of tyrosine phenol lyase vigor.
(6) while the supernatant after taking sample acquired by step (4) the differential responses time to centrifuge is carried out at the same time efficient liquid
Analysis of hplc.The liquid phase testing conditions of Sodium Pyruvate are as follows：Liquid chromatogram is Shimadzu LC-16 (Japan), chromatographic column
For：C18 columns (Welch, 5 250 × 4.6mm of μ m)；Column temperature：34℃；Flow velocity：1mL/min；Sample size：10mL；Detection wavelength：
UV 210nm；Mobile phase：20mM KH2PO4(HCl tune pH to 2.6)：Methanol=9:1.Under above-mentioned chromatographic condition, Sodium Pyruvate
Appearance time be 3.37min.It takes supernatant to be measured to dilute 20 times, is analyzed using high performance liquid chromatography, calculate recombination
Conversion ratio of the bacterium to substrate.
This high-throughput screening method and traditional HPLC analytical method comparing result such as table 1, with the reaction time
Extension, catechol conversion ratio improves, and Sodium Pyruvate concentration reduces, and absorbance declines, and by measured by chromogenic reaction
Conversion ratio and the conversion ratio positive correlation measured by high performance liquid chromatography, test method is accurate, and error range is within 3%, card
The feasibility of 96 orifice plate enzyme linked immunosorbent assays is illustrated.
The comparison of the surveyed Sodium Pyruvate conversion ratio of distinct methods under the 1 same reaction time of table
By the comparison (being shown in Table 2) of 2 kinds of method detection times, the time difference of sample pre-treatments ignores.
2 this method of table, the comparison of high performance liquid chromatography detection time
| This method | High performance liquid chromatography | |
| Detection time (57 samples)/min | 1 | 1140 |
This high-throughput screening method (selects 96 orifice plates), and each sample makees parallel three times, one piece of 96 orifice plate 57 sample of detection
Product, detection time are about 1min.The program of high performance liquid chromatography is each sample 20min, and 57 samples of detection need about
1140min.Therefore, the high-throughput screening method that the present invention is established, greatly reduces screening time, improves screening efficiency.
The verification that embodiment 6 screens multiple unknown strains
(1) it under aseptic condition, takes unknown single bacterium colony to be screened (fund containing tyrosine phenol lyase) to be inoculated in and is trained containing seed
It is for use after activation in 96 orifice plates for supporting base；
(2) bacterium solution after activation is inoculated in by volumetric concentration 2% containing fermentation medium in 96 orifice plates, 28 DEG C of cultures
10h, 1000-3000 × g centrifuge 30min, obtain thalline；
(3) be added substrate reactions liquid 400 μ L in 96 orifice plates that step (2) obtains thalline, 30 DEG C, 150rpm shaking tables it is anti-
It answers, separately sampled after reacting 30min, samples taken places 5-10min at 95 DEG C and terminates reaction, 3000 × g centrifugations
10min.Above-mentioned substrate reactions liquid (pH 8.0) final concentration group becomes：Catechol 5g/L, Sodium Pyruvate 8g/L, ammonium acetate 50g/
L, sodium sulfite 1g/L, EDTA-2Na 2g/L, PLP 1mM, solvent are ultra-pure water；
(4) supernatant after taking step (3) to centrifuge carries out chromogenic reaction 1h, and absorbance is measured in 465nm with microplate reader, on
It is 1ml to state coloring reaction system, is successively separately added into 100 μ l 250g/L NaOH aqueous solutions, 20 μ L are by step (3) differential responses
The supernatant after sample centrifugation acquired by time, 670 μ l ultra-pure waters, 10 μ L salicylides, 200 μ l 250g/L NaOH are water-soluble
Liquid is uniformly mixed.The Sodium Pyruvate concentration and light absorption value correspondence curve Y=-0.01076X+ obtained according to embodiment 4
1.2423, the concentration for obtaining Sodium Pyruvate in supernatant is calculated, conversion ratio of the recombinant bacterium to substrate is further obtained, obtains junket ammonia
The sour higher bacterial strain of phenols cracking enzyme activity.
(5) supernatant after taking the sample acquired by step (3) to centrifuge is carried out at the same time efficient liquid phase chromatographic analysis.Pyruvic acid
Sodium liquid phase testing conditions are as follows：Liquid chromatogram is Shimadzu LC-16 (Japan), and chromatographic column is：C18 columns (Welch, 5 μ ms
250×4.6mm)；Column temperature：34℃；Flow velocity：1mL/min；Sample size：10mL；Detection wavelength：UV210nm；Mobile phase：20mM
KH2PO4(HCl tune pH to 2.6)：Methanol=9:1.Under above-mentioned chromatographic condition, the appearance time of Sodium Pyruvate is 3.37min.It takes
Supernatant to be measured dilutes 20 times, is analyzed using high performance liquid chromatography, calculates conversion ratio of the unknown bacterium to substrate.
The comparison of the surveyed Sodium Pyruvate conversion ratio of 3 96 orifice plate the first row of table, 12 plants of unknown strains distinct methods
High flux screening is carried out to strain to be tested, the results show that turn that the chromogenic reaction established through the invention measures
Rate and the conversion ratio positive correlation measured by high performance liquid chromatography, test method are accurate.
Tyrosine phenol lyase source according to the present invention is not limited only to the Fusobacterium nucleatum source that embodiment is previously mentioned
Enzyme, but the tyrosine phenol lyase and its mutant to other sources are generally applicable in.
The present invention is not specifically limited text.The present invention can make in the range of claims are summarized
Various changes, these changes are all within the scope of the present invention.
Sequence table
<110>Zhejiang Polytechnical University
<120>A kind of high-throughput screening method of tyrosine phenol lyase high dynamic strain
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1383
<212> DNA
<213>Unknown (Unknown)
<400> 1
atgagatttg aagattatcc agcagagcca tttagaatta aaagtgtaga aactgttaaa 60
atgattgata aggcagcaag agaagaagta attaaaaaag caggatataa tactttctta 120
attaactctg aagatgttta cattgattta ttaactgata gtggaactaa tgctatgagt 180
gataaacaat ggggtggatt aatgcaaggt gatgaagctt atgcaggaag tagaaatttc 240
ttccacttag aagaaactgt aaaagaaata tttgggttta aacatatagt tcctactcac 300
caaggaagag gagcagaaaa tattttatct caaatagcta taaaacctgg acaatatgtt 360
cctggaaata tgtattttac aactactaga tatcaccaag aaagaaatgg tggaatattt 420
aaagatatta tcagagatga ggcacatgat gctactctta atgttccttt caaaggagat 480
attgacttaa ataaattaca aaaattaata gatgaagttg gagcagaaaa cattgcttat 540
gtttgtttag ctgtaactgt aaaccttgct ggtggacaac cagtttctat gaaaaatatg 600
aaagcagtta gagaactaac taaaaaacat ggaataaaag ttttctatga tgcaactaga 660
tgtgttgaaa atgcttactt cattaaagaa caagaagaag gatatcaaga taaaactata 720
aaggaaatag tgcatgaaat gtttagctat gctgatggat gtactatgag tggtaaaaaa 780
gattgtcttg ttaatatagg tggattttta tgtatgaatg atgaagattt attcttagct 840
gcaaaagaaa tagttgttgt ttatgaaggt atgccatctt atggtggact tgctggtaga 900
gatatggaag ctatggcaat agggttaaga gaatctttac aatatgaata cattagacat 960
agaattttac aagttagata cttaggagaa aaattaaaag aagctggtgt acctatactt 1020
gaaccagttg gaggacatgc tgtattccta gatgctagaa gattctgtcc tcatatccca 1080
caagaagaat tcccagctca agctcttgca gcagctatct atgttgaatg tggtgtaaga 1140
actatggaaa gaggaataat ttctgctggt agagatgtaa aaactggtga aaaccataaa 1200
cctaaactag aaactgttag agttactatt ccaagaagag tttatactta taaacatatg 1260
gatgtagtag cagaaggtat aatcaaatta tataaacata aagaagatat aaaaccatta 1320
gaatttgtat atgaaccaaa acaattaaga ttctttacag ctagatttgg aataaaaaaa 1380
taa 1383
Claims (10)
1. a kind of high-throughput screening method of tyrosine phenol lyase high dynamic strain, it is characterised in that the method is：(1) turn
Change reaction：The wet thallus that the fermented culture of strain to be tested obtains is added as strain to be tested sample in substrate reactions liquid, in 10-
30 DEG C, 100-300rpm shaking tables reaction 20-120min, reaction solution is centrifuged, supernatant is obtained；The substrate reactions liquid final concentration
Group becomes：Catechol 2-15g/L, Sodium Pyruvate 2-20g/L, ammonium acetate 5-50g/L, sodium sulfite 0.1-2g/L, EDTA-
2Na 0.1-2g/L, phosphopyridoxal pyridoxal phosphate 0.2-2mM, pH 7.0-8.0, solvent is ultra-pure water；(2) chromogenic reaction：Take step (1)
Supernatant is added in chromogenic reaction liquid, is stored at room temperature colour developing, and light absorption value is surveyed at 465nm and is obtained according to Sodium Pyruvate standard curve
The content of Sodium Pyruvate in supernatant is obtained, and then obtains the vigor of tyrosine phenol lyase in strain to be tested sample, to screen
Go out tyrosine phenol lyase high dynamic strain；The chromogenic reaction liquid by 10-250g/L sodium hydrate aqueous solutions and salicylide and
Ultra-pure water is with volume ratio 3:0.1-3:5-10 is formed；The standard curve is in the case where strain to be tested detects the same terms, by substrate
Reaction solution carries out chromogenic reaction with the supernatant after the recombination bacillus coli conversion reaction of the gene containing tyrosine phenol lyase, with third
Ketone acid na concn is abscissa, is drawn as ordinate using light absorption value at 465nm.
2. the high-throughput screening method of the high dynamic strain containing tyrosine phenol lyase as described in claim 1, it is characterised in that institute
Stating substrate reactions liquid final concentration group becomes：Catechol 4-8g/L, Sodium Pyruvate 4-10g/L, ammonium acetate 30-50g/L, sulfurous acid
Sodium 0.5-1g/L, EDTA-2Na 1-2g/L, phosphopyridoxal pyridoxal phosphate 0.5-1mM, pH 7.0-8.0, solvent is ultra-pure water.
3. the high-throughput screening method of the high dynamic strain containing tyrosine phenol lyase as described in claim 1, it is characterised in that institute
Chromogenic reaction liquid is stated by 250g/L sodium hydrate aqueous solutions and salicylide and ultra-pure water with volume ratio 3:0.1:6.7 composition.
4. the high-throughput screening method of the high dynamic strain containing tyrosine phenol lyase as described in claim 1, it is characterised in that step
Suddenly (2) described chromogenic reaction carries out as follows：Successively it is separately added into 1ml 250g/L NaOH aqueous solutions, 200 μ L steps
(1) supernatant, 6.7ml ultra-pure waters, 100 μ L salicylides, 2ml 250g/L NaOH aqueous solutions are uniformly mixed, and constitute 10ml colour developings
Reaction system is stored at room temperature colour developing, and light absorption value is surveyed at 465nm.
5. the high-throughput screening method of the high dynamic strain containing tyrosine phenol lyase as described in claim 1, it is characterised in that institute
Sodium Pyruvate standard curve is stated to be prepared as follows：(1) conversion reaction：By the recombination large intestine of the gene containing tyrosine phenol lyase
Bacillus wet thallus is added in substrate reactions liquid, and 30 DEG C, 150rpm shaking tables reaction 5min-5h, timing sampling are added 1M HCl and terminate
After reaction, 10min is centrifuged in 3000rpm, obtains the supernatant of differential responses time；The substrate reactions liquid final concentration composition
For：Catechol 5g/L, Sodium Pyruvate 5g/L, ammonium acetate 50g/L, sodium sulfite 1g/L, EDTA-2Na2g/L, phosphopyridoxal pyridoxal phosphate
1mM, solvent are ultra-pure water, pH 7.0-8.0；Final concentration of 4g/L is added in the wet thallus；
(2) chromogenic reaction：1mL 250g/L NaOH aqueous solutions are sequentially added, what 200 μ L steps (1) differential responses times obtained
Supernatant, 6.7mL ultra-pure waters, 100 μ L salicylides, 2mL 250g/L NaOH aqueous solutions, 900 μ L ultra-pure waters are uniformly mixed, structure
At coloring reaction system 10mL, 2h is placed at room temperature, and absorbance is measured in 465nm with microplate reader, using absorbance as ordinate, with
A concentration of abscissa of Sodium Pyruvate obtains Sodium Pyruvate standard curve.
6. the high-throughput screening method of the high dynamic strain containing tyrosine phenol lyase as claimed in claim 5, it is characterised in that institute
The recombination bacillus coli for stating the gene containing tyrosine phenol lyase is that the channel genes of nucleotide sequence shown in SEQ ID NO.1 are big
What enterobacteria obtained.
7. the high-throughput screening method of the high dynamic strain containing tyrosine phenol lyase as described in claim 1, it is characterised in that wait for
Surveying strain fermentation method is：Strain to be tested is seeded to fermentation medium, at 20-37 DEG C, 100-300rpm shaking table cultures 6-
12h obtains wet thallus；The fermentation medium group becomes：Peptone 5-20g/L, yeast powder 2-10g/L, sodium chloride 2-10g/
L, IPTG 10-30g/L, solvent are water, and pH value is natural.
8. the high-throughput screening method of the high dynamic strain containing tyrosine phenol lyase as claimed in claim 7, it is characterised in that institute
It states strain to be tested and first carries out seed activation culture before fermentation, then seed liquor is seeded to the inoculum concentration of volumetric concentration 2-5%
Fermentation medium, the seed activation cultural method are：Strain to be tested is seeded to seed culture medium, at 30-37 DEG C, 100-
300rpm shaking table culture 10-14h obtain seed liquor；The seed culture medium group becomes：Peptone 5-20g/L, yeast powder 2-
10g/L, sodium chloride 2-10g/L block that enzyme element 50-100 μ g/mL, and solvent is water, and pH value is natural.
9. the high-throughput screening method of the high dynamic strain containing tyrosine phenol lyase as described in claim 1, it is characterised in that institute
Method is stated to be reacted in 96 orifice plates.
10. the high-throughput screening method of the high dynamic strain containing tyrosine phenol lyase as claimed in claim 9, it is characterised in that institute
The method of stating is：(1) strain to be tested is inoculated in the deep-well plates I equipped with seed culture medium, in 30-37 DEG C, 100-300rpm shakes
Bed culture 10-14h, obtains seed liquor；(2) seed liquor in deep-well plates I is accordingly inoculated into the depth equipped with fermentation medium
In orifice plate II, in 20-37 DEG C, 100-300rpm shaking table cultures 6-12h；Deep-well plates II is positioned over orifice plate centrifuge, 1000-
3000 × g centrifuges 10-30min, discards supernatant liquid；(3) 100-400 μ L substrate reactions liquids, 10-30 are added in deep-well plates II
DEG C, 100-300rpm shaking tables react 20min-2h, and 100-400 μ L 1M HCl are added and terminate reaction；Deep-well plates II is positioned over hole
Plate centrifuge, 1000-3000 × g centrifuge 10-30min, obtain supernatant；(4) it is separately added into 1ml to III priority of deep-well plates
250g/L NaOH aqueous solutions, 200 μ L, 6.7ml ultra-pure waters of step (3) supernatant, 100 μ L salicylides, 2ml 250g/L NaOH
Aqueous solution is uniformly mixed, and constitutes 10ml coloring reaction systems, is placed 20min-2h at room temperature and is carried out chromogenic reaction, uses microplate reader
Light absorption value at 465nm is detected, the content of Sodium Pyruvate in supernatant is obtained according to Sodium Pyruvate standard curve, screening, which obtains, contains junket
The bacterial strain that propylhomoserin phenols cracking enzyme activity improves.
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| CN110331153A (en) * | 2019-06-24 | 2019-10-15 | 浙江工业大学 | A kind of gram Lyu Wall Salmonella tyrosine phenol lyase mutant and its application |
| CN112662732A (en) * | 2020-12-04 | 2021-04-16 | 浙江工业大学 | High-throughput rapid screening method of pantothenate synthetase mutants |
| CN114250237A (en) * | 2020-09-23 | 2022-03-29 | 浙江工业大学 | Tyrosine phenol lyase mutant, engineering bacteria and application of tyrosine phenol lyase mutant in catalytic synthesis of levodopa |
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| CN109580604A (en) * | 2018-11-28 | 2019-04-05 | 江南大学 | A kind of method of high-throughput detection naringenin |
| CN109929889A (en) * | 2019-03-29 | 2019-06-25 | 安徽华恒生物科技股份有限公司 | A kind of preparation method of l-tyrosine |
| CN110331153A (en) * | 2019-06-24 | 2019-10-15 | 浙江工业大学 | A kind of gram Lyu Wall Salmonella tyrosine phenol lyase mutant and its application |
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