CN104232734A - High-throughput screening method of high-yield microbial strains for adenosine - Google Patents
High-throughput screening method of high-yield microbial strains for adenosine Download PDFInfo
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Abstract
The invention discloses a high-throughput screening method of high-yield microbial strains for adenosine. The method comprises steps as follows: inoculating single colonies into a deep-pore plate I containing a liquid seed culture medium for shaking culture; then correspondingly inoculating a seed solution in pores of the deep-pore plate I into a deep-pore plate II containing a fermentation culture medium; after shaking culture, centrifugalizing the deep-pore plate II, taking a supernatant, and measuring the content of the adenosine; adding the fermentation liquor supernatant, a buffer solution and moderate adenosine deaminase into micro-pores of an ELISA plate for a warm-bath reaction at the temperature of 30-40 DEG C for 10-40 min; after the reaction, adding a reaction liquid A and a reaction liquid B immediately for a warm-bath reaction at the temperature of 30-40 DEG C for 20-50 min, then measuring absorbance values of the reaction liquids by an ELISA, and calculating the yield of the adenosine according to a standard curve and the dilution ratio of the fermentation liquor supernatant. The purposes of high-throughput culture, high-throughput preparation of the fermentation liquor and high-throughput measurement of the adenosine are achieved with the method.
Description
Technical field
The present invention relates to a kind of high-throughput screening method of adenosine high yield microorganism strains, belong to Microbial Breeding and fermentation engineering field.
Background technology
Adenosine (adenosine) i.e. adenosine is a kind of endogenous nucleoside spreading all over human body cell, directly can enter cardiac muscle and generate adenylic acid (AMP) through phosphorylation, participate in energy metabolism of myocardial, also participate in dilating coronary blood vessel simultaneously, increase volume of blood flow.Adenosine to cardiovascular system unify human body other systems many and organize and all have physiological action; it is the conventional medicine of emergency Treatment tachyarrhythmia and pharmacological stress test; confirm that it has cardioprotection; the tolerance of cardiac muscle to ischemic can be strengthened; reduce the reperfusion injury of cardiac muscle, reduce infarct size.Adenosine simultaneously or a kind of important medicine intermediate, can synthesize multiple medical nucleotide class material, as 8-nitrogen adenosine, Triphosaden (ATP), adenylic acid (AMP), vidarabine etc.
At present, the production of adenosine is mainly that raw material carries out chemosynthesis with inosine, production cost is still higher, for 35-40 ten thousand yuan/about t, then cost is very low to adopt saccharine material direct fermentation to produce adenosine, if fermentation level reaches more than 20g/L, extract total recovery more than 80%, then production cost can be controlled in 100,000 yuan/below t.Therefore, if the fermentation method industrial production of adenosine succeeds and the raising of fermentation level, as Inosine Fermentation, very large pushing effect will be played to the nucleosides fermentation industry of China.And the core of industrial fermentation production adenosine is the microbial strains of excellent.
In microbial metabolism, adenosine can be released in substratum as product, in order to verify the ability of microorganisms producing adenosine, needs the content measuring adenosine in substratum.The common method that current adenosine measures mainly comprises Paper Chromatography and high performance liquid chromatography.It is not high that Paper Chromatography detects adenosine precision, and organic solvent used is as acetone etc., generally has volatility and has certain toxicity.High performance liquid chromatography can detect micro-adenosine, and precision and accuracy are very high, but expensive equipment, operation and maintenance are loaded down with trivial details.In addition, also have and cause resonant light scattering signal to amplify the adenosine new detecting method set up based on adenosine specific recognition fit with it and the self-assembly of adenosine fit coupling golden nanometer particle, but still be in the laboratory study stage.Although patent CN103320326A establishes a kind of high-throughput screening method, namely compared by full wavelength scanner.This method affects very large by medium component, and detects sensitive not.Therefore, seek easy, quick, accurate and widely used adenosine analytical method extremely to pay attention to.
Adopt high throughput method screening adenosine superior strain, can increase work efficiency, reduce costs, breakneck acceleration can be improved significantly again.Develop a kind of easy, fast and accurately method detect the adenosine content in fermented liquid, the bacterial strain producing adenosine for high flux screening carries out adenosine and produces significant.
Summary of the invention
The workload existed in order to the ordinary method overcoming screening adenosine superior strain is large, the cycle long and high in cost of production problem, the invention provides a kind of microminiaturization based on deep-well plates and cultivates and the high-throughput screening method detected based on microplate reader milligram ammonia.The method of high flux screening adenosine superior strain is as follows:
(1) picking adenosine produces single bacterium colony of bacterium, is inoculated in and is equipped with in the deep-well plates I of liquid seed culture medium, cover deep-well plates lid, in 30-40 DEG C, 300-800 rpm shaking table cultivation 8-14 h;
(2) corresponding for the seed liquor in deep-well plates I hole being inoculated into is equipped with in the deep-well plates II of fermention medium, 30-40 DEG C, 300-800 rpm shaking table cultivation 8-48 h;
(3) deep-well plates II is positioned over orifice plate whizzer, the centrifugal 5-30 min of 5000-10000 × g, suitably dilutes fermented liquid supernatant with milligram ammonia gradient dilution method;
(4) in the micropore of enzyme plate, the fermented liquid supernatant of suitably dilution, damping fluid and appropriate adenosine deaminase is added, not add the experiment of adenosine deaminase for blank, in 30-40 DEG C of temperature bath reaction 10-40 min;
(5) reaction adds reaction solution A and reaction solution B after terminating immediately, and the addition sequence of reaction solution A and B be reaction solution A front, B rear, then in 30-40 DEG C of temperature bath reaction 20-50 min;
(6) after step (5) reaction terminates, adopt the absorbancy of microplate reader assaying reaction liquid, the extension rate according to adenosine typical curve and fermented liquid supernatant calculates adenosine output;
(7) according to adenosine detected result, select corresponding bacterial strain to carry out shaking flask and sieve again, obtain adenosine superior strain,
Wherein, described reaction solution A comprises sodium hydroxide 20-30 g/L, Whitfield's ointment 45-75 g/L and sodium nitroprusside 1-3 g/L, reaction solution B comprises clorox 40-60 ml/L, and the determined wavelength that step (5) reaction terminates the blue material of rear generation is 695-700 nm.
In another preference, described reaction solution A comprises phenol 15-30 ml/L and sodium nitroprusside 0.8-3 g/L, reaction solution B is for comprising sodium hydroxide 10-15 g/L and clorox 20-40 ml/L, and the determined wavelength that step (5) reaction terminates the blue material of rear generation is 625-630 nm.
In another preference, single bacterium colony that described adenosine produces bacterium is the mutant strain that nature is contained bacterium sample or obtained by mutafacient system such as physical chemistry.
In another preference, described deep hole culture plate is 96 hole depth orifice plates, and the volume in each hole is 3 mL, but adds the substratum of 0.5-2.5 mL in every hole.
In another preference, described solution seed culture medium is: yeast powder 2-10 g/L, peptone 5-15 g/L, sodium-chlor 5-15 g/L, pH7.0,121 DEG C of sterilizing 20 min.
In another preference, described fermention medium is: glucose 10 g/L, Na
2hPO
412H
2o 8.96 g/L, KH
2pO
41.5 g/L, NaCl 2.5 g/L, urea 2 g/L, L-Trp 0.05 g/L, 1000 × micro-1 mL, MgSO
47H
20 0.49 g/L, CaCl
20.011 g/L,
Wherein, described 1000 × trace element suite is divided into: MnCl
21 g/L, ZnCl
21.7 g/L, CuCl
22H
2o 0.43 g/L, CoCl
26H
2o 0.6 g/L, FeCl
38.125 g/L, Na
2moO
42H
2o 0.6 g/L,
Described each component sterilising conditions is: glucose 110 DEG C of sterilizing 20 min, urea and tryptophane filtration sterilization, other component 121 DEG C of sterilizing 20 min.
In another preference, can not contain thalline in described supernatant liquor, the supernatant liquor after dilution mixes with described damping fluid, and in the supernatant liquor after dilution, the concentration of adenosine is 0.01-10 mM.
In another preference, described enzyme reaction damping fluid used is the one among Tris-HCl damping fluid, potassium phosphate buffer, sodium phosphate buffer and HEPES damping fluid, and the pH of described damping fluid is 7.0-8.0, and concentration is 0.01-0.1 M.
In another preference, described adenosine deaminase derives from people or intestinal bacteria, adds 1-20 U adenosine deaminase during reaction.
In another preference, the volume ratio of described step (4) fermented supernatant fluid and damping fluid is 1:1-4, and/or
The volume ratio of described step (5) reaction solution A and B is 1:1-4.
In another preference, the foundation of the adenosine typical curve in described detection sample in adenosine content and described fermention medium is carried out simultaneously.
The definition that adenosine deaminase enzyme is lived: at the temperature of specifying and pH condition, per minute transforms the enzyme amount of 1 μm of ol adenosine release generation needed for ammonia, is a Ge Meihuo unit (U).
Detection method of the present invention, can detect adenosine content in fermented liquid simply, quickly and accurately, overcome apparatus expensive in prior art, complex operation, measures grade consuming time not enough.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
The method of high flux screening adenosine superior strain provided by the invention is as follows:
(1) picking adenosine produces single bacterium colony of bacterium, is inoculated in and is equipped with in the deep-well plates I of liquid seed culture medium, cover deep-well plates lid, in 30-40 DEG C, 300-800 rpm shaking table cultivation 8-14 h;
(2) corresponding for the seed liquor in deep-well plates I hole being inoculated into is equipped with in the deep-well plates II of fermention medium, 30-40 DEG C, 300-800 rpm shaking table cultivation 8-48 h;
(3) deep-well plates II is positioned over orifice plate whizzer, the centrifugal 5-30 min of 5000-10000 × g, suitably dilutes fermented liquid supernatant with milligram ammonia gradient dilution method;
(4) in the micropore of enzyme plate, the fermented liquid supernatant of suitably dilution, damping fluid and appropriate adenosine deaminase is added, not add the experiment of adenosine deaminase for blank, in 30-40 DEG C of temperature bath reaction 10-40 min;
(5) reaction adds reaction solution A and reaction solution B after terminating immediately, and the addition sequence of reaction solution A and B be reaction solution A front, B rear, then in 30-40 DEG C of temperature bath reaction 20-50 min;
(6) after step (5) reaction terminates, adopt the absorbancy of microplate reader assaying reaction liquid, the extension rate according to adenosine typical curve and fermented liquid supernatant calculates adenosine output;
(7) according to adenosine detected result, select corresponding bacterial strain to carry out shaking flask and sieve again, obtain adenosine superior strain,
Wherein, described reaction solution A comprises sodium hydroxide 20-30 g/L, Whitfield's ointment 45-75 g/L and sodium nitroprusside 1-3 g/L, reaction solution B comprises clorox 40-60 ml/L, and the determined wavelength that step (5) reaction terminates the blue material of rear generation is 695-700 nm.
In another preference, described reaction solution A comprises phenol 15-30 ml/L and sodium nitroprusside 0.8-3 g/L, reaction solution B is for comprising sodium hydroxide 10-15 g/L and clorox 20-40 ml/L, and the determined wavelength that step (5) reaction terminates the blue material of rear generation is 625-630 nm.
In another preference, single bacterium colony that described adenosine produces bacterium is the mutant strain that nature is contained bacterium sample or obtained by mutafacient system such as physical chemistry.
In another preference, described deep hole culture plate is 96 hole depth orifice plates, and the volume in each hole is 3 mL, but adds the substratum of 0.5-2.5 mL in every hole.
In another preference, described solution seed culture medium is: yeast powder 2-10 g/L, peptone 5-15 g/L, sodium-chlor 5-15 g/L, pH7.0,121 DEG C of sterilizing 20 min.
In another preference, described fermention medium is: glucose 10 g/L, Na
2hPO
412H
2o 8.96 g/L, KH
2pO
41.5 g/L, NaCl 2.5 g/L, urea 2 g/L, L-Trp 0.05 g/L, 1000 × micro-1 mL, MgSO
47H
20 0.49 g/L, CaCl
20.011 g/L,
Wherein, described 1000 × trace element suite is divided into: MnCl
21 g/L, ZnCl
21.7 g/L, CuCl
22H
2o 0.43 g/L, CoCl
26H
2o 0.6 g/L, FeCl
38.125 g/L, Na
2moO
42H
2o 0.6 g/L,
Described each component sterilising conditions is: glucose 110 DEG C of sterilizing 20 min, urea and tryptophane filtration sterilization, other component 121 DEG C of sterilizing 20 min.
In another preference, can not contain thalline in described supernatant liquor, the supernatant liquor after dilution mixes with described damping fluid, and in the supernatant liquor after dilution, the concentration of adenosine is 0.01-10 mM.
In another preference, described enzyme reaction damping fluid used is the one among Tris-HCl damping fluid, potassium phosphate buffer, sodium phosphate buffer and HEPES damping fluid, and the pH of described damping fluid is 7.0-8.0, and concentration is 0.01-0.1 M.
In another preference, described adenosine deaminase derives from people or intestinal bacteria, adds 1-20 U adenosine deaminase during reaction.
In another preference, the volume ratio of described step (4) fermented supernatant fluid and damping fluid is 1:1-4, and/or
The volume ratio of described step (5) reaction solution A and B is 1:1-4.
In another preference, the foundation of the adenosine typical curve in described detection sample in adenosine content and described fermention medium is carried out simultaneously.
In the present invention, described adenosine deaminase (ADA) derive from people or intestinal bacteria one or more.The acquisition of ADA has no particular limits, and can adopt various method known in the art.In preference, following methods is adopted to obtain ADA:
Be that template PCR obtains with genome of E.coli
addgene, and be connected to pET28 carrier (purchased from American invitrogen company) and transform BL21 (DE3) bacterial strain, add isopropylthiogalactoside (IPTG) and carry out abduction delivering, centrifugal receipts bacterium after expression 2h, ultrasonication is carried out after adding Lysis Buffer (containing PMSF and DTT), after supernatant and Balanced His Purification Resin spend the night and combine, after adding Wash Buffer repeated washing 3-4 time, add Elution Buffer eluted protein.ADA albumen due to purifying contains the imidazoles (250 mM) of high density, therefore needs to carry out dialysis treatment.Elutriant is placed in dialysis tubing and puts into the beaker filling dialyzate, 4 DEG C of dialysed overnight.The ADA protein solution of having dialysed is added 30% glycerine and be packed as 200 μ L/ pipes be placed on-20 DEG C for subsequent use.
The above-mentioned feature that the present invention mentions, or the feature that embodiment is mentioned can arbitrary combination.All features that this case specification sheets discloses can with any composition forms and use, each feature disclosed in specification sheets, anyly can be provided identical, alternative characteristics that is impartial or similar object replaces.Therefore apart from special instruction, the feature disclosed is only general example that is impartial or similar features.
The advantage that the present invention is compared with prior art had is as follows:
1, flux is detected high.So-called " high-throughput " (High throughput screening, HTS) refer to and utilize and the instrument of batch operation and the operating system that can carry out automated analysis can perform the process of experiment, and gather experimental data by the detector of rapid sensitive and carry out a kind of method of batch processing analysis.This law relates to a kind of using ADA enzyme as toolenzyme and by the absorbance of indophenol blue of reaction generation and the detection method of the output phase coupling of adenosine.This law can be carried out in the micropore of 200 μ L, utilizes 96 orifice plates and microplate reader can realize high-throughout operation, can meet the requirement of upper batch sample detection during industrial production.
2, testing cost is low.It is standing and cheap that equipment required in measuring method provided by the invention and reagent are laboratory.Compared with high performance liquid chromatography (HPLC) etc., this method eliminates expensive equipment handover charge, and can reduce the testing cost of sample further during high-throughput operation.
3, detection time is short.The detection method that present method relates to minute is about one hour, utilizes 96 orifice plates once to test the result that can obtain a large amount of sample.This law Simultaneously test standard model (referred to as mark product) and sample, in sample, the content of adenosine is by reading.In addition, this method only needs to carry out simple centrifuging and taking high fermentation liquid to sample, compared with high performance liquid chromatography (HPLC), this method substantially reduces time required for sample of detection, thus can obtain experimental result fast and and guide subsequent experimental.
4, operation steps is simple, requires low, can grasp fast experimental installation and operator.In present method, instrument is that the process software Excel etc. of multichannel pipettor, whizzer, microplate reader and data is the technical ability comparatively commonly used in laboratory, only need train simply and can grasp at short notice.
5, experimental data is accurate, and collimation is good.It is basically identical that the result that in the fermented liquid measured by the method, the content of adenosine and HPLC measure compares rear discovery, difference is within operate miss scope, and repeatability is good between sample, the difference that same sample is measured for several times remains within 5%, has higher directive significance to production.
accompanying drawing illustrates:
Fig. 1 is H
2the typical curve of adenosine in O;
Fig. 2 is the typical curve of adenosine in M9 substratum;
Fig. 3 is the result figure that application the inventive method and HPLC method detect the content of adenosine in M9 substratum fermented liquid;
Fig. 4 is fermented liquid supernatant recovery testu result figure;
Fig. 5 is that the inventive method and HPLC method detect the adenosine yield result comparison diagram applying the adenosine Producing Strain that the inventive method filters out.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.Unless otherwise indicated, otherwise per-cent and number calculate by weight.Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the same meaning be familiar with.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.
embodiment 1
A high-throughput screening method for adenosine high yield microorganism strains, is characterized in that being undertaken by following step:
(1) picking adenosine produces single bacterium colony of bacterium, is inoculated in and is equipped with in the deep-well plates I of liquid seed culture medium, cover deep-well plates lid, in 30-40 DEG C, 300-800 rpm shaking table cultivation 8h;
(2) the seed liquor correspondence in deep-well plates I hole is inoculated into and is equipped with in the deep-well plates II of fermention medium, 40 DEG C, 300 rpm shaking tables cultivate 20 h;
(3) deep-well plates II is positioned over orifice plate whizzer, centrifugal 30 min of 8000 × g, suitably dilute fermented liquid supernatant with milligram ammonia gradient dilution method;
(4) in the micropore of enzyme plate, the fermented liquid supernatant of suitably dilution, damping fluid and appropriate adenosine deaminase is added, not add the experiment of adenosine deaminase for blank, in 35 DEG C of temperature bath reaction 20 min; The volume ratio of fermented supernatant fluid and damping fluid is 1:1-4;
(5) reaction adds reaction solution A and reaction solution B after terminating immediately, and the addition sequence of reaction solution A and B be reaction solution A front, B rear, then in 35 DEG C of temperature bath reaction 30 min; The volume ratio of reaction solution A and B is 1:1-4.
(6) after step (5) reaction terminates, adopt the absorbancy of microplate reader assaying reaction liquid, the extension rate according to adenosine typical curve and fermented liquid supernatant calculates adenosine output;
(7) according to adenosine detected result, select corresponding bacterial strain to carry out shaking flask and sieve again, obtain adenosine superior strain,
Wherein, described reaction solution A comprises: sodium hydroxide 20 g/L, Whitfield's ointment 45 g/L and sodium nitroprusside 1-3 g/L; Reaction solution B comprises: clorox 40 ml/L, and the determined wavelength that step (5) reaction terminates the blue material of rear generation is 695-700 nm, or
Described reaction solution A comprises phenol 15-ml/L and sodium nitroprusside 2 g/L, reaction solution B for comprising sodium hydroxide 10 g/L and clorox 20 ml/L, and the determined wavelength that step (5) reaction terminates the blue material of rear generation is 625-630 nm.
Described deep hole culture plate is 96 hole depth orifice plates, and the volume in each hole is 3 mL, but adds the substratum of 2 mL in every hole.Described solution seed culture medium is: yeast powder 2 g/L, peptone 5 g/L, sodium-chlor 5 g/L, pH7.0,121 DEG C of sterilizing 20 min.Described fermention medium is: glucose 10 g/L, Na
2hPO
412H
2o 8.96 g/L, KH
2pO
41.5 g/L, NaCl 2.5 g/L, urea 2 g/L, L-Trp 0.05 g/L, 1000 × micro-1 mL, MgSO
47H
20 0.49 g/L, CaCl
20.011 g/L, described 1000 × trace element suite is divided into: MnCl
21 g/L, ZnCl
21.7 g/L, CuCl
22H
2o 0.43 g/L, CoCl
26H
2o 0.6 g/L, FeCl
38.125 g/L, Na
2moO
42H
2o 0.6 g/L,
Described each component sterilising conditions is: glucose 110 DEG C of sterilizing 20 min, urea and tryptophane filtration sterilization, other component 121 DEG C of sterilizing 20 min.
embodiment 2
h
2
the typical curve of adenosine in O.
Preparation reaction solution A and reaction solution B:
Reaction solution A: sodium hydroxide 26 g/L, Whitfield's ointment 68 g/L and sodium nitroprusside 2 g/L;
Reaction solution B: clorox 45 ml/L.
The final concentration that adenosine mark product are diluted to is 0.01 mM by adenosine mark product deionized water dissolving, 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM.
The adenosine standard substance of 25 μ L different concns are added, thawed on ice after the PBS damping fluid of 100 μ L 0.01M and 3U ADA(take out from-20 DEG C in the micropore of enzyme plate), in 37 DEG C of temperature bath reaction 30 min; Add 50 μ L reaction solution A and 50 μ L reaction solution B with the volley of rifle fire immediately after reaction terminates, in 37 DEG C of temperature bath reaction 30 min, then adopt the absorbance of microplate reader assaying reaction liquid at 696 nm places.Each sample does 3 repetitions.After processing data with Excel, respectively with adenosine concentration and OD
696value is transverse and longitudinal coordinate drawing standard curve, as shown in Figure 1.
embodiment 3
the typical curve of adenosine in M9 substratum.
1000 × trace element: MnCl
21 g/L; ZnCl
21.7 g/L; CuCl
22H
2o 0.43 g/L; CoCl
26H
2o 0.6 g/L; FeCl
38.125 g/L; Na
2moO
42H
2o 0.6 g/L.
M9 culture medium prescription: glucose 10 g/L; Na
2hPO
412H
2o 8.96 g/L; KH
2pO
41.5 g/L; NaCl 2.5 g/L; Urea 2 g/L; L-Trp 0.05 g/L; 1000 × micro-1 mL; MgSO
47H
2o 0.49 g/L; CaCl
20.011 g/L.
The compound method of reaction solution A and B is with embodiment 1.
Adenosine mark product M9 dissolves, and it is 0.01 mM that adenosine mark product M9 is diluted to final concentration, 0.05 mM, 0.1 mM, 0.25 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM.。
The adenosine standard substance of 25 μ L different concns are added, thawed on ice after the PBS damping fluid of 100 μ L 0.01M and 3U ADA(take out from-20 DEG C in the micropore of enzyme plate), in 37 DEG C of temperature bath reaction 30 min; Add 50 μ L reaction solution A and 50 μ L reaction solution B with the volley of rifle fire immediately after reaction terminates, in 37 DEG C of temperature bath reaction 30 min, then adopt the absorbance of microplate reader assaying reaction liquid at 696 nm places.Each sample does 3 repetitions.After processing data with Excel, respectively with adenosine concentration and OD
696value is transverse and longitudinal coordinate drawing standard curve, as shown in Figure 2.
embodiment 4
interference--free experiments
By the salt ion in conventional substratum and other composite partss, the accuracy impact on aforesaid method is studied the present embodiment.2 mM adenosines are added in system, not add any interference component as a control group, to add in table 1 after various composition as experimental group respectively, the adenosine relative value of consequence that control group measures is set to 100, after the measurement result of experimental group and control group compare, draws relative value.
Experimental result is as shown in table 1.
Table 1 different components is on the impact of ADA detected result
From above-mentioned detected result, the component in the conventional substratum of great majority does not affect the inventive method, and method of the present invention has stronger immunity from interference.The M9 substratum of the present embodiment due to containing glucose, SO
4 2-, Mg
2+, Na
+, Cl
-, H
2pO
4-do not have influential salt ion Deng to aforesaid method, therefore method of the present invention is specially adapted to the mensuration of the adenosine content in the fermented liquid obtained with the strain fermentation of this culture medium culturing.
embodiment 5
utilize ADA method and HPLC method to detect adenosine content in M9 fermention medium to compare.
In the present embodiment, seed culture medium used is yeast powder 5 g/L, peptone 10 g/L, sodium-chlor 10 g/L.Fermention medium used is with the M9 substratum in embodiment 3, and fermented bacterium is for producing adenosine subtilis.HPLC detects instrument and condition: Agilent 1200 series high performance liquid chromatograph (high pressure binary pump, UV-detector, automatic sampler, column oven, Chem station workstation); Chromatographic column: Agilent C18 post (250 mm × 416mm, 5 μm); Detector: UV-detector; Determined wavelength: 280 nm; Moving phase: water (pH=3.0): acetonitrile=94:6; Flow velocity: 0.8 mL/min; Column temperature: 35 DEG C: sample size: 10 μ L.
Get-80 DEG C of preservation of bacteria strain streak inoculations dull and stereotyped in activation, 37 DEG C of constant temperature quiescent culture 16 h.Being inoculated into by 1% inoculum size is equipped with in 250 mL Erlenmeyer flasks of 50 mL seed culture mediums, and 37 DEG C, 200 r/min, cultivate 12 h.Then be 0.2 be inoculated into and be equipped with in 250 mL Erlenmeyer flasks of 50 mL fermention mediums by initial OD 600,37 DEG C, 200 r/min, cultivate 48 h.Get a sample every 8 h, detect the adenosine content in fermented liquid respectively by ADA method and HPLC method, the results are shown in Figure 3.
By comparing found that of two kinds of methods, the result of two kinds of methods is basically identical, shows that in the present invention, detection method used has higher accuracy.
embodiment 6
recovery testu.
Add the adenosine standard substance of 0-2 mM in the 24 h fermentation broth samples of the present embodiment in embodiment 5, then the absorbance of detection reaction liquid, the results are shown in Figure 4.
As can be seen from Figure 4, closely, the detected value adding the adenosine of different concns in fermented liquid presents good linear relationship (R for the adenosine content that the detection method in the present invention detects and the adenosine content added
2=0.9951).The slope of straight line represents the rate of recovery, and the rate of recovery of present method is 100.7%.These results illustrate that the method has good accuracy and repeatability.
embodiment 7
utilize ADA method high flux screening adenosine superior strain.
The present embodiment starting strain used is the product adenosine subtilis in embodiment 5.First bacterial strain is activated by the seed culture medium in this bacterial strain embodiment 5, then carry out mutagenic exposure 1-3 min with the mutation induced by laser device of 402 nm.Mutagenic strain is with after the dull and stereotyped sorting of conventional LB, and random picking 95 single colony inoculations are that in the 96 hole depth well culture plates of 3 mL, 37 DEG C, 200 r/min cultivate 16 h to the cumulative volume that 1 mL seed culture medium is housed.Then the cumulative volume by 1% inoculum size switching 1 mL fermention medium being once housed is the 96 hole depth well culture plates of 3 mL, and 37 DEG C, 200 r/min cultivate 24 h.Then the adenosine content in fermented liquid supernatant is detected.
What table 2 showed is the adenosine content of fermented liquid supernatant in deep hole culture plate, the adenosine output of 4 bacterial strains of the adenosine high yield that Fig. 5 expression detection method and HPLC detection detection method simultaneously filter out.Result shows the good relationship of two methods.
Adenosine content in table 2 96 hole depth orifice plate
Claims (10)
1. a high-throughput screening method for adenosine high yield microorganism strains, is characterized in that being undertaken by following step:
(1) picking adenosine produces single bacterium colony of bacterium, is inoculated in and is equipped with in the deep-well plates I of liquid seed culture medium, cover deep-well plates lid, in 30-40 DEG C, 300-800 rpm shaking table cultivation 8-14 h;
(2) corresponding for the seed liquor in deep-well plates I hole being inoculated into is equipped with in the deep-well plates II of fermention medium, 30-40 DEG C, 300-800 rpm shaking table cultivation 8-48 h;
(3) deep-well plates II is positioned over orifice plate whizzer, the centrifugal 5-30 min of 5000-10000 × g, suitably dilutes fermented liquid supernatant with milligram ammonia gradient dilution method;
(4) in the micropore of enzyme plate, the fermented liquid supernatant of suitably dilution, damping fluid and appropriate adenosine deaminase is added, not add the experiment of adenosine deaminase for blank, in 30-40 DEG C of temperature bath reaction 10-40 min;
(5) reaction adds reaction solution A and reaction solution B after terminating immediately, and the addition sequence of reaction solution A and B be reaction solution A front, B rear, then in 30-40 DEG C of temperature bath reaction 20-50 min;
(6) after step (5) reaction terminates, adopt the absorbancy of microplate reader assaying reaction liquid, the extension rate according to adenosine typical curve and fermented liquid supernatant calculates adenosine output;
(7) according to adenosine detected result, select corresponding bacterial strain to carry out shaking flask and sieve again, obtain adenosine superior strain,
Wherein, described reaction solution A comprises: sodium hydroxide 20-30 g/L, Whitfield's ointment 45-75 g/L and sodium nitroprusside 1-3 g/L; Reaction solution B comprises: clorox 40-60 ml/L, and the determined wavelength that step (5) reaction terminates the blue material of rear generation is 695-700 nm, or
Described reaction solution A comprises phenol 15-30 ml/L and sodium nitroprusside 0.8-3 g/L, reaction solution B is for comprising sodium hydroxide 10-15 g/L and clorox 20-40 ml/L, and the determined wavelength that step (5) reaction terminates the blue material of rear generation is 625-630 nm.
2. screening method according to claim 1, is characterized in that single bacterium colony of described adenosine production bacterium is the mutant strain that nature is contained bacterium sample or obtained by mutafacient system such as physical chemistry.
3. screening method according to claim 1, it is characterized in that described deep hole culture plate is 96 hole depth orifice plates, the volume in each hole is 3 mL, but adds the substratum of 0.5-2.5 mL in every hole.
4. screening method according to claim 1, is characterized in that described solution seed culture medium is: yeast powder 2-10 g/L, peptone 5-15 g/L, sodium-chlor 5-15 g/L, pH7.0,121 DEG C of sterilizing 20 min.
5. screening method according to claim 1, is characterized in that described fermention medium is: glucose 10 g/L, Na
2hPO
412H
2o 8.96 g/L, KH
2pO
41.5 g/L, NaCl 2.5 g/L, urea 2 g/L, L-Trp 0.05 g/L, 1000 × micro-1 mL, MgSO
47H
20 0.49 g/L, CaCl
20.011 g/L,
Wherein, described 1000 × trace element suite is divided into: MnCl
21 g/L, ZnCl
21.7 g/L, CuCl
22H
2o 0.43 g/L, CoCl
26H
2o 0.6 g/L, FeCl
38.125 g/L, Na
2moO
42H
2o 0.6 g/L,
Described each component sterilising conditions is: glucose 110 DEG C of sterilizing 20 min, urea and tryptophane filtration sterilization, other component 121 DEG C of sterilizing 20 min.
6. screening method according to claim 1, it is characterized in that can not containing thalline in described supernatant liquor, the supernatant liquor after dilution mixes with described damping fluid, and in the supernatant liquor after dilution, the concentration of adenosine is 0.01-10 mM.
7. screening method according to claim 1, it is characterized in that described enzyme reaction damping fluid used is the one among Tris-HCl damping fluid, potassium phosphate buffer, sodium phosphate buffer and HEPES damping fluid, the pH of described damping fluid is 7.0-8.0, and concentration is 0.01-0.1 M.
8. screening method according to claim 1, is characterized in that described adenosine deaminase derives from people or intestinal bacteria, adds 1-20 U adenosine deaminase during reaction.
9. screening method according to claim 1, it is characterized in that the volume ratio of described step (4) fermented supernatant fluid and damping fluid is 1:1-4, and/or the volume ratio of described step (5) reaction solution A and B is 1:1-4.
10. screening method according to claim 1, is characterized in that the foundation of the adenosine typical curve in described detection sample in adenosine content and described fermention medium is carried out simultaneously.
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| CN108642130A (en) * | 2018-03-29 | 2018-10-12 | 浙江工业大学 | A kind of high-throughput screening method of tyrosine phenol lyase high dynamic strain |
| CN108642130B (en) * | 2018-03-29 | 2021-10-15 | 浙江工业大学 | High-throughput screening method for high-activity strain of tyrosine phenol lyase |
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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 |
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