CN109342415A - A kind of method of high-throughput detection eriodictyol - Google Patents
A kind of method of high-throughput detection eriodictyol Download PDFInfo
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- CN109342415A CN109342415A CN201811435786.4A CN201811435786A CN109342415A CN 109342415 A CN109342415 A CN 109342415A CN 201811435786 A CN201811435786 A CN 201811435786A CN 109342415 A CN109342415 A CN 109342415A
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Abstract
The invention discloses a kind of methods of high-throughput detection eriodictyol, belong to synthesising biological technical field.The color developing agent that the present invention uses is sodium hydroxide or potassium hydroxide, after color development treatment, purple is presented in eriodictyol, concentration is higher, and purple is deeper, and it is faster to develop the color, time of repose is longer, color can also deepen, end of reaction in general 5min, even if still directly can select superior strain with visual method in the presence of interfering substance naringenin.The reaction solution also can be used microplate reader and do further detection.In aqueous solution, it is proposed that handled using NaOH, best detection wavelength 550-580nm, suggest handling using KOH in colourless synthetic media, best detection wavelength 560-570nm.This method simple and effective, anti-interference, precision is high, and time and human cost are low, is suitable for high flux screening eriodictyol superior strain.
Description
Technical field
The present invention relates to a kind of methods of high-throughput detection eriodictyol, belong to synthesising biological technical field.
Background technique
Eriodictyol is synthesized after 3 ' position hydroxylatings via naringenin, and health care product is important.Eriodictyol is mostly from plant
Middle extraction, low output, price are higher.And when Microbe synthesis, the enzyme of catalysis naringenin synthesis eriodictyol is P450 enzyme, needs Huang
3 ' position hydroxylase of ketone (F3 ' H) and its coenzyme (CPR) collaboration transmitting electronics could complete hydroxylating, operate more complex.It is micro- being transformed
When biosynthesis eriodictyol, its content mostly is detected using high performance liquid chromatography (HPLC), is needed before detection using 0.22 μm
Filter filtering, then can just be obtained after 30min using methanol-water (45:55, the V/V) solution of chromatographically pure as mobile phase
Obtain testing result.Therefore, HPLC detection method flux is low, time and human cost are high.When carrying out approach transformation, especially with
It when machine is mutated, can generate thousands of to several hundred million even more mutons, relying solely on the means such as HPLC is to be difficult to realize quickly examine
It surveys.Moreover, have naringenin in intermediate product when microbial fermentation produces eriodictyol, or directly use naringenin as
Precursor, the presence of these precursors, it is also possible to influence the detection of corresponding product.Therefore it provides a kind of fast and accurately high-throughput
The method for detecting eriodictyol is of great significance for quickly detecting mountain balsam phenol content and screening eriodictyol superior strain.
Summary of the invention
To solve the above-mentioned problems, the present invention uses potassium hydroxide or sodium hydroxide as color developing agent, can with it is water-soluble
Eriodictyol in liquid, colourless synthetic media or fermentation liquid reacts, and occurs apparent chromogenic reaction, root in 5min
Eriodictyol superior strain can be quickly screened according to color change.
The first purpose of the invention is to provide a kind of methods of high-throughput detection eriodictyol, and the method includes following steps
It is rapid:
(1) color developing agent potassium hydroxide is added into the prepare liquid containing eriodictyol or sodium hydroxide carries out chromogenic reaction, is obtained
To reaction solution；
(2) with the concentration of eriodictyol in microplate reader detection reaction solution；The chromogenic reaction and microplate reader detection are in height
It is carried out on flux orifice plate.
In one embodiment of the invention, the high pass orifice includes 96 deep-well plates, 96 shallow bore hole plates, 48 deep holes
Plate, 24 deep-well plates, 12 deep-well plates.
In one embodiment of the invention, the prepare liquid containing eriodictyol includes aqueous solution containing eriodictyol, nothing
The synthetic media or bacterial strain fermentation liquor of color.
In one embodiment of the invention, the colourless synthetic media containing eriodictyol includes but is not limited to
SD, YNB, MOPS culture medium.
In one embodiment of the invention, chromogenic agent 0.1-10M.
In one embodiment of the invention, the final concentration of 0.05-9M of color developing agent in detection architecture.
In one embodiment of the invention, when detecting the concentration of eriodictyol in reaction solution, the wave-length coverage of detection is
500-600nm。
Preferably, in aqueous solution, eriodictyol is handled using NaOH, treated, and reaction solution best detection wavelength is 550-
580nm。
Preferably, in colourless synthetic media, eriodictyol, reaction solution optimum detection that treated are handled using KOH
Wavelength is 560-570nm.
It is after being transformed a second object of the present invention is to provide a kind of method of high flux screening eriodictyol superior strain
Bacterial strain to be screened fermented after, staticly settle thallus or centrifugation obtain fermented supernatant fluid, add color developing agent potassium hydroxide
Or sodium hydroxide carries out chromogenic reaction, obtains reaction solution；With the concentration of eriodictyol in microplate reader detection reaction solution；The hair
Ferment, centrifugation, colour developing, microplate reader detection are carried out in high pass orifice.
In one embodiment of the invention, the transformation includes physical mutagenesis, chemical mutagenesis or metabolic engineering.
Present invention finds potassium hydroxide and sodium hydroxide can be used as color developing agent, quickly detected in 5min aqueous solution or
The concentration of eriodictyol in person's fermentation liquid.Eriodictyol concentration is higher, and purple is deeper, develops the color faster.And in common interference object shaddock ped
In the presence of element, still accuracy with higher, and microplate reader can be used and more accurately detected.What the present invention used
Color developing agent is easily obtained and makes, and can by the speed and the depth of color change come it is quick determine product concentration,
It can be further detected and confirmed by microplate reader.When being transformed or be mutated in microorganism, fermentation can be processed in batches
96 orifice plate of high throughput of liquid, one piece of single treatment addition, 96 bacterial strain fermentation liquors only needs 30-60s, and a hour can be handled
5000-10000 single colonie, substantially increases screening efficiency.
Detailed description of the invention
Fig. 1: with the eriodictyol and naringenin mixed liquor of the NaOH solution processing various concentration of 4M in YNB culture medium.
Fig. 2: in aqueous solution with the eriodictyol and naringenin mixed liquor of the NaOH solution processing various concentration of 4M.
Fig. 3: with the eriodictyol and naringenin mixed liquor of the KOH solution processing various concentration of 4M in YNB culture medium.
Fig. 4: in aqueous solution with the eriodictyol and naringenin mixed liquor of the KOH solution processing various concentration of 4M.
Fig. 5: 50mg/L eriodictyol, the naringenin of 50mg/L, the eriodictyol of 50mg/L and 50mg/L naringenin it is mixed
Liquid is closed in YNB culture medium with NaOH solution treated full wavelength scanner figure.
Fig. 6: 50mg/L eriodictyol, the naringenin of 50mg/L, the eriodictyol of 50mg/L and 50mg/L naringenin it is mixed
It closes liquid and uses NaOH solution in aqueous solution treated full wavelength scanner figure.
Fig. 7: 50mg/L eriodictyol, the naringenin of 50mg/L, the eriodictyol of 50mg/L and 50mg/L naringenin it is mixed
Liquid is closed in YNB culture medium with KOH treated full wavelength scanner figure.
Fig. 8: 50mg/L eriodictyol, the naringenin of 50mg/L, the eriodictyol of 50mg/L and 50mg/L naringenin it is mixed
It closes liquid and uses KOH in aqueous solution treated full wavelength scanner figure.
Fig. 9: in YNB culture medium, after the sodium hydroxide solution processing of 4M, there are situations for various concentration naringenin
Under, the light absorption value of reaction solution under 540nm.
Figure 10: in aqueous solution, after the sodium hydroxide solution processing of 4M, in the case of various concentration naringenin exists
The light absorption value of reaction solution, A:550nm under 550-580nm wavelength；B:560nm；C:570nm；D:580nm.
Figure 11: in YNB culture medium, after the potassium hydroxide solution processing of 4M, there are situations for various concentration naringenin
Under, the light absorption value of reaction solution, A:550nm under 550,560 and 570nm wavelength；B:560nm；C:570nm.
Figure 12: in aqueous solution, after the potassium hydroxide solution processing of 4M, in the case of various concentration naringenin exists,
The light absorption value of reaction solution, A:560nm under 560 and 570nm wavelength；B:570nm.
Specific embodiment
(1) culture medium
YNB culture medium: YNB culture medium (Yeast nitrogen base, yeast nitrogen culture medium, the no ammonia of 1.72g/L
Base acid and ammonium sulfate), the amino acid of 50mg/L is added as needed in the ammonium sulfate of 5g/L；
YPD culture medium: 20g/L glucose, 20g/L peptone, 10g/L yeast powder.
(2) high performance liquid chromatography detection eriodictyol content method
Agilent 1100, mobile phase: 45% methanol aqueous solution adds 0.3% phosphoric acid, organic membrane filter after mixing.Stream
Dynamic phase 1mL/min, 30 DEG C of column temperature, Detection wavelength 290nm, 10 μ L of volume.Sample runing time 30min.
Embodiment 1
1 high-throughput chromogenic reaction
Naringenin, eriodictyol mother liquor are configured, is dissolved in 100% ethanol solution, is configured to the titer of 4g/L.Then match
The naringenin and eriodictyol mixed liquor of various concentration are set, uses water or YNB culture medium dissolved dilution respectively, and constant volume is to 100 μ
Then L adds the sodium hydroxide solution of the 4M of 100 μ L and the potassium hydroxide solution of 4M respectively.Face is observed after normal-temperature reaction 5min
Colour response.As shown in Figs 1-4, eriodictyol concentration is higher, and purple is deeper for reaction solution color variation, develops the color faster.
2 reaction solution full wavelength scanners
From the reaction solution crossed by 4M potassium hydroxide and 4M naoh treatment, the final concentration of 50mg/L of naringenin is chosen,
96 hole Board positions of the final concentration of 50mg/L of eriodictyol, naringenin-each 50mg/L of eriodictyol final concentration are placed in microplate reader and do entirely
Length scanning, scanning range 230-998nm, step-length 2nm.After full wavelength scanner, scanning result is drawn, is examined beyond light absorption value
Upper limit value 4 is surveyed, label is without exception.Chaff interferent is found according to full wavelength scanner result, and the smallest wavelength is interfered to detection substance
Range.It can be found that eriodictyol, after naoh treatment, in aqueous solution, best detection wavelength is between 500-650nm；
And in colourless synthetic media, best detection wavelength is between 500-550nm.Eriodictyol is after potassium hydroxide treatment, in water
In solution, best detection wavelength is between 500-650nm；In simple culture media, best detection wavelength be 500-600nm it
Between.All-wave length surface sweeping result is shown in Fig. 5-8.
The selection in 3 best detection wavelength sections
After the detection interval of above-mentioned reaction solution determines, in order to be more convenient to quantify to concentration in reaction solution using microplate reader
Detection, further detects the best detection wavelength having determined, in the presence of determining chaff interferent naringenin, with substance to be detected
The better Detection wavelength of eriodictyol actual concentrations matching degree.
Eriodictyol reaction solution does section scanning, and according to full wavelength scanner as a result, selecting different sweep intervals, step-length is
10nm.The results show that with after naoh treatment, when reaction solution is YNB, best detection wavelength 540nm；Reaction solution is water
When, best detection wavelength 550-580nm.And with after potassium hydroxide treatment, when reaction solution is YNB, best detection wavelength is
Between 550-570nm；When reaction solution is water, best detection wavelength is between 560-570nm, as shown in Figure 10 to Figure 12.
Embodiment 2
Saccharomyces cerevisiae C800 (CENPK2-1D, Δ gal80::G418) is for fermenting and constructing plasmid.E. coli jm109
It saves and expands for plasmid.pY26-PGPD-F3’H-PTEF- CPR is shuttle plasmid, as control plasmid and is set out plasmid,
Screening label in saccharomyces cerevisiae is uracil.pY26-PGPD-F3’H-PTEF- CPR can be simultaneously in saccharomyces cerevisiae and large intestine bar
Duplication is expanded in bacterium, wherein F3 ' H gene is originated by promoter GPD and transcribed, and CPR gene is originated by promoter TEF and transcribed.
1 plasmid construction and high-throughput testing process
Promoter 20 of saccharomyces cerevisiae varying strength are chosen, is divided into two groups of AB, every group 10, every Zu Zhongyou senior middle school
Low three gears promoter may be implemented the varying strength expression of corresponding gene, generate 100 kinds of combinations (10 × 10).Wherein A group starts
Son is for starting F3 ' H gene, and B group promoter is for starting CPR gene.
After promoter, gene, plasmid primer amplification, promoter, gene and plasmid have about 35bp's between any two
Then homologous region is transformed into saccharomyces cerevisiae C800 simultaneously, in saccharomyces cerevisiae, these segments will voluntarily occur homologous heavy
Group can form 100 kinds of combinations.Then the yeast of conversion is coated in selective plate YNB, does not add urine in the medium
Pyrimidine can make the thalli growth for reassembling into function at single colonie, as recombination single colonie.And fail to the matter of homologous recombination
Grain can not be grown.
When primary dcreening operation, the YNB culture medium for being not added with uracil is divided in 48 deep-well plates by every hole 1.5mL, in culture medium
Naringenin containing 500mg/L.The successful single colonie of the above-mentioned conversion of picking is seeded in deep-well plates and contact plate is stayed on YNB plate
Kind, 30 DEG C, 220rpm cultivates 48h, stands 2h, precipitates thallus completely, using 100 μ L of volley of rifle fire Aspirate supernatant in 98 shallow bore hole plates
In, the 4M KOH of 100 μ L is then added using the volley of rifle fire, after standing about 5min, according to shade, marks superior strain number,
The relatively high preceding 10 plants of bacterium of yield are chosen on the plate of reservation, carry out secondary screening.96 shallow bore hole plates after reaction can also be set
Detect light absorption value at 560nm, relatively high preceding 10 holes of label light absorption value, the single colonie on corresponding plate enters next round
Secondary screening.By primary dcreening operation, 10 high yield single colonies can be once chosen from 1000 recombination single colonies.
When secondary screening, 10 high yield single bacteriums for being inoculated with acquisition fall within shaking flask in the YNB of 10mL in 250mL, and 30 DEG C,
220rpm, 14-16h is to the logarithm middle and later periods for culture, at this time bacterium colony OD600Between 6-8.The YPD of 20mL is transferred to 2% (V/V)
In in the shaking flask of 250mL, while adding the naringenin of final concentration 250mg/, 30 DEG C, 220rpm cultivates to 12h, for 24 hours and 36h
Add the naringenin of final concentration 250mg/L again afterwards.It is sampled after culture 72h, detects yield using high performance liquid chromatography.
2. high-throughput testing result
1 plant of highest bacterium colony of yield is finally obtained from 1000 single colonies (10 × combinatorial libraries) by primary dcreening operation and secondary screening,
Plasmid is extracted, sequencing obtains optimum start-up sub-portfolio.
The starting sub-portfolio of best production eriodictyol is pY26-PINO1-F3’H-PTDH1-CPR.Wherein F3 ' H gene is by starting
Sub- INO1 starting transcription, CPR gene are originated by promoter TDH1 and are transcribed.Plasmid eriodictyol yield in shaking flask of setting out is
57.8mg/L, and eriodictyol yield is 672mg/L under the same conditions by the high-throughput best plasmid obtained, yield provides
10.6 times.
The above result shows that the method detects the concentration of eriodictyol in fermentation liquid, have flux high, speed is fast, accuracy
Height, simple and convenient feature.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of method of high-throughput detection eriodictyol, which is characterized in that the described method comprises the following steps:
(1) color developing agent potassium hydroxide is added into the prepare liquid containing eriodictyol or sodium hydroxide carries out chromogenic reaction, is obtained anti-
Answer liquid；
(2) with the concentration of eriodictyol in microplate reader detection reaction solution, the chromogenic reaction and microplate reader detection are in high throughput
It is carried out on orifice plate.
2. the method according to claim 1, wherein the high pass orifice include 96 deep-well plates, 96 shallow bore hole plates,
48 deep-well plates, 24 deep-well plates, 12 deep-well plates.
3. the method according to claim 1, wherein the prepare liquid containing eriodictyol includes the water containing eriodictyol
Solution, colourless synthetic media or bacterial strain fermentation liquor.
4. the method according to claim 1, wherein chromogenic agent is 0.1-10M.
5. method according to claim 1 or 4, which is characterized in that the final concentration of 0.05-9M of color developing agent in detection architecture.
6. method according to claim 1 or 3, which is characterized in that in detection reaction solution when the concentration of eriodictyol, detect wave
Long range is 500-600nm.
7. method according to claim 1 or 3, which is characterized in that the eriodictyol after potassium hydroxide treatment, in aqueous solution
Detection wavelength range be 560-570nm, Detection wavelength range in colourless synthetic media is 550-570nm.
8. a kind of method of high flux screening eriodictyol superior strain, which is characterized in that the method is by improved wait sieve
After selecting bacterial strain to be fermented, staticly settles thallus and be perhaps centrifuged acquisition fermented supernatant fluid addition color developing agent potassium hydroxide or hydrogen
Sodium oxide molybdena carries out chromogenic reaction, obtains reaction solution；With the concentration of eriodictyol in microplate reader detection reaction solution；It is described fermentation, centrifugation,
Colour developing, microplate reader detection are carried out in high pass orifice.
9. according to the method described in claim 8, it is characterized in that, the rebuilding approach include physical mutagenesis, chemical mutagenesis or
Metabolic engineering.
10. the eriodictyol superior strain that method described in application claim 8 or 9 is screened.
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Cited By (2)
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| CN112391362A (en) * | 2020-11-04 | 2021-02-23 | 江南大学 | Flavone 3 beta-hydroxylase mutant with improved catalytic activity and application thereof |
| CN112391300A (en) * | 2020-11-04 | 2021-02-23 | 江南大学 | Silybum marianum-derived flavone 3 beta-hydroxylase and application of coenzyme thereof |
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Application publication date: 20190215 |