CN106083989B - A kind of macro protein extraction purification process of high-temperature daqu leaching liquor - Google Patents
A kind of macro protein extraction purification process of high-temperature daqu leaching liquor Download PDFInfo
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Abstract
The present invention relates to a kind of method for extraction and purification of macro albumen of high-temperature daqu leaching liquor.Method is that spray sterile water mixing placement bio-incubator step-up temperature method progress activation culture is spare after high-temperature daqu crushes；Acetic acid-sodium acetate buffer solution and phenylmethylsulfonyl fluoride solution is added, oscillation extracts overnight after mixing, and filtering, centrifugation obtains high-temperature daqu leaching liquor；TCA- acetone soln, ammonium acetate methanol solution, acetone soln, Tris saturated phenol/sodium lauryl sulfate buffer, ammonium acetate methanol solution, methanol and acetone soln are sequentially added, A precipitating, B precipitating, phenol layer, C precipitating, D precipitating and the macro protein example of high-temperature daqu are accordingly obtained；Sample dissociation liquid, ice-bath ultrasonic hydrotropy is added, centrifugation abandons sediment and obtains the macro protein sample liquid of high-temperature daqu.The macro protein sample liquid of high-temperature daqu of this method preparation can be applied to the dielectrophoresis of the macro albumen of high-temperature daqu, obtain high-resolution, the Two-dimensional Gel Electrophoresis of high duplication.
Description
Technical field
The invention belongs to macro proteomic techniques fields.A kind of extraction more particularly to macro albumen of high-temperature daqu leaching liquor is pure
Change method.
Background technique
High-temperature daqu is that the knee-piece of bricked is pressed into through crushing and water-adding using wheat as primary raw material, in certain temperature, humidity
Under the conditions of it is open cultivate, koji-making culture maximum temperature is up to 60 ~ 68 DEG C, from environment for producing Daqu, bent female, in raw material microorganism
Growth and breeding metabolism generates various enzyme systems on knee-piece, so that high-temperature daqu has liquefaction power, saccharifying power and breaks down proteins power.
High-temperature daqu is mainly used for the production of Maotai-flavor liquor, is typical case with Maotai.The complexity that microorganism produces in high-temperature daqu is a variety of
Enzyme system or zymoprotein system are that Maotai-flavor liquor flavor substance forms major impetus, and influences the important of Maotai-flavor liquor quality
Factor.
Macro protein group is in 2004, and Rodriguez is proposed according to the concept of macro genome (metagenome),
It refers in environment the zoic protein group summation of institute in multiple-microorganism group.In the research of the macro protein science of yeast, river
Southern university has made the research of exploration in the macro protein science of yeast and bidirectional electrophoresis technique.The macro protein science of high-temperature daqu
Current studies in China data is studied also to be not directed to.Condition due to high-temperature daqu in more water high temperature is cultivated, and complexity is both generated
Enzyme is formed simultaneously the impurity of a variety of influence enzyme analyses.Especially it is anti-that Mei Lade has occurred in high-temperature daqu in yeast making process
It answers.Maillard reaction makes to generate between amino-containing compound and carbonyl-containing compound through condensation, polymerization in high-temperature daqu
A large amount of melanoidin substances.The presence of melanoidin pigment brings and asks greatly very much to the quantitative of macro protein sample, purifying, dielectrophoresis
Topic.
Therefore, high-temperature daqu Metaproteomics research must be set up a kind of high-resolution, the dielectrophoresis of high duplication
Technical method is the guarantee of effective Metaproteomics analysis.The bidirectional electrophoresis technique of the macro albumen of high-temperature daqu will be helpful to analyze
The dynamic change of the macro protein group of high-temperature daqu, and energy effective ratio is compared with the macro protein group for analyzing high-temperature daqu and strengthening porcelain
Composed structure difference provides the guidance of science for the improvement of high-temperature daqu production technology.To understand complexity between functional microorganism
The interaction of multi-enzyme system is the basis for improving production technology and improving Maotai-flavor liquor quality.And high-resolution, Gao Chongfu
The bidirectional electrophoresis technique of property is built upon on the basis of the macro protein group sample of good high-temperature daqu.So preparing good
The macro protein group sample of high-temperature daqu is to generate high-resolution, the key technology of the bidirectional electrophoresis technique map of high duplication, with
Guarantee to realize that effective Metaproteomics of sample are analyzed.
There are the impurity and a large amount of melanoidins of a variety of Two-dimensional Electrophoresis Analysis for influencing macro albumen for high-temperature daqu by the present invention
Pigment problem is tested, the method for improving macro protein extraction repeatedly by various aspects, the basic melanoidin pigment for solving high-temperature daqu
Interference problem, prepare the macro protein group sample of good high-temperature daqu for dielectrophoresis, establish a kind of high-resolution, Gao Chong
The bidirectional electrophoresis technique method of renaturation carries out the research of the macro protein science of high-temperature daqu.
Summary of the invention
High-temperature daqu preparation process had not only generated complicated enzyme simultaneously but also had formed the impurity of a variety of influence enzyme analyses.Especially
Maillard reaction has occurred in yeast making process and generates a large amount of melanoidin substances for high-temperature daqu.Seriously affect the effect of dielectrophoresis
Fruit.The present invention, using high-temperature daqu as material, carries out activation culture with high-temperature daqu, improves protein extraction for where problem
Method, the interference problem of the basic melanoidin pigment for solving high-temperature daqu, constructs a kind of high-resolution, the high temperature of high duplication is big
Bent leaching liquor extracts the sample of macro albumen for high-resolution, the dielectrophoresis of high duplication.
The technical solution for using high-temperature daqu leaching liquor to extract macro albumen in order to achieve the object of the present invention is as follows:
1, the activation culture of high-temperature daqu:
On the knee-piece of high-temperature daqu, takes high-temperature daqu 300g to smash it through 40 mesh sieve according to five point sampling and obtain height
Warm yeast starter powder, spray 20-40g sterile water mix, and place bio-incubator step-up temperature method and carry out 24 hours activation cultures, that is, exist
Activation culture 8 hours under the conditions of 30 DEG C, are warming up to 35 DEG C of activation cultures 6 hours, then are warming up to 40 DEG C of activation cultures 6 hours, most
It is spare after 4 hours to be warming up to 45 DEG C of activation cultures again afterwards.
2, prepared by high-temperature daqu leaching liquor:
High-temperature daqu starter powder of the 30g Jing Guo activation culture is weighed, the acetic acid-sodium acetate buffer solution and 90 μ L benzene of 90ml is added
Methanesulfonyl fluoride solution (PMSF solution), oscillation mixing extract overnight under the conditions of being placed on 4 DEG C.Then with 4 layers of filtered through gauze,
Filtrate is centrifuged 20 min, and taking supernatant is high-temperature daqu leaching liquor.
3, Tris- acetone-phenol improved method extracts
Take 10ml high-temperature daqu leaching liquor in centrifuge tube, -20 DEG C of pre-cooling 40mlTCA- acetone solns of addition mix.Mixing
It after liquid oscillation shakes up, is placed 2 hours in -20 DEG C of refrigerators, is centrifuged 30 min, abandon supernatant, collect A precipitating；
In A precipitating, the submergence of 5ml ammonium acetate methanol solution is added, after oscillation 3-5 minutes, is placed in -20 DEG C of refrigerators
30min is centrifuged 10 min.Supernatant is abandoned, B precipitating is collected；
In B precipitating, the cleaning of 10ml acetone soln is added, is subsequently placed in vacuum drying instrument, after so that acetone is volatilized completely,
Tris saturated phenol/sodium lauryl sulfate buffer (SDS Buffer) of 5ml 1:1 volume ratio is added, vibrates 15-20 minutes
Afterwards, it is centrifuged, collects light upper-layer phenol layer；
Ammonium acetate methanol solution, phenol layer: ammonium acetate methanol solution volume ratio 1:5 are added in phenol layer.It stands overnight, is centrifuged
10 min collect C precipitating；
10ml methanol is added in C precipitating, oscillation cleaning 5 minutes, is centrifuged 10min, collects D precipitating；
In D precipitating, the cleaning of 10ml acetone soln is added, acetone is made to volatilize completely, remainder is the macro protein of high-temperature daqu
Sample.
Above-described oscillation is carried out on vortex mixer；The centrifugation is under the conditions of being kept for 4 DEG C with 12000
R/min is centrifuged.
4, ultrasonic treatment
In the macro protein example of high-temperature daqu, 0.5-1.0ml sample dissociation liquid is added, submerges the macro protein of high-temperature daqu
Sample, ice-bath ultrasonic hydrotropy 30min, in the case where being kept for 4 DEG C, 12000 r/min are centrifuged 15 min, and taking supernatant is that high-temperature daqu is macro
Protein sample liquid.
Reagent solution involved in technical solution of the present invention is formulated as follows:
(1) acetic acid-sodium acetate buffer solution: weighing 8.1 g anhydrous sodium acetates and be dissolved in suitable quantity of water, constant volume to 1000
ML is made into 0.1mol/L sodium acetate solution.It takes 5.73mL glacial acetic acid to add water constant volume to 1000 mL, is made into 0.1mol/L acetic acid
Solution.Taking sodium acetate solution and acetic acid solution mixing to be adjusted to pH value respectively is 4.6~4.8.
(2) TCA acetone soln: 50 g of trichloroacetic acid (TCA), 350 μ L of beta -mercaptoethanol add acetone solution, constant volume arrives
500 mL are saved backup in -20 DEG C of refrigerators.
(3) sodium lauryl sulfate buffer (SDS Buffer): 30% sucrose, 2%SDS, 0.1M Tris-HCL pH value
8.0,5% beta -mercaptoethanol, 1% protease inhibitor cocktail.It is saved backup in -20 DEG C of refrigerators.
(4) acetone soln: 350 μ L beta -mercaptoethanols add acetone solution, constant volume to 500mL.It is protected in -20 DEG C of refrigerators
It deposits.
(5) ammonium acetate methanol solution: 0.1mol ammonium acetate is dissolved in methanol, and constant volume is made into 0.1mol/L to 1000 mL
Ammonium acetate methanol solution saves backup in -20 DEG C of refrigerators.
(6) sample dissociation liquid: urea 10.5g, thiocarbamide 3.8g, CHAPS 1g, IPG Buffer(pH 3-10) 500 μ L,
Constant volume is to 25mL after DTT154mg is dissolved in water.Sample dissociation liquid 1mL is dispensed in every 1.5mL EP pipe, in -20 DEG C of refrigerators
It saves backup.
Above is referred to reagent be purchased from GE company.The macro protein sample liquid of high-temperature daqu obtained by above technical scheme
The dielectrophoresis that can be applied to the macro albumen of high-temperature daqu, obtains high-resolution, and the Two-dimensional Gel Electrophoresis of high duplication carries out high
The research of the warm macro protein science of yeast.Concrete application process is as follows:
The 350 macro protein sample liquid of μ L high-temperature daqu extracted are risen the mode of loading with IPG adhesive tape bubble, are risen Pan Zhongpao in bubble
Rise after 18h, carries out isoelectric focusing, current limit 50 μ A, 20 DEG C of operating temperature.Concrete operations according to GE company " dielectrophoresis
Handbook " program carries out isoelectric focusing, SDS-PAGE electrophoresis, gel-colored according to this, and laser scanner ImageScaner III takes pictures
That is the dielectrophoresis spectrogram of the macro protein sample of high-temperature daqu.
Summarize the macro albumen bidirectional electrophoresis technique scheme of high-temperature daqu: high-temperature daqu needs to handle by activation culture；With
The preparation of pH4.6-4.8 acetic acid-sodium acetate buffer solution progress high-temperature daqu leaching liquor；It is extracted using Tris- acetone-phenol improved method
The macro protein group sample of high-temperature daqu.The method for extraction and purification of the macro albumen of high-temperature daqu leaching liquor of the invention, dielectrophoresis are solidifying
Glue clear background, the interference problem of the basic melanoidin pigment for solving high-temperature daqu, it is clear to take pictures to obtain high-resolution, repetitive rate
The macro albumen dielectrophoresis spectrogram of high high-temperature daqu.And it is suitable for next step Mass Spectrometric Identification and analyzes work.
Detailed description of the invention
Fig. 1 is that the macro protein sample liquid of high-temperature daqu extracted using document report Tris- acetone method carries out dielectrophoresis
Map.
Fig. 2 is that the macro protein sample liquid of high-temperature daqu extracted using method of the present invention carries out Two-dimensional Gel Electrophoresis.
Specific embodiment
Embodiment 1
The technical solution for using high-temperature daqu leaching liquor to extract macro albumen in order to achieve the object of the present invention is as follows:
1, the activation culture of high-temperature daqu:
On the knee-piece of high-temperature daqu, takes high-temperature daqu 300g to smash it through 40 mesh sieve according to five point sampling and obtain height
Warm yeast starter powder, spray 40g sterile water mix, and place bio-incubator step-up temperature method and carry out 24 hours activation cultures, i.e., 30
DEG C activation culture 8 hours, the 35 DEG C of activation cultures that heat up 6 hours, the 40 DEG C of activation cultures that heat up 6 hours, heated up 45 DEG C of activation cultures 4
It is spare after hour.
2, prepared by high-temperature daqu leaching liquor:
High-temperature daqu powder of the 30g Jing Guo activation culture is weighed, the acetic acid-sodium acetate buffer solution of the pH value 4.6 of 90ml is added
With 90 μ L phenylmethylsulfonyl fluoride solution (PMSF solution), oscillation mixing extracts overnight under the conditions of being placed on 4 DEG C.Then with 4 layers of yarn
Cloth filtering, filtrate are centrifuged 20 min, and taking supernatant is high-temperature daqu leaching liquor.
3, Tris- acetone-phenol improved method extracts
Take 10ml high-temperature daqu leaching liquor in centrifuge tube, -20 DEG C of pre-cooling 40mlTCA- acetone solns of addition mix.Mixing
It after liquid oscillation shakes up, is placed 2 hours in -20 DEG C of refrigerators, is centrifuged 30 min, abandon supernatant, collect A precipitating；
In A precipitating, the submergence of 5ml ammonium acetate methanol solution is added, after oscillation 3-5 minutes, is placed in -20 DEG C of refrigerators
30min is centrifuged 10 min.Supernatant is abandoned, B precipitating is collected；
In B precipitating, the cleaning of 10ml acetone soln is added, is subsequently placed in vacuum drying instrument, after so that acetone is volatilized completely,
Tris saturated phenol/sodium lauryl sulfate buffer (SDS Buffer) of 5ml 1:1 volume ratio is added, after oscillation 15 minutes,
Centrifugation, collects light upper-layer phenol layer；
Ammonium acetate methanol solution, phenol layer: ammonium acetate methanol solution volume ratio 1:5 are added in phenol layer.It stands overnight, is centrifuged
10 min collect C precipitating；
10ml methanol is added in C precipitating, oscillation cleaning 5 minutes, is centrifuged 10min, collects D precipitating；
In D precipitating, the cleaning of 10ml acetone soln is added, acetone is made to volatilize completely, remainder is the macro protein of high-temperature daqu
Sample.
The above oscillating operation is carried out on vortex mixer；Centrifugally operated be keep 4 DEG C at 12000 r/min from
The heart.
4, ultrasonic treatment
1.0ml sample dissociation liquid is added in the macro protein example of high-temperature daqu, submerges the macro protein example of high-temperature daqu,
Ice-bath ultrasonic hydrotropy 30min, in the case where being kept for 4 DEG C, 12000 r/min are centrifuged 15 min, and taking supernatant is the macro albumen sample of high-temperature daqu
Product liquid.
5, the macro protein sample liquid of high-temperature daqu is used to prepare Two-dimensional Gel Electrophoresis:
The macro protein sample liquid of the high-temperature daqu that said extracted is purified, the two-way electricity applied to the macro albumen of high-temperature daqu
Swimming, obtains high-resolution, the Two-dimensional Gel Electrophoresis of high duplication, and concrete application is as follows:
The 350 macro protein sample liquid of μ L high-temperature daqu extracted are risen the mode of loading with IPG adhesive tape bubble, are risen Pan Zhongpao in bubble
Rise after 18h, carries out isoelectric focusing, current limit 50 μ A, 20 DEG C of operating temperature.Concrete operations according to GE company " dielectrophoresis
Handbook " program carries out isoelectric focusing, SDS-PAGE electrophoresis, gel-colored according to this, and laser scanner ImageScaner III takes pictures
That is the dielectrophoresis spectrogram of the macro protein sample of high-temperature daqu.As shown in Fig. 2.
The present embodiment reports Tris- acetone method using open source literature, is extracted the macro protein sample liquid of high-temperature daqu and adopts
Dielectrophoresis, which has been carried out, with identical Two-dimensional Gel Electrophoresis obtains Two-dimensional Gel Electrophoresis.As shown in Fig. 1.
Comparative drawings figs 1 and attached drawing 2, it is known that: Fig. 1 is since Melanoidins interfere, and pigementation region is big, clear protein site
Few, resolution ratio is low；Fig. 2 excludes Melanoidins interference substantially, and pigementation region is small, and clear protein site is more, high resolution, can answer
For protein science research；
Above is referred to reagent be purchased from GE company.
Reagent solution involved in the present embodiment is formulated as follows:
(1) acetic acid-sodium acetate buffer solution: weighing 8.1 g anhydrous sodium acetates and be dissolved in suitable quantity of water, constant volume to 1000
ML is made into 0.1mol/L sodium acetate solution.It takes 5.73mL glacial acetic acid to add water constant volume to 1000 mL, is made into 0.1mol/L acetic acid
Solution.Taking sodium acetate solution and acetic acid solution mixing to be adjusted to pH value respectively is 4.6~4.8.
(2) TCA acetone soln: 50 g of trichloroacetic acid (TCA), 350 μ L of beta -mercaptoethanol add acetone solution, constant volume arrives
500 mL are saved backup in -20 DEG C of refrigerators.
(3) sodium lauryl sulfate buffer (SDS Buffer): 30% sucrose, 2%SDS, 0.1M Tris-HCL pH value
8.0,5% beta -mercaptoethanol, 1% protease inhibitor cocktail.It is saved backup in -20 DEG C of refrigerators.
(4) acetone soln: 350 μ L beta -mercaptoethanols add acetone solution, constant volume to 500mL.It is protected in -20 DEG C of refrigerators
It deposits.
(5) ammonium acetate methanol solution: 0.1mol ammonium acetate is dissolved in methanol, and constant volume is made into 0.1mol/L to 1000 mL
Ammonium acetate methanol solution saves backup in -20 DEG C of refrigerators.
(6) sample dissociation liquid: urea 10.5g, thiocarbamide 3.8g, CHAPS 1g, IPG Buffer(pH 3-10) 500 μ L,
Constant volume is to 25mL after DTT154mg is dissolved in water.Sample dissociation liquid 1mL is dispensed in every 1.5mL EP pipe, in -20 DEG C of refrigerators
It saves backup.
Above is referred to reagent be purchased from GE company.
Claims (6)
1. a kind of method for extraction and purification of the macro albumen of high-temperature daqu leaching liquor, it is characterised in that:
1) activation culture of high-temperature daqu:
On the knee-piece of high-temperature daqu, taking high-temperature daqu 300g to smash it through 40 mesh sieve according to five point sampling, to obtain high temperature big
Starter powder, spray 20-40g sterile water mix, and it is spare to place 24 hours activation cultures of bio-incubator step-up temperature method progress；
2) prepared by high-temperature daqu leaching liquor:
High-temperature daqu starter powder of the 30g Jing Guo activation culture is weighed, the acetic acid-sodium acetate buffer solution and 90 μ L benzyls of 90ml is added
Sulfuryl fluoride solution, oscillation mix be placed on 4 DEG C under the conditions of extract overnight, then use 4 layers of filtered through gauze, filtrate centrifugation 20 min,
Taking supernatant is high-temperature daqu leaching liquor；
3) Tris- acetone-phenol improved method extracts
Take 10ml high-temperature daqu leaching liquor in centrifuge tube, -20 DEG C of pre-cooling 40mlTCA- acetone solns of addition mix；Oscillation shakes up
It is placed 2 hours in -20 DEG C of refrigerators afterwards, is centrifuged 30 min, abandon supernatant, collect A precipitating；
In A precipitating, the submergence of 5ml ammonium acetate methanol solution is added, after oscillation 3-5 minutes, places 30min in -20 DEG C of refrigerators,
10 min are centrifuged, B precipitating is collected；
In B precipitating, the cleaning of 10ml acetone soln is added, is subsequently placed in vacuum drying instrument, after so that acetone is volatilized completely, is added
Tris saturated phenol/sodium lauryl sulfate buffer of 5ml 1:1 volume ratio, after oscillation 15-20 minutes, light color is collected in centrifugation
Upper-layer phenol layer；
Ammonium acetate methanol solution is added in phenol layer, phenol layer: ammonium acetate methanol solution volume ratio 1:5 is stood overnight, centrifugation 10
Min collects C precipitating；
10ml methanol is added in C precipitating, oscillation cleaning 5 minutes, is centrifuged 10min, collects D precipitating；
In D precipitating, the cleaning of 10ml acetone soln is added, so that acetone is volatilized completely, obtains the macro protein example of high-temperature daqu；
4) ultrasonic treatment
In the macro protein example of high-temperature daqu, 0.5-1.0ml sample dissociation liquid is added, submerges the macro protein sample of high-temperature daqu
Product, ice-bath ultrasonic hydrotropy 30min are centrifuged 15 min, abandon sediment and obtain the macro protein sample liquid of high-temperature daqu；
The step-up temperature method refers under the conditions of 30 DEG C activation culture 8 hours, is warming up to 35 DEG C of activation cultures 6 hours, then
It is warming up to 40 DEG C of activation cultures 6 hours, the 45 DEG C of activation cultures that finally heat up again 4 hours；
The sample dissociation liquid is prepared by following procedure: urea 10.5g, thiocarbamide 3.8g, CHAPS 1g, pH 3-10
500 μ L, DTT 154mg of IPG Buffer be dissolved in water after constant volume to 25mL, dispense sample dissociation in every 1.5mL EP pipe
Liquid 1mL is saved backup in -20 DEG C of refrigerators.
2. a kind of method for extraction and purification of macro albumen of high-temperature daqu leaching liquor according to claim 1, it is characterised in that institute
The oscillation stated is carried out on vortex mixer；The centrifugation be keep 4 DEG C under the conditions of with 12000 r/min carry out from
The heart.
3. a kind of method for extraction and purification of macro albumen of high-temperature daqu leaching liquor according to claim 1, it is characterised in that institute
The acetic acid-sodium acetate buffer solution stated is prepared by following procedure: it weighs 8.1 g anhydrous sodium acetates and is dissolved in suitable quantity of water, it is fixed
Hold 1000 mL, is made into 0.1mol/L sodium acetate solution；It takes 5.73mL glacial acetic acid to add water constant volume to 1000 mL, is made into
0.1mol/L acetic acid solution；Taking sodium acetate solution and acetic acid solution mixing to be adjusted to pH value respectively is 4.6~4.8.
4. a kind of method for extraction and purification of macro albumen of high-temperature daqu leaching liquor according to claim 1, it is characterised in that institute
The TCA- acetone soln stated is prepared by following procedure: 50 g of trichloroacetic acid, and 350 μ L of beta -mercaptoethanol adds acetone solution,
Constant volume saves backup in -20 DEG C of refrigerators to 500 mL；
Sodium lauryl sulfate buffer is prepared by following procedure: 30% sucrose, 2%SDS, 0.1M Tris-HCL pH value
8.0,5% beta -mercaptoethanol, 1% protease inhibitor cocktail saves backup in -20 DEG C of refrigerators.
5. a kind of method for extraction and purification of macro albumen of high-temperature daqu leaching liquor according to claim 1, it is characterised in that institute
The acetone soln stated is prepared by following procedure: 350 μ L beta -mercaptoethanols, adds acetone solution, constant volume to 500mL ,-
It is saved in 20 DEG C of refrigerators.
6. a kind of method for extraction and purification of macro albumen of high-temperature daqu leaching liquor according to claim 1, it is characterised in that institute
The ammonium acetate methanol solution stated is prepared by following procedure: 0.1mol ammonium acetate being dissolved in methanol, constant volume to 1000
ML is made into 0.1mol/L ammonium acetate methanol solution, saves backup in -20 DEG C of refrigerators.
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| CN114539348B (en) * | 2022-01-25 | 2024-04-05 | 四川省农业科学院水稻高粱研究所 | Protein extraction method for fermented grain microorganism macro proteomics detection |
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