CN105319316B - A kind of method of quick detection threonine content in fermentation liquor - Google Patents
A kind of method of quick detection threonine content in fermentation liquor Download PDFInfo
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Abstract
The invention discloses a kind of methods of quickly detection threonine content in fermentation liquor, after react after sample solution is diluted with the solution mixing containing NADH, PLP, alcohol dehydrogenase and DTT, then with the solution reaction containing LTA, pass through and detect calculating OD340Rate is reduced, the content for obtaining threonine is compared with standard curve.Method of the invention can simply, quickly and accurately detect the content of threonine in fermentation liquid, and detection flux is high, expense is low, the time is short, error is small.
Description
Technical field
The present invention relates to microbiological industries, in particular to produce L-threonine using the industrial microorganism of genetic engineering transformation
Field, and in particular to a method of quickly detection threonine content in fermentation liquor.
Background technique
Fermentation method is industrially mostly used to produce threonine at present.The threonine production level of American-European countries will be apparently higher than me
State, such as the ADM company of German goldschmidt chemical corporation, the U.S. are transformed strain using advanced molecule manipulation technology, make
The yield and conversion ratio of threonine have huge raising.World strain fermentation level is 150g/L, fermentation period 36- at present
48h, high conversion rate is in 50%.Domestic either fermentation yield or conversion ratio is below world level.
On the one hand the acquisition of threonine Producing Strain is to carry out rationality transformation to bacterial strain using the means of genetic engineering, another
Aspect is the bacterial strain by screening output increased after physical mutagenesis or chemical mutagenesis, and the latter is obtain superior strain one
Kind important means.
The detection of threonine is one of main bottleneck in screening process in fermentation liquid.It revives ammonia in detection fermentation liquid at present
It mainly include paper chromatography and high performance liquid chromatography etc. when acid content.Paper chromatography is less using equipment, low in cost, still
Measurement needs 3-4 hours every time, and accuracy rate is relatively low, and error cannot rapidly provide guidance up to 10% or so
The data of experiment.High performance liquid chromatography can provide accurate experimental result, but expensive equipment used by this method, cannot
It is widely popularized.
In addition, the method for measurement threonine content further includes perchloric acid potentiometric titration, it is solid that this method can only be confined to measurement
Random sample product not can be carried out the measurement of threonine content in fermentation liquor.
Therefore, it is still necessary to develop a kind of method of quickly detection threonine content in fermentation liquor for this field.
Summary of the invention
The purpose of the present invention is to provide it is a kind of can simply, fast and accurately detect threonine content in fermentation liquor
Method.
The first aspect of the present invention provides a kind of method of threonine content in test sample, and the method includes following
Step:
(i) sample is uniformly mixed with the first reaction solution, obtains the first mixed solution；
(ii) recovery temperature is to 15-30 DEG C after first mixed solution being placed in 4 ± 1 DEG C of placement 10-90min；
(iii) the first mixed solution is uniformly mixed to obtain the second mixed solution with the second reaction solution；
(iv) OD of second mixed solution is detected340, it is primary every detection in 1-5 minutes, it detects 5-7 times altogether；
(v) with detection time and corresponding OD340Mapping calculates the OD that slope is sample340Reduce rate；
(vi) OD that will be calculated340Reduction rate be compared with standard curve, obtain ammonia of reviving in the fermentation liquid
Acid content,
Wherein, first reaction solution contains HEPES buffer, NADH, PLP, alcohol dehydrogenase, DTT and water；
In first mixed solution containing 10-500mM HEPES buffer, 100 μM of -1000 μM of NADH, 10 μM -
100 μM of PLP, 1U-50U alcohol dehydrogenases and 0.5mM-8mM DTT；
Contain 1-20U LTA in second reaction solution, surplus is water.
In another preferred example, 50-400mM HEPES buffer, 100 μM~800 are contained in first mixed solution
μM NADH, 20 μM~80 μM PLP, 1U~35U alcohol dehydrogenases and 0.5mM~5mM DTT.
In another preferred example, it is uniformly mixed, dilutes with first reaction solution after diluting sample in step (i)
The concentration of threonine is 0.1-10mM in sample afterwards.
In another preferred example, the volume ratio of the sample after dilution and the first reaction solution is 1:1-5.
In another preferred example, the volume ratio of first mixed solution and second reaction solution is 1-5:1.
In another preferred example, the standard curve is obtained by following steps:
(a) the different standard Threonine solution of several concentration that concentration range is 0.1mM~8mM is reacted with first respectively
Solution is uniformly mixed, and obtains the first mixed solution；
(b) recovery temperature is to 15-30 DEG C after first mixed solution being placed in 4 ± 1 DEG C of placement 10-90min；
(c) the first mixed solution is uniformly mixed to obtain the second mixed solution with the second reaction solution；
(d) OD of second mixed solution is detected340, it is primary every detection in 1-5 minutes, it detects 5-7 times altogether；
(e) with minute and corresponding OD340For coordinate mapping, calculating slope is OD340Reduce rate；
(f) with the concentration of standard Threonine solution and corresponding OD340Reduction rate is coordinate, and drafting obtains the standard
Curve,
Wherein, the first reaction solution described in step (a) contain HEPES buffer, NADH, PLP, alcohol dehydrogenase,
DTT and water；
Contain 10-500mM HEPES buffer, 100 μM -1000 μM in first mixed solution that step (a) obtains
NADH, 10 μM of -100 μM of PLP, 1U-50U alcohol dehydrogenases and 0.5mM-8mM DTT；
Contain 1-20U LTA in second reaction solution, surplus is water.
In another preferred example, 50-400mM HEPES buffer, 100 μM~800 are contained in first mixed solution
μM NADH, 20 μM~80 μM PLP, 1U~35U alcohol dehydrogenases and 0.5mM~5mM DTT.
In another preferred example, 50-200mM HEPES buffer, 100 μM~500 are contained in first mixed solution
μM NADH, 20 μM~80 μM PLP, 1U~5U alcohol dehydrogenases and 0.5mM~1.5mM DTT.
In another preferred example, the volume ratio of step b) the Plays Threonine solution and the first reaction solution is 1:1-
5；And/or
The volume ratio of first mixed solution described in the step d) and the second reaction solution is 1-5:1.
In another preferred example, the foundation of threonine content and the standard curve carries out simultaneously in the detection sample.
In another preferred example, the LTA is from pseudomonas aeruginosa, Escherichia coli, bacillus subtilis, wine brewing ferment
One or more of mother, Candida, candida albicans.
In another preferred example, step (a) prepares the different standard Threonine solution of 8 concentration, and concentration is respectively
0.2mM、0.5mM、1mM、2mM、3mM、4mM、5mM、6mM。
Detection method of the invention, can simply, quickly and accurately detect threonine content in fermentation liquor, overcome existing
The deficiencies of equipment is expensive in technology, cumbersome, and measurement is time-consuming.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, exist
This no longer tires out one by one states.
Detailed description of the invention
Fig. 1 is 1 testing result figure of embodiment.
Fig. 2 is the testing result figure of embodiment 3.
Fig. 3 is that threonine concentration and NADH reduce rate curve.
Fig. 4 is the range of linearity that two enzymes method detects threonine content.
Fig. 5 is that threonine content and NADH reduce rate curve in M9+ yeast powder culture medium.
Fig. 6 is that threonine content and NADH reduce rate curve in LB culture medium.
Fig. 7 is to detect threonine content result figure, each time point using the method for the present invention (enzyme coupling method) and HPLC method
Locating the first column (Zuo Zhu) is HPLC testing result, and the second column (right column) is enzyme coupling method testing result of the present invention.
Specific embodiment
Present inventor is unexpected for the first time to develop a kind of quickly detection hair threonine by depth studying extensively
The method of content is mutually coupled using LTA enzyme as toolenzyme and by the reduction rate of NADH and the yield of threonine.L-threonine aldehyde
Contracting enzyme (L-threonine aldolase, LTA) can be catalyzed L-threonine and generate glycine and acetaldehyde and its back reaction, in LTA
And in the presence of pyridoxal phosphate (PLP), threonine can be broken down into acetaldehyde and glycine, and acetaldehyde is by alcohol dehydrogenase (ADH)
It is reduced to ethyl alcohol, while nicotinamide adenine dinucleotide (NADH) is oxidized to NAD+, pass through OD340The change of nm detection NADH
Change the content that rate determines threonine.On this basis, the present invention is completed.
Detection method
The method of threonine content in test sample provided by the invention, comprising the following steps:
(i) sample is uniformly mixed with the first reaction solution, obtains the first mixed solution；
(ii) recovery temperature is to 15-30 DEG C after first mixed solution being placed in 4 ± 1 DEG C of placement 10-90min；
(iii) the first mixed solution is uniformly mixed to obtain the second mixed solution with the second reaction solution；
(iv) OD of second mixed solution is detected340, it is primary every detection in 1-5 minutes, it detects 5-7 times altogether；
(v) with detection time and corresponding OD340Mapping calculates the OD that slope is sample340Reduce rate；
(vi) OD that will be calculated340Reduction rate be compared with standard curve, obtain ammonia of reviving in the fermentation liquid
Acid content,
Wherein, first reaction solution contains HEPES buffer, NADH, PLP, alcohol dehydrogenase, DTT and water；
In first mixed solution containing 10-500mM HEPES buffer, 100 μM of -1000 μM of NADH, 10 μM -
100 μM of PLP, 1U-50U alcohol dehydrogenases and 0.5mM-8mM DTT；
Contain 1-20U LTA in second reaction solution, surplus is water.
In first mixed solution containing 50-200mM HEPES buffer, 100 μM~500 μM NADH, 20 μM~
80 μM of PLP, 1U~5U alcohol dehydrogenase and 0.5mM~1.5mM DTT.
In another preferred example, 100mM HEPES buffer, 300 μM of NADH, 50 are contained in first mixed solution
μM PLP, 3U alcohol dehydrogenase and 1mM DTT.
In another preferred example, the pH value of the HEPES buffer be 7.8-8.2, preferably 8.0.
In another preferred example, 2-15U LTA is contained in second reaction solution, surplus is water.
In another preferred example, it is uniformly mixed, dilutes with first reaction solution after diluting sample in step (i)
The concentration of threonine is 0.1-10mM in sample afterwards.
In another preferred example, the concentration for diluting threonine in post-fermentation liquid is 0.5-5mM.
In another preferred example, the volume ratio of the sample after dilution and the first reaction solution is 1:1-5.
In another preferred example, the volume ratio of the sample after the dilution and the first reaction solution is 1:1.5-3, preferably
For 1:2.
In another preferred example, the volume ratio of first mixed solution and second reaction solution is 1-5:1.
In another preferred example, the volume ratio of first mixed solution and second reaction solution is 1-3:1, preferably
It is 1.5:1.
In another preferred example, the volume ratio of first reaction solution and second reaction solution is 1:0.5-2, compared with
It is goodly 1:0.5-1.5, is more preferably 1:1.
In the present invention, the standard curve is obtained by following steps:
(a) the different standard Threonine solution of several concentration that concentration range is 0.1mM~8mM is reacted with first respectively
Solution is uniformly mixed, and obtains the first mixed solution；
(b) recovery temperature is to 15-30 DEG C after first mixed solution being placed in 4 ± 1 DEG C of placement 10-90min；
(c) the first mixed solution is uniformly mixed to obtain the second mixed solution with the second reaction solution；
(d) OD of second mixed solution is detected340, it is primary every detection in 1-5 minutes, it detects 5-7 times altogether；
(e) with minute and corresponding OD340For coordinate mapping, calculating slope is OD340Reduce rate；
(f) with the concentration of standard Threonine solution and corresponding OD340Reduction rate is coordinate, and drafting obtains the standard
Curve,
Wherein, the first reaction solution described in step (a) contain HEPES buffer, NADH, PLP, alcohol dehydrogenase,
DTT and water；
Contain 10-500mM HEPES buffer, 100 μM -1000 μM in first mixed solution that step (a) obtains
NADH, 10 μM of -100 μM of PLP, 1U-50U alcohol dehydrogenases and 0.5mM-8mM DTT；
Contain 1-20U LTA in second reaction solution, surplus is water.
In another preferred example, the standard Threonine solution, concentration range are 0.2mM~6mM.
In another preferred example, step (a) prepares the different standard Threonine solution of 8 concentration, and concentration is respectively
0.2mM、0.5mM、1mM、2mM、3mM、4mM、5mM、6mM。
In first mixed solution containing 50-200mM HEPES buffer, 100 μM~500 μM NADH, 20 μM~
80 μM of PLP, 1U~5U alcohol dehydrogenase and 0.5mM~1.5mM DTT.
In another preferred example, 100mM HEPES buffer, 300 μM of NADH, 50 are contained in first mixed solution
μM PLP, 3U alcohol dehydrogenase and 1mM DTT.
In another preferred example, 2-15U LTA is contained in second reaction solution, surplus is water.
The volume ratio of step b) the Plays Threonine solution and the first reaction solution is 1:1-5.
The volume ratio of first mixed solution described in the step d) and the second reaction solution is 1-5:1.
In another preferred example, standard Threonine solution described in the step b) and the volume ratio of the first reaction solution are
1:1.5-3 preferably 1:2.
In another preferred example, the volume ratio of the first mixed solution described in the step d) and second reaction solution
For 1-3:1, preferably 1.5:1.
In another preferred example, during standard curve obtains, first reaction solution and second reaction solution
Volume ratio be 1:0.5-2, preferably 1:0.5-1.5, be more preferably 1:1.
The foundation of threonine content and the standard curve carries out simultaneously in the detection sample.
In the present invention, the foundation for detecting threonine content and the standard curve in sample carries out simultaneously, specifically, simultaneously
Step (i) and (a) are carried out, while carrying out step (ii) and (b), while carrying out step (iii) and (c), while carrying out step
(iv) and (d).
In the present invention, the LTA derives from pseudomonas aeruginosa, Escherichia coli, bacillus subtilis, saccharomyces cerevisiae, vacation
One or more of silk yeast, candida albicans., LTA's is not particularly limited, and can be used known in the art
Various methods.In a preferred example, LTA is obtained using following methods:
Lta gene is obtained by template PCR of pseudomonas aeruginosa gene group, and is connected to pET28 carrier (purchased from beauty
Invitrogen company, state) conversion BL21 (DE3) bacterial strain, isopropylthiogalactoside IPTG is added and carries out inducing expression, table
Bacterium is received in centrifugation after up to 2h, is added Lysis Buffer (containing PMSF and DTT) and is carried out ultrasonication afterwards, supernatant with it is Balanced
After His Purification Resin combines overnight, it is added after Wash Buffer is washed repeatedly 3-4 times and Elute Buffer elution albumen is added.
Since the LTA albumen of purifying contains the imidazoles (250mM) of high concentration, dialysis treatment need to be carried out.Eluent is placed in dialysis
It is put into the beaker for filling dialyzate in bag, 4 DEG C of dialysed overnights.Simultaneously by the glycerol of the LTA protein solution dialysed addition 30%
Be packed as 200 μ l/ pipes be placed on -20 DEG C it is spare.
In another preferred example, standard curve is obtained using following methods:
It first prepares a series of standard Threonine solution of various concentrations, the first reaction solution is added and obtains the after mixing
One mixed solution contains HEPES buffer (pH8.0), NADH, 50 μM of PLP, alcohol dehydrogenases in first reaction solution
(ADH) and DTT.
First mixed solution is restored into room temperature after 4 DEG C of placement 30min-1h, is put into microplate reader after suitable LTA is added
It is interior, an OD is read every 2min340Numerical value is read 3~7 times altogether.Using the time as abscissa, with OD340Reading is ordinate, meter
The slope of light absorption value regression curve is calculated, as light absorption value reduces rate (OD340Reduce rate).
With the corresponding light absorption value OD of the concentration of standard Threonine solution340Reduction rate is coordinate, and it is bent to draw standard
Line.
Prepared standard Threonine solution concentration is between 0.2mM~6mM.
NADH concentration is 100 μM~1000 μM in first mixed solution, and PLP concentration is 10 μM~100 μM, and ethyl alcohol is de-
Hydrogen enzyme concentration is 1U~50U, and DTT concentration is 1mM~5mM.Preferably, containing 100mM HEPES in the first mixed solution
Buffer (pH8.0), 300 μM of NADH, 50 μM of PLP, 3U alcohol dehydrogenases (ADH) and 1mM DTT.
The reading OD340nmTime is 6min~20min.
The feature that the features described above or embodiment that the present invention mentions are mentioned can be in any combination.Disclosed in this case specification
All features can be used in combination with any composition form, each feature disclosed in specification, can by it is any provide it is identical,
The alternative characteristics of impartial or similar purpose replace.Therefore except there is special instruction, revealed feature is only impartial or similar spy
The general example of sign.
Advantage for present invention is as follows compared with prior art:
1 detection flux is high.So-called " high throughput " (High throughput screening, HTS) refers to that utilization can batch
The tool of operation and the operating system that can be automated analysis execute the process of experiment, and are adopted by the detector of rapid sensitive
Collection experimental data and a kind of method for carrying out batch processing analysis.This law is a kind of using LTA enzyme as toolenzyme and by NADH
The detection method that reduction rate and the yield of threonine are mutually coupled.This law can carry out in the micropore of 200 μ L, utilize 96 orifice plates
High-throughput operation may be implemented with microplate reader, can satisfy the requirement of upper batch sample detection when industrial production.
2 testing costs are low.Equipment needed for measuring method provided by the invention and reagent are that laboratory is standing and valence
Lattice are cheap.Compared with high performance liquid chromatography (HPLC) or amino-acid analyzer etc., this method eliminate expensive derivative reagent and
The expenses such as equipment handover charge, and the testing cost of sample can be further decreased when high-throughput operation.
3 detection times are short.Minute of this method is about a hour, and once being tested using 96 orifice plates can be obtained
The result of a large amount of samples.This law measures standard sample (referred to as mark product) and sample simultaneously, and the content of threonine passes through in sample
Reading.In addition this method only needs to carry out simple centrifuging and taking high fermentation liquid to sample, with common paper chromatography
Or high performance liquid chromatography (HPLC) is compared, this method substantially reduces the time required for test sample, so as to quick
It obtains experimental result and and guides subsequent experimental.
4 operating procedures are simple, and required reagent is non-toxic, require experimental facilities and operator low, can quickly grasp.
Drug involved in this method and reagent are non-toxic to humans, and multichannel pipettor, centrifuge, microplate reader and data place
Managing software Excel etc. is the more common technical ability in laboratory, only need to simply train and can grasp in a short time.
5 experimental datas are accurate, and collimation is good.The content of threonine and HPLC are measured in the fermentation liquid measured by this method
Result be compared after find almost the same, difference connects within operating error, and repeatability is good between sample, with
The difference that product measure for several times is maintained within 5%, has relatively high directive significance to production.
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip
Part such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and
Number is calculated by weight.
Unless otherwise defined, it anticipates known to all professional and scientific terms as used herein and one skilled in the art
Justice is identical.In addition, any method similar to or equal to what is recorded and material can be applied to the method for the present invention.Wen Zhong
The preferred implement methods and materials are for illustrative purposes only.
Embodiment 1
Detect the content of threonine in M9 culture medium
The formula of M9 culture medium is as follows:
5 × M9 salting liquid: Na2HPO47H2O12.8g, KH2PO43.0g, NaCl0.5g, NH4Cl1.0g is configured first,
Distilled water 200mL is added to dissolve, 121 DEG C sterilize 15 minutes.
According still further to following recipe configuration M9 culture medium: the MgSO42mL of 5 × M9 salting liquid 200mL, 1M, 20% grape
The CaCl20.1mL of sugar juice 20mL, 1M add sterilizing distilled water to 1000mL.
Configure reaction solution Solution I and Solution II.(formula of Solution I and Solution II are each sample
Product aequum)
Solution I (80 μ l): including HEPES, NADH, ADH, PLP, DTT and H2O
Solution II (80 μ l): include LTA (3U), surplus H2O
Mark product and sample:
It takes a certain amount of sample or mark product and adds H2O is supplied as 40 μ l.Its product deionized water dissolving of getting the bid, sample
For the M9 culture medium containing unknown threonine concentration.
Final concentration of 0.2mM, 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM, the 6mM that threonine mark product are diluted to, and will
Threonine concentration is diluted between 1~4mM in sample, and Solution I and sample or mark product is added in 96 orifice plates, mixed with the volley of rifle fire
It is even, it include HEPES (150mM), NADH (200 μM), ADH (3U), PLP (20 μM), DTT (1mM) in reaction system.It is quiet at 4 DEG C
Taking-up is returned to room temperature after setting 30min.
LTA is melted on ice after -20 DEG C of taking-ups.Appropriate H is added according to the formula of Solution II2By it after O mixing
Return back to room temperature.Solution II is added to 96 orifice plates.
After opening microplate reader sets program, 96 orifice plates are put into microplate reader, program setting are as follows: automatic mixing 5 times, often
Start to read after secondary 3s, 37 DEG C of incubation 2min, reads a numerical value every 2min, read 7 times altogether.With minute and OD340Value is seat
Plot, calculating slope are OD340Reduce rate.
It is handled with data of the Excel to each micropore, using the concentration of threonine mark product as abscissa, with corresponding
OD340Reducing rate is that ordinate draws standard curve, as a result as shown in Figure 1.
According to the OD of sample340Rate and standard curve are reduced, reads the concentration value of threonine in sample, and according to dilution
The content of initial threonine in multiple conversion sample.
Embodiment 2
Detect the content of threonine in LB culture medium
LB culture medium prescription are as follows: tryptone 10g/L, yeast extract 5g/L, NaCl10g/L.
Configuring reaction solution Solution I and Solution II, (formula of Solution I and Solution II are each sample
Aequum).
Solution I (80 μ l): including HEPES, NADH, ADH, PLP, DTT and H2O
Solution II (80 μ l): include LTA (6U), surplus H2O
Mark product and sample: a certain amount of sample or mark product are taken and add H2O is supplied as 40 μ l.Its acceptance of the bid product deionization
Water dissolution, sample are the LB culture medium containing unknown threonine concentration.
Threonine mark product are diluted to final concentration of 0.2mM, 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, and by sample
Threonine concentration is diluted between 1~4mM in product, and Solution I and sample or mark product is added in 96 orifice plates, mixed with the volley of rifle fire
It is even, HEPES (250mM), NADH (500 μM), ADH (10U), PLP (50 μM) and DTT (2.5mM) are included in reaction system.4℃
It is taken out after lower standing 30min and is returned to room temperature.
LTA is melted on ice after -20 DEG C of taking-ups.Appropriate H is added according to the formula of Solution II2By it after O mixing
Return back to room temperature.Solution II is added to 96 orifice plates.
After opening microplate reader sets program, 96 orifice plates are put into microplate reader, program setting are as follows: automatic mixing 5 times, often
Start to read after secondary 3s, 37 DEG C of incubation 2min, reads a numerical value every 2min, read 7 times altogether.With minute and OD340Value is seat
Plot, calculating slope are OD340Reduce rate.
It is handled with data of the Excel to each micropore, using the concentration of threonine mark product as abscissa, with corresponding
OD340Reducing rate is that ordinate draws standard curve.According to the OD of sample340Rate and standard curve are reduced, is read in sample
The concentration value of threonine, and according to the content of initial threonine in extension rate conversion sample.
Embodiment 3
Detect the content of threonine in threonine fermentation liquid
The formula of threonine fermentation liquid is as follows:
Glucose 70g/L, (NH4)2SO410g/L, KH2PO42g/L, MgSO4·7H2O0.5g/L, FeSO4·7H2O5mg/
L, MnSO4·4H2O5mg/L, methionine 800mg/L, pH are adjusted to 6.0
Configure reaction solution Solution I and Solution II.(formula of Solution I and Solution II are each sample
Product aequum)
Solution I (80 μ l): including HEPES, NADH, ADH, PLP, DTT and H2O
Solution II (80 μ l): include LTA (12U), surplus H2O
Mark product and sample: a certain amount of sample or mark product are taken and add H2O is supplied as 40 μ l.Its acceptance of the bid product deionization
Water dissolution, sample are the Threonine Fermentation culture medium containing unknown threonine concentration.
Threonine mark product are diluted to final concentration of 0.2mM, 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM, and by sample
Threonine concentration is diluted between 1~4mM in product, and Solution I and sample or mark product is added in 96 orifice plates, mixed with the volley of rifle fire
It is even, HEPES (400mM), NADH (800 μM), ADH (30U), PLP (80 μM) and DTT (4mM) are included in reaction system.At 4 DEG C
Taking-up is returned to room temperature after standing 30min.
LTA is melted on ice after -20 DEG C of taking-ups.Appropriate H is added according to the formula of Solution II2By it after O mixing
Return back to room temperature.Solution II is added to 96 orifice plates.
After opening microplate reader sets program, 96 orifice plates are put into microplate reader, program setting are as follows: automatic mixing 5 times, often
Start to read after secondary 3s, 37 DEG C of incubation 2min, reads a numerical value every 2min, read 7 times altogether.With minute and OD340Value is seat
Plot, calculating slope are OD340Reduce rate.
It is handled with data of the Excel to each micropore, using the concentration of threonine mark product as abscissa, with corresponding
OD340Reducing rate is that ordinate draws standard curve.According to the OD of sample340Rate and standard curve are reduced, is read in sample
The concentration value of threonine, and according to the content of initial threonine in extension rate conversion sample.
Experimental result is as shown in Figure 2.The result shows that M9 culture medium influences the method without apparent, all deviations all exist
Within tolerance interval, definite ingredients in M9 culture medium are therefore glucose and part salt ion can use the method inspection
Survey the content of threonine in M9 culture medium.
Embodiment 4
Linear Experiment
In order to determine the range of two enzymes method detection threonine of the invention, according to the method for embodiment 1 to various concentration
Threonine (0~70mM) mark product are detected, as a result as shown in Figure 3 and Figure 4.
The result shows that the content of threonine is good with the reduction Rate-dependency of NADH within the scope of 0mM~70mM, and revive
The content of propylhomoserin has good linear relationship with the reduction rate of NADH within the scope of 0~6mM.
Embodiment 5
Sensitivity experiment
The present embodiment investigates the sensitivity of two enzymes method detection threonine.
The high fermentation liquid using M9+ yeast powder culture medium and LB culture medium culture Escherichia coli is had collected respectively, and will
It is diluted to various concentration, is detected according to the method for embodiment 3 using two enzymes method, as a result as shown in Figure 5 and Figure 6 respectively.
The result shows that two enzymes method of the present invention can detecte out the difference of 0.05mM threonine in fermentation liquid.The method of the present invention
With very high sensitivity, small difference can detecte out.
Embodiment 6
Interference--free experiments
By in common culture medium salt ion and other composite parts the influence of the accuracy of the above method is ground
Study carefully.2mM threonine is added in system, not add any interference component as a control group, adds various composition in table 1 respectively
It is used as experimental group afterwards, the threonine relative value of consequence that control group measures is set as 100, the measurement result and control group of experimental group
Relative value is obtained after being compared.
Experimental result is as shown in table 1.
Influence of 1 component of table to two enzymes method result
When relative value is in 100 ± 5 ranges, it is believed that it is experimental error, the substance of addition does not influence the method,
More than thinking have an impact to the method after this range.By above-mentioned testing result it is found that component pair in most of common culture mediums
The method of the present invention does not influence, and method of the invention has strong anti-interference ability.
M9 culture medium is due to containing glucose, SO in common culture medium4 2-, Mg2+, Na+, NH4 +, Cl-, H2PO4 -Deng to upper
The method of stating does not have influential salt ion, therefore method of the invention is particularly suitable for obtained with the strain fermentation of M9 culture medium culture
Fermentation liquid in threonine content measurement.
Embodiment 7
Using enzyme coupling method compared with HPLC measurement result
The present embodiment from 70L fermentor every 2 hours taking-up samples, (alternatively referred to as using enzyme coupling method of the invention
Two enzymes method) and two methods of known HPLC the content of threonine is measured and is compared, as a result as shown in Figure 7.For
Facilitate and compare, the result of the HPLC of different time points is set as 100%, after the result of enzyme coupling method measurement is compared with HPLC
Result shown in figure.
By comparing two methods as a result, it has been found that, two enzymes method measured value/HPLC measured value in 88%~128% float
Dynamic, the result of two methods is almost the same, shows two enzymes method accuracy with higher.
In conclusion two enzymes method of the present invention be it is a kind of it is quickly accurate, can be used in detection hair high-throughput in industrial production
The method of threonine content, has a good application prospect in zymotic fluid.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document
It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can
To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims
It encloses.
Claims (10)
1. a kind of method of threonine content in test sample, which is characterized in that the described method comprises the following steps:
(i) sample is uniformly mixed with the first reaction solution, obtains the first mixed solution；
(ii) recovery temperature is to 15-30 DEG C after first mixed solution being placed in 4 ± 1 DEG C of placement 10-90min；
(iii) the first mixed solution is uniformly mixed to obtain the second mixed solution with the second reaction solution；
(iv) OD of second mixed solution is detected340, it is primary every detection in 1-5 minutes, it detects 5-7 times altogether；
(v) with detection time and corresponding OD340Mapping calculates the OD that slope is sample340Reduce rate；
(vi) OD that will be calculated340Reduction rate be compared with standard curve, obtain threonine in the sample and contain
Amount,
Wherein, first reaction solution contains HEPES buffer, NADH, PLP, alcohol dehydrogenase, DTT and water；
Contain 10-500mM HEPES buffer, 100 μM of -1000 μM of NADH, 10 μM -100 μM in first mixed solution
PLP, 1U-50U alcohol dehydrogenase and 0.5mM-8mM DTT；
Contain 1-20U LTA in second reaction solution, surplus is water.
2. the method as described in claim 1, which is characterized in that reacted after diluting sample in step (i) with described first
Solution is uniformly mixed, and the concentration of threonine is 0.1-10mM in the sample after dilution.
3. method according to claim 2, which is characterized in that the volume ratio of sample and the first reaction solution after dilution is 1:
1-5。
4. the method as described in claim 1, which is characterized in that the body of first mixed solution and second reaction solution
Product is than being 1-5:1.
5. the method as described in claim 1, which is characterized in that obtain the standard curve by following steps:
(a) by the different standard Threonine solution of several concentration that concentration range is 0.1mM~8mM respectively with the first reaction solution
It is uniformly mixed, obtains the first mixed solution；
(b) recovery temperature is to 15-30 DEG C after first mixed solution being placed in 4 ± 1 DEG C of placement 10-90min；
(c) the first mixed solution is uniformly mixed to obtain the second mixed solution with the second reaction solution；
(d) OD of second mixed solution is detected340, it is primary every detection in 1-5 minutes, it detects 5-7 times altogether；
(e) with minute and corresponding OD340For coordinate mapping, calculating slope is OD340Reduce rate；
(f) with the concentration of standard Threonine solution and corresponding OD340Reduction rate is coordinate, and drafting obtains the standard curve,
Wherein, the first reaction solution described in step (a) contain HEPES buffer, NADH, PLP, alcohol dehydrogenase, DTT and
Water；
Contain 10-500mM HEPES buffer, 100 μM -1000 μM in first mixed solution that step (a) obtains
NADH, 10 μM of -100 μM of PLP, 1U-50U alcohol dehydrogenases and 0.5mM-8mM DTT；
Contain 1-20U LTA in second reaction solution, surplus is water.
6. method as claimed in claim 1 or 5, which is characterized in that contain 50-400mM in first mixed solution
HEPES buffer, 100 μM~800 μM NADH, 20 μM~80 μM PLP, 1U~35U alcohol dehydrogenases and 0.5mM~5mM
DTT。
7. method as claimed in claim 5, which is characterized in that step a) the Plays Threonine solution reacts molten with first
The volume ratio of liquid is 1:1-5；And/or
The volume ratio of first mixed solution described in the step c) and the second reaction solution is 1-5:1.
8. method as claimed in claim 1 or 5, which is characterized in that threonine content and the standard in the test sample
The foundation of curve carries out simultaneously.
9. method as claimed in claim 1 or 5, which is characterized in that the LTA from pseudomonas aeruginosa, Escherichia coli,
One or more of bacillus subtilis, saccharomyces cerevisiae, Candida, candida albicans.
10. method as claimed in claim 5, which is characterized in that it is molten that step (a) prepares the different standard threonine of 8 concentration
Liquid, concentration are respectively 0.2mM, 0.5mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM.
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