CN107084867B - A kind of extracting method of filamentous fungi solid fermentation product total protein - Google Patents
A kind of extracting method of filamentous fungi solid fermentation product total protein Download PDFInfo
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Abstract
The invention discloses a kind of extracting methods of filamentous fungi solid fermentation product total protein.The technological core of this method is the method for combining bead mechanical breaking-wall method and clasmatosis buffer solution (SI); it destroys the thick and solid tough and tensile cell membrane of filamentous fungi and releases intracellular total protein, and the trichoderma solid fermentation product total protein of high quality is extracted under the protection of Extraction buffer (SII) and protease inhibitors.Present invention employs a kind of simple and practicable methods, the thick and solid tough and tensile, intracellular protein of filamentous fungal cell wall is overcome to be difficult to discharge, the interference of high organic and humic acid material in solid fermentation object, the total protein of high quality can be directly extracted from trichoderma solid fermentation product with reference to semi-permeable membrane dialysis, the research for filamentous fungi proteomics in solid fermentation process provides technical guarantee.
Description
Technical field
The invention belongs to bio-science fields, are related to a kind of extracting method of filamentous fungi solid fermentation product total protein.
Background technology
China is a large agricultural country, and cultivated area accounts for 9% or so of global cultivated area.In recent years, China's Traditional Agricultural
Industry is to modern efficient, agricultural is increased production and increasing peasant income provide guarantee, but agricultural chemicals (agriculture for intensive agriculture being changed into
Medicine, chemical fertilizer etc.) a large amount of applications also bring the problem of serious to agricultural production.It is to realize that chemical fertilizer subtracts using microbial organic fertilizer
Apply, promote the effective way of China's agricultural sustainable development.Plant rhizosphere inhabites substantial amounts of beneficial microbe, they pass through not
Same mode promotes plant root growth and plant is induced to generate defense reaction, enhances the resistivity to external environment.Research
It was found that trichoderma is widely present in as a kind of beneficial microbe in soil, symbiosis can be formed after plant root with plant by colonizing
Body promotes the growth of plant by secreting secondary metabolite and plant growth regulator.Research finds Trichoderma harzianum biology
Organic fertilizer can be good at promoting the growth of plant, by optimizing the solid fermentation parameter of Guizhou trichoderma, probing into biological organic fertilizer
Effect, have great importance for improving crop yield, improving crop quality, and provide theoretical foundation for actual production.
Trichoderma is a kind of common biocontrol agent, while can promote the growth of plant, is especially had to the growth-promoting of root system of plant notable
Effect.In recent years, prebiotic trichoderma had as microorganism because of the advantages that its is environmental-friendly, safe and non-toxic, parasitic plant pathogen
It is gradually widely used in the agricultural production at home and abroad of machine fertilizer, is taken promoting plant growth and prevention and control soil-borne disease etc.
Obtained good effect.
In the concrete application and production process of trichoderma, biological control and plant growth-promoting effect and the close phase of its zymotechnique
It closes, fermentation can be mainly divided into two kinds of different techniques of liquid and solid fermentation.Liquid fermentation is with the liquid containing nutriment
Body culture medium carrys out the process of fermented and cultured microorganism；And solid fermentation is under the conditions of certain moisture content, microorganism is being suitble to support
It is grown on the solid state substrate divided and generates the process of metabolite.Numerous studies show solid-fermented technique than liquid fermentation skill
Art has the advantages of more.Firstly, since solid matrix nutrient used is more abundant, in the characteristic of yield, production capacity and product
Liquid fermentation will be significantly better than；Secondly, most solid fermentation is all to utilize the agricultural of low cost or industrial or agricultural by-product
As culture substrate, fund and operating cost are greatly reduced；Finally, in solid fermentation process, mobility is relatively low, required equipment
Simply, meanwhile, the cost that the downstream engineering of liquid fermentation is brought is reduced.
Proteomics research is for specific environment, different condition, different cell types or particular growth stage of development
The all protein expressed in cell and tissue, the main differential expression including protein, functional protein identification and quantitative analysis,
Posttranslational modification, physiological function and its interactive network etc..Proteomics can be from the level of gross protein, more accurately
Go find and inquire into the essence of the rule of vital movement and its important physiology course.At present, proteomics research into
For the hot spot of world research, it is not only the expansion of genome times afterwards comprehensively research, is even more the core of current life science research
Intracardiac appearance.The technology that proteomics research is related to mainly includes electrophoretic techniques, tree species for bio-energy source, and the two is indispensable.
Two dimensional gel electrophoresis (2D-PAGE) is most widely used protein stripping technique, mainly according in test protein not
Same isoelectric point and molecular weight, the total protein mixture for making hydrophobicity different with relative abundance is sufficiently separated, this technology
The advantages of be that it can study the posttranslational modification of albumen, but the reappearance of this technology is not high and is easy to cause albumen and loses
It loses, has higher requirement to the extraction of total protein.ITRAQ technologies (isotope relative index and absolute quantitation technology) are in recent years
A kind of new proteomics quantitative study technology out newly developed is generally possible to identify 500 to 600 kinds of albumen, and fixed
The difference of different sample room protein expressions is measured, is a kind of method of proteomics research most widely used at present.Biology
Mass-spectrometric technique is to carry out ingredient and structural analysis by the mass-to-charge ratio (m/z) of determination sample, is the main skill of identification of proteins
Art, it has many advantages, such as that high sensitivity, accuracy are good and easy to automate.
With scientific and technological progress and the development of society, the mankind are increasing to the research of fungi, particularly in agricultural research
It is used for the trichoderma of plants probiotics in journey, is played an important role in terms of plant growth-promoting with biological and ecological methods to prevent plant disease, pests, and erosion.But early period is a large amount of
Research tightly rests on genome and transcript profile level, and the horizontal progress of protein groups is slow, and most important one restriction factor is
It is difficult to obtain the total protein of high quality.Compared with other species, the proteomics research of filamentous fungi is also in a starting
Stage is badly in need of a kind of efficiently quick total protein extraction method to break through protein science research.
The content of the invention：
The purpose of the present invention is to solve in protein science research process, the technical bottleneck of filamentous fungi total protein extraction
Problem provides a kind of extracting method of filamentous fungi tunning total protein rapidly and efficiently.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of extracting method of filamentous fungi solid fermentation product total protein, comprises the following steps：
(1) filamentous fungi solid fermentation product is placed in the ball milling instrument tank body of zirconium oxide inner wall, adds in and shift to an earlier date precooling
Lysate SI and zirconia ball are vortexed uniform；It is vortexed after adding in DDT solution, then carries out ice bath, ice bath terminates mixed after freezing
It closes and is ground on ball milling instrument；
(2) extracting solution SII is added in after grinding, by solid fermentation product and zirconium oxide bead ice bath after mixing；Ice
It is transferred on ball milling instrument and is ground after bath；
(3) mixture ground in step (2) is transferred in centrifuge tube, is centrifuged at 4 DEG C；It, will be upper after centrifugation
It is transferred to clearly in new centrifuge tube, and abandons precipitation；
(4) after supernatant being carried out ice bath, according to 1:The ratio of 4 (v/v) adds in 100%TCA solution and is uniformly mixed, will be mixed
Close liquid be stored in -20 DEG C under the conditions of 2 it is small when, then place it on ice until liquefaction；
(5) after supernatant completely liquefaction, in 4 DEG C of centrifugations, supernatant is abandoned, centrifuge tube is upside down on blotting paper, removed more
Remaining liquid；The acetone for shifting to an earlier date precooling is added in into precipitation, and is beaten with liquid-transfering gun come resorption by pellet resuspended among acetone,
After precipitation is blown and beaten mixing, 4 DEG C centrifuge, and remove the liquid on upper strata；Step 2-5 times that supernatant is abandoned in acetone resuspension-centrifugation-is repeated,
After acetone color less depth, upper strata acetone is removed, extra acetone is volatilized clean, solid powder is filamentous fungi
Solid powder is stored in -20 DEG C by the total protein in solid fermentation product.
The preferred trichoderma of the filamentous fungi；Further preferred filamentous fungi is Guizhou trichoderma.Guizhou of the present invention
Trichoderma is suitable for all Guizhou trichodermas, is particularly suitable for growth-promoting functions, for producing the Guizhou trichoderma of bio-feritlizer, such as
Voluntarily separation and commercially available Trichoderma harziaum are suitable for the method for the present invention；It is most preferably suited to Guizhou trichoderma NJAU4742
(Trichoderma guizhouense), bacterial strain preserving number are CGMCC No.12166.
The filamentous fungi solid fermentation product is that filamentous fungi is inoculated in agricultural wastes straw to carry out solid
Fermentation stops culture after mycelia is covered in solid fermentation matrix, obtains the filamentous fungi solid fermentation product；The agriculture
Any one of industry wastes straw in rice straw, wheat stalk or maize straw.
In the method for the invention, after stalk is dried, it is cut into the small fragment of 1-2cm and is ground into 40 purposes with pulverizer
Powder.The different stalk powders of 10g are weighed in the big triangular flask of 1L, it is prepared that 23mL is then added in into triangular flask
Mandels salting liquids (initial aqueous rate about 70%) wait culture medium is high at 121 DEG C after solid contents and the abundant mixing of salting liquid
Temperature sterilizing 30min is spare.
The preparation method of SI solution described in step (1) is preferred：8M urea, 2M thiocarbamides, 1mM EDTA, 10mM PBS,
4 DEG C of refrigerators preserve after 0.1%Triton-X-100, fully dissolving, use preceding addition Roche Protease inhibitor cocktail；On
It is mass volume ratio concentration (w/v) to state percent concentration.
DDT solution preparation methods described in step (1) are preferred：1.55gDTT is settled to 10mL with deionized water, -20 DEG C
It saves backup；It is 10min that freezing mixing and ball milling instrument working procedure, which is arranged to grinding net time, suspends 1 minute within every 2 minutes, grinding
Temperature setting is 4 DEG C.
SII solution formulas described in step (2) are：3%CHAPS, 1%SDS, 0.1% NaTDC, 0.2mg/mL
BSA。
Extracting method of the present invention, further preferably includes the following steps：
(1) cultured filamentous fungi solid fermentation product is weighed in the ball milling instrument zirconium oxide inner wall jar cleaned up,
And add in the Zirconia beads in the lysate SI for shifting to an earlier date precooling with 0.5mm；Tank body is placed in vortex instrument, with 1800-
Solid fermentation product is uniformly mixed by 2200rpm/min with zirconium oxide bead；Enter DDT solution after opening tank body and be vortexed 2 minutes, it will
Tank body ice bath 15min；Tank body is ground on freezing mixing and ball milling instrument after ice bath；Wherein filamentous fungi solid is sent out
The mass volume ratio of ferment product and lysate SI are 1g：3-7mL；The quality of filamentous fungi solid fermentation product and Zirconia beads
Than for 1:0.8-1.2；The mass volume ratio of filamentous fungi solid fermentation product and DDT solution is 1g：3-7μL；
(2) tank body is placed in 15min on ice after grinding, open tank body and adds in extracting solution SII, tank body is placed in whirlpool
It revolves on instrument, is uniformly mixed solid fermentation product with zirconium oxide bead with middling speed, by tank body ice bath 30 minutes；By tank after ice bath
Body is transferred on ball milling instrument and is ground, and grinding temperature is arranged to 4 DEG C, and grinding net time is 10min, suspends 1 point within every 2 minutes
Clock；The mass volume ratio of filamentous fungi solid fermentation product and extracting solution SII are 1.8-2.2g：1mL；
(3) ground mixture is transferred in the centrifuge tube cleaned up, is centrifuged under the conditions of 4 DEG C of 8000rpm
10min；After centrifugation, supernatant is transferred in new centrifuge tube, and abandons precipitation, total protein is present in supernatant；
(4) supernatant is subjected to ice bath after ten minutes, according to 1:The ratio of 4 (v/v) adds in 100%TCA solution and centrifuges
Pipe, which turns upside down, makes it be sufficiently mixed uniformly；When 2-4 is small under the conditions of mixed liquor is stored in -20 DEG C；After cooling time has arrived,
And it places it on ice until liquefaction；
(5) after supernatant completely liquefaction, 10min is centrifuged under the conditions of 4 DEG C of 8000rpm；Supernatant is abandoned after centrifugation, it will
Centrifuge tube is upside down on blotting paper, removes extra liquid；The acetone for shifting to an earlier date precooling is added in into precipitation, and with liquid-transfering gun back and forth
Suction is beaten by pellet resuspended among acetone, and after precipitation is blown and beaten mixing, 10min is centrifuged under the conditions of 4 DEG C of 8000rpm, is removed
The liquid on upper strata；It repeats the above steps 3 times, after acetone color less depth, removes upper strata acetone, will be more using nitrogen evaporator
Remaining acetone volatilization is clean, and solid powder is the total protein in solid fermentation product, and solid powder is stored in -20 DEG C.
When supernatant is removed in the method for the invention, after centrifugation, have to as far as possible remove supernatant totally, otherwise hold
Destructible albumen precipitation also influences whether the quality of albumen, if liquid is organic reagent, as far as possible with nitrogen evaporator by organic examination
Agent is cleaned out；When on the other hand, with pipette tips Aspirate supernatant, it is impossible to be drawn onto the precipitation of lower floor；It is added in ice-cold acetone
0.01% beta -mercaptoethanol can protect Filamentous true total protein not oxidized, but definitely cannot excessively add in, and can excessively make egg
White matter is denatured and is in unstable transition, accelerates the decomposition of protein.
Advantageous effect：
The present invention is matrix using agricultural wastes straw, carries out solid fermentation after being inoculated with trichoderma, and utilizes mechanical breaking-wall method
And the method that suitable extractant phase combines, the trichoderma total protein of high quality, beneficial effect of the invention are extracted from solid matrix
Fruit is：
(1) method of this method combination bead mechanical breaking-wall method and clasmatosis buffer solution (SI), can sufficiently destroy
The thick and solid tough and tensile cell membrane of filamentous fungi simultaneously releases intracellular total protein, can greatly improve the extracted amount of albumen.
(2) after agricultural crop straw inoculation trichoderma carries out solid fermentation, substantial amounts of organic matter and humic acids are contained in matrix
Substance, this method can remove organic matter and humic by adding a certain proportion of surfactant (3%CHAPS, 1%SDS)
The interference of acid.
(3) this method can be greatly improved by optimizing the pH value of Extraction buffer (SII) and adding a certain amount of BSA
The rate of recovery of the intracellular protein discharged after filamentous fungi extracellular protein and broken wall is filamentous fungi protein in solid fermentation process
The research that group is learned provides technical guarantee.
(4) 0.01% beta -mercaptoethanol is added in cold acetone, intracellular protein can be protected not oxidized, enabling mirror
Determine and quantify more filamentous fungi total protein quantity.
Description of the drawings
The total protein electrophoretogram of the Guizhou trichoderma NJAU4742 of Fig. 1 Different Extraction Methods extraction.M is standard protein
marker；1 swimming lane is the protein electrophoresis that conventional method one is extracted as a result, 2 swimming lanes are the total protein electrophoresis that conventional method two is extracted
As a result；3 swimming lanes are the total protein electrophoresis result that conventional method three is extracted；4 swimming lanes are the total protein electrophoresis that conventional method four is extracted
As a result；5-1,5-2 swimming lane are the total protein electrophoresis result of method used in the present invention extraction.
The total protein electrophoretogram of Guizhou trichoderma NJAU4742 tunnings in Fig. 2 different substrates.M is standard protein
marker；1 swimming lane extracts Guizhou trichoderma NJAU4742 rice straw tunning total proteins for optimization method of the present invention；2 be normal
Rule method three extracts Guizhou trichoderma NJAU4742 rice straw tunning total proteins；3 extract Guizhou for optimization method of the present invention
Trichoderma NJAU4742 wheat stalk tunning total proteins；4 extract Guizhou trichoderma NJAU4742 wheat stalks for conventional method three
Tunning total protein；5 extract Guizhou trichoderma NJAU4742 maize straw tunning total proteins for optimization method of the present invention；6
Guizhou trichoderma NJAU4742 maize straw tunning total proteins are extracted for conventional method three.
The total protein two dimensional electrophoresis figure of Fig. 3 Guizhou trichoderma NJAU4742.(rice straw is fermentation substrate, present invention optimization
Method) Fig. 4 Guizhou trichoderma NJAU4742 total protein two dimensional electrophoresis figure.(rice straw be fermentation substrate, conventional method three)
Biomaterial preservation information
NJAU4742, Classification And Nomenclature Guizhou trichoderma (Trichoderma guizhouense), is preserved in China Microbiological bacterium
Kind preservation administration committee common micro-organisms center, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese section
Institute of microbiology of institute, preservation date be on April 11st, 2016, bacterial strain preserving number CGMCC No.12166.
Specific embodiment：
Following embodiment illustrates this hair by taking Guizhou trichoderma NJAU4742 (Trichoderma guizhouense) as an example
Bright technical solution, but therefore do not limit protection scope of the present invention.In fact, inventor is according to the method for the present invention, with more
Kind voluntarily separation and the extraction of commercially available filamentous fungi progress solid fermentation product total protein, obtains similar to the present invention
Effect, but in view of patent discloses sufficient requirement, below only with Guizhou trichoderma NJAU4742 (Trichoderma
Guizhouense exemplified by), the extraction of the extraction total protein of the filamentous fungi solid fermentation product total protein of the present invention is illustrated.
Reagent used in this example is enumerated as follows, and all reagents are bought from regular Reagent Company：
Urea (U0631, SIGMA), thiocarbamide (T7750, SIGMA), EDTA (798681, ALDRICH), Triton-X-100
(T9284, SIGMA-ALDRICH), protease inhibitors (Roche Protease inhibitor cocktail), CHAPS (226947,
ALDRICH), SDS (74255, SIGMA), BSA (D0565, SIGMA), NaTDC (D6750, SIGMA-ALDRICH)；
The prefabricated adhesive tape of IPG (Bio-Rad companies of the U.S.), ReadyStrip IPG Strips, pH 4-7,7cm, 163-2001-20 DEG C of ice
Case preserves；Carrier ampholyte (Bio-Rad companies of the U.S.), Bio-Lyte 3/10Ampholyte, 40%, 10ml, 163-
1112,4 DEG C of refrigerators preserve；Protein quantification kit Protein Assay Kit II.
The conidial preparations of 1 Guizhou trichoderma NJAU4742 of embodiment and solid fermentation
1. Guizhou trichoderma NJAU4742 brief introductions
Bacterial strain used in the present invention be Guizhou trichoderma NJAU4742 (Trichoderma guizhouense), the bacterial strain
Belong to Trichoderma harzianum branch in evolution, mycelia being capable of hyperparasite the pathogen of Botrytis cinerea, Alternaria alternata bacterium, sclerotinite, miliary damping-off by force
Bacterium, Sclerotium rolfsii and Fusarium oxysporum；Meanwhile can be very good to colonize in soil and plant rhizosphere, promote plant growth, suppression
Plant disease processed occurs, and improves the yield of crops.
2. conidial preparation
The Guizhou trichoderma NJAU4742 strains of activation are inoculated into the triangle of the 250ml equipped with 50ml solid PDA mediums
In bottle, after then being cultivated 3-4 days under the conditions of 50 DEG C, 0.9% NaCl solution of 10ml high-temperature sterilizations is added under aseptic condition,
After 120rpm about 30min, mycelia and sporangium are filtered to remove with two layers of sterile gauze, and spore suspension is counted with blood counting chamber
The spore concentration of the inside.
3. the solid fermentation of Guizhou trichoderma NJAU4742
After rice straw is dried, it is cut into the small fragment of 1-2cm and the size of 40 mesh is ground into pulverizer, weigh 10g
Then rice straw powder adds in the prepared Mandels salting liquids [0.3g of 23mL in the big triangular flask of 1L into triangular flask
L-1Urea, 1.4gL-1(NH4)2SO4, 2.0gL-1KH2PO4, 0.3gL-1CaCl2, 0.3gL-1MgSO4, 0.25gL-1
Yeast extract, 0.75gL-1Peptone, 5mgL-1FeSO4·7H2O, 20mgL-1CoCl2, 1.6mgL-1MnSO4With
1.4mg·L-1ZnSO4].By culture medium, high-temperature sterilization 30min is spare at 121 DEG C after grade solid contents and the abundant mixing of salting liquid.
The Guizhou trichoderma NJAU4742 spore suspensions that will be prepared, according to 107The inoculum concentration of cfu/g dw is inoculated into preparation
In good solid fermentation culture medium.After being inoculated with, triangular flask is placed in standing in 28 DEG C of constant incubator and is protected from light culture, is cultivated
Time is 5-7 days, which will be for the extraction of follow-up total protein.
Total protein in 2 distinct methods of embodiment extraction Guizhou trichoderma NJAU4742 solid fermentation products
2.1 common fungus total protein extraction methods one
Quickly Guizhou trichoderma NJAU4742 solid fermentation products are ground into uniformly using mortar under the conditions of liquid nitrogen frozen
Powder, the powder for taking 2g ground is transferred in the centrifuge tube of clean 50mL, adds in (20mM Tris- in 10mL lysates
HCl pH 7.4,150mM NaCl, 1%NP-40,0.5%Sodium deoxycholate, 0.1%SDS, protease inhibitors
Mixture), 1h is incubated on ice, during which overturns mixing every 5min, 4 DEG C, take supernatant, supernatant after 24 000r/min centrifugations 30min
The total protein as extracted, protein concentration is as shown in table 1 in supernatant, and electrophoresis result is as shown in Figure 1.
2.2 common fungus total protein extraction methods two
Quickly Guizhou trichoderma NJAU4742 solid fermentation products are ground into uniformly using mortar under the conditions of liquid nitrogen frozen
Powder, the powder for taking 2g ground is transferred in the centrifuge tube of clean 50mL, adds in (50mM Tris- in 10mL lysates
Hcl, 1mM EDTA, 50mM NaCl, 0.1%Triston X-100,1mM DTT, 1mM PMSF, pH8.0), system is carried out
Mixing；During which every 5min mixings once ice bath 30min, and crushes thalline with ultrasonic cell disruption instrument in ice bath
(750W, ultrasonic 6s are spaced 1s, ultrasound 20 times)；After ultrasound, at 4 DEG C, 12000rpm pelleted by centrifugation 20min, centrifugation terminates
Afterwards, careful taking-up supernatant, supernatant are total protein solution, and protein concentration is as shown in table 1 in supernatant, and electrophoresis result is as schemed
Shown in 1.
2.3 common fungus total protein extraction methods three
Quickly Guizhou trichoderma NJAU4742 solid fermentation products are ground into uniformly using mortar under the conditions of liquid nitrogen frozen
Powder, the powder for taking 2g ground is transferred in the centrifuge tube of clean 50mL, adds in the 10%TCA- acetone solns of precooling
(DTT containing 20mM) is stood overnight for -20 DEG C after mixing.In 4 DEG C, 13400r/min centrifugation 25min, supernatant is abandoned, into precipitation again
Acetone soln (DTT containing 20mmol/L) 1mL washings of precooling are added in, 4 DEG C, 12000r/min centrifugation 25min abandon supernatant, repeat
2 times.Mud chamber's warm air is done, precipitation is dried to fine powder.The 400 μ L of dielectrophoresis sample lysate containing urea, thiocarbamide are added in,
Room temperature extracts 2h, and 4 DEG C of sample solution, 13 400r/min centrifugation 20min, supernatant is total protein solution, and albumen is dense in supernatant
Degree is as shown in table 1, and electrophoresis result is as shown in Figure 1.
2.4 common fungus total protein extraction methods four
Quickly Guizhou trichoderma NJAU4742 solid fermentation products are ground into uniformly using mortar under the conditions of liquid nitrogen frozen
Powder, the powder for taking 2g ground is transferred in the centrifuge tube of clean 50mL, and it is fine then to add in 300mg glusulases, 100mg
The DDT100 μ L of the plain enzyme of dimension and 1M, add 25mM phosphate buffers (pH7.9) to total volume 20mL, are sufficiently mixed with vortex instrument
37 DEG C of water-bath 3h afterwards.4 DEG C, 12000rpm abandons supernatant after centrifuging 5min, removes extra enzyme after being repeated 2 times；Add in lysate
(8M urea, 200mM sodium carbonate) 400 μ L, iceberg incubation 20min, 4 DEG C of sample, 12000r/min are centrifuged after mixing
25min, supernatant are total protein solution, and protein concentration is as shown in table 1 in supernatant, and electrophoresis result is as shown in Figure 1.
2.5 fungi total protein extraction methods of the present invention
The cultured Guizhou trichoderma NJAU4742 solid fermentation products of 2g are weighed in the ball milling instrument zirconium oxide cleaned up
Wall jar (50mL), and add in 10mL and shift to an earlier date in the lysate SI of precooling and the Zirconia beads of 2g 0.5mm；Tank body is placed in
In vortex instrument, solid matrix is uniformly mixed with zirconium oxide bead with 2000rpm/min；Enter 10 μ L DDT solution simultaneously after opening tank body
It is vortexed 2 minutes, by tank body ice bath 15min；By tank body mounted in freezing mixing and ball milling instrument after ice bath (German Lay is speeded, MM400)
On be ground.Tank body is placed in 15min on ice after grinding, open tank body and adds in 1mL extracting solution SII, tank body is put
In in vortex instrument, solid matrix is uniformly mixed with zirconium oxide bead with middling speed, by tank body ice bath 30 minutes；By tank after ice bath
Body is transferred on ball milling instrument and is ground, and grinding temperature is arranged to 4 DEG C, and grinding net time is 10min, suspends 1 point within every 2 minutes
Clock.Ground mixture is transferred in the centrifuge tube of the 50mL cleaned up, 10min is centrifuged under the conditions of 4 DEG C of 8000rpm；
After centrifugation, supernatant is transferred in new centrifuge tube, and abandons precipitation；Supernatant is subjected to ice bath after ten minutes, according to
1:The ratio of 4 (v/v) adds in 100%TCA solution (500g TCA are added in 227mL deionized waters, 4 DEG C of preservations) and centrifuge tube
It turns upside down uniformly mixed, it is made to be sufficiently mixed uniformly；Under the conditions of mixed liquor is stored in -20 DEG C 4 it is small when；Cooling time arrives
After, and place it on ice until liquefaction, unsuitable artificial heating hydrotropy；After supernatant completely liquefaction, at 4 DEG C
10min is centrifuged under the conditions of 8000rpm；Supernatant is abandoned after centrifugation, centrifuge tube is upside down on blotting paper, removes extra liquid
Body；Toward precipitation in add in 1mL shift to an earlier date precooling acetone (chromatographically pure, use it is preceding addition 0.01% beta -mercaptoethanol, protect intracellular egg
It is white not oxidized), and beaten with the liquid-transfering gun of 1mL come resorption by pellet resuspended among acetone, it tries not with very big power
Gas so that mixed liquor is as far as possible uniform.After precipitation is blown and beaten mixing, 10min is centrifuged under the conditions of 4 DEG C of 8000rpm, removes upper strata
Liquid (must not remove the precipitation of lower floor)；It repeats the above steps 3 times, after acetone color less depth, in removal
Layer acetone, will be precipitated and dissolved among PBS (pH6.0) solution of 400mL, and supernatant is total protein solution, albumen in supernatant
Concentration is as shown in table 1, and electrophoresis result is as shown in Figure 1.
Total protein result in the different protein extracting method extraction Guizhou trichoderma NJAU4742 solid fermentation products of table 1
* protein content is mgg-1Dw, expression is the total protein concentration extracted in every gram of dry fermentation product.
3 Guizhou trichoderma NJAU4742 of embodiment is in rice straw, wheat stalk and maize straw in solid fermentation product
The comparison of the extraction result of total protein
The solid fermentation of 3.1 Guizhou trichoderma NJAU4742
After different stalks (rice straw, wheat stalk and maize straw) is dried, the small fragment for being cut into 1-2cm is used in combination
Pulverizer crushes, and weighs the different solid fermentation matrix of 10g in the big triangular flask of 1L, then adds in and prepare into triangular flask
Mandels salting liquids (preparation method is shown in embodiment 1).By culture medium at 121 DEG C after grade solid contents and the abundant mixing of salting liquid
Lower high-temperature sterilization 30min is spare.
The Guizhou trichoderma NJAU4742 spore suspensions that will be prepared, according to 107The inoculum concentration of cfu/g dw is inoculated into preparation
In good solid fermentation culture medium.After being inoculated with, triangular flask is placed in standing in 28 DEG C of constant incubator and is protected from light culture, is fermented
Time is 5-7 days, which will be for the extraction of follow-up total protein.
3.2 Guizhou trichoderma NJAU4742 are in the protein extraction of different stalk top fermentation products
The pre-treatment of the solid fermentation product of Guizhou trichoderma NJAU4742 difference stalks according to the method in embodiment 2 into
Row；After culture, Guizhou trichoderma NJAU4742 is extracted in different stalk top fermentation objects according to the method for the present invention in embodiment 2
Total protein, and in common fungus total protein extraction method three as a comparison.
3.3 Guizhou trichoderma NJAU4742 are analyzed in the quantitative and SDS-PAGE of different stalk fermentation product setting egg(s) white matters
Respectively with common fungus total protein extraction method three in embodiment 2 and the method for the present invention extraction Guizhou trichoderma
NJAU4742 fermentate total proteins are respectively compared the extraction efficiency of the two, and the results are shown in Table 2 for extraction.Further use SDS-
The method of PAGE, intuitively to observe the extraction effect of albumen in different disposal, electrophoresis result in figure as shown in Fig. 2, each swim
Road applied sample amount is all 10 μ L (not waiting Tot Prots in equal volume).Binding protein extracts solubility and SDS-PAGE electrophoresis results, from figure
In it is known that the present invention method extraction Guizhou trichoderma NJAU4742 solid fermentation objects total protein effect it is more preferable, wherein
Total protein concentration in wheat stalk fermentate is up to 10.34mgg-1dw；And common fungus total protein extraction method three is being extracted
Maximum protein concentration (4.78mgg is obtained during wheat stalk fermentate-1Dw), the total protein of this method extraction is conventional method
2.16 times of the total protein of three extractions further illustrate that the improved total protein extraction amount of this method significantly rises.
The different protein extracting methods of table 2 extract Guizhou trichoderma NJAU4742 in rice straw, wheat stalk and maize straw
Total protein result in solid fermentation product
* protein content is mg/g dw, and expression is the total protein concentration extracted in every gram of dry fermentation product.
The Guizhou trichoderma NJAU4742 wheat stalk solid fermentation product total proteins of 4 Different Extraction Method of embodiment extraction
Two-dimensional Electrophoresis Analysis
Due to total protein in the Guizhou trichoderma NJAU4742 wheat stalk solid fermentation products using the method for the present invention extraction
Quality highest, therefore the Guizhou trichoderma that the method for the present invention and conventional method three are extracted is compared using the method for dielectrophoresis
Total protein in NJAU4742 wheat stalk tunnings, the detailed process of dielectrophoresis are as follows：
1st, first to isoelectric focusing
(1) the aquation sample-loading buffer (I) (without DTT, without Bio-Lyte) 1 of -20 DEG C of freezen protectives is taken from refrigerator
Tubule (1mL/ pipes), puts room-temperature dissolution；0.01g DTT, Bio-Lyte 4-6, each 2.5 μ L of 5-7 are added in tubule, it is fully mixed
400 μ L aquation sample-loading buffers are taken out after even from tubule, add in 100 μ L samples, abundant mixing.
(2) the prefabricated adhesive tape of the IPG of -20 DEG C of freezen protectives (17cm pH 4-7) is taken from refrigerator, in being placed at room temperature for 10 minutes；
Along edge to the left side for focusing on disk or aquation disk bracket groove, the right side linearly adds in sample；At slot both ends, each 1cm or so be not loaded, in
Between sample liquid have to link up.Pay attention to：Bubble is not generated.Otherwise the distribution of protein in adhesive tape is influenced；When all
After protein example is all had been added in focusing disk or aquation disk, with the protection in the prefabricated IPG adhesive tape of removal of tweezers gently
Layer.
(3) positive and negative anodes of adhesive tape are distinguished, it is molten that IPG adhesive tape glue lightly is placed face down on sample in focusing disk or aquation disk
On liquid so that the anode (indicating+) of adhesive tape corresponds to the anode for focusing on disk.Ensure that adhesive tape is contacted with electrode seal；Sample is not made
Product solution is got in the plastic support film at the adhesive tape back side, because these solution will not be absorbed by adhesive tape；Equally being also noted that does not make
Solution below adhesive tape generates bubble；If having generated bubble, one end of adhesive tape is lightly lifted with tweezers, moves up and down glue
Item, until bubble is rushed to beyond adhesive tape.
(4) 2-3mL mineral oil is covered in every adhesive tape, prevents the evaporation of liquid in adhesive tape hydration process.It needs slow
Mineral oil is added in, along adhesive tape, mineral oil is made drop by drop slowly to be added in plastic support film, to good positive and negative electrode, closed the lid
Son sets isoelectric focusing program as follows：
The placed adhesive tape number of selection；The carrying current (50-70A/ roots) of every adhesive tape is set；During setting isoelectric focusing
Temperature (16 DEG C).Focus on the adhesive tape terminated.Be balanced immediately, second to SDS-PAGE electrophoresis, adhesive tape is otherwise placed in sample
In aquation disk, -20 DEG C of refrigerators preserve.
2nd, second to SDS-PAGE
(1) 10% acrylamide gel is prepared
With 80mL gel solutions, per clotting glue 40mL, solution is injected separately into glass plate interlayer, the sky of 1cm is stayed on top
Between, with MilliQ water, ethyl alcohol or water-saturated n-butanol front cover, keep glue surface smooth.Polyase 13 0 minute.General gel and top liquid
After body layering, show that gel polymerize substantially.After gel sets, MilliQ water, ethyl alcohol or the water on separation gel surface is gone to satisfy
And n-butanol, it is rinsed with MilliQ water.The adhesive tape taken out from -20 DEG C of refrigerators prior to being placed at room temperature for 10 minutes, makes its dissolving.
(2) adhesive tape level pad I is prepared
Dry thick filter paper is first placed on the table, and the adhesive tape glue surface focused on is placed on upward on dry thick filter paper.By another
Thick filter paper MilliQ water-soakeds squeeze and remove excessive moisture, are then placed directly in adhesive tape, gently blot mineral oil in adhesive tape and
Redundant sample.This vertical stripe occurred when can reduce gel-colored.Adhesive tape is transferred in swelling disk, each piece glue of slot
Item adds in 5mL adhesive tape level pads I in the slot for having adhesive tape.Sample hydration disk is placed on horizontal shaker and slowly rocks 15
Minute.
(3) adhesive tape level pad II is prepared
After balancing for the first time, the adhesive tape level pad I in sample hydration disk is thoroughly outwelled or sopped up.And use filter paper
Draw extra equilibrium liquid (adhesive tape is erected on filter paper, so as not to loss albumen or damage gel surface).Add adhesive tape balance
Buffer solution II continues slowly to rock on horizontal shaker 15 minutes.
Liquid extra between glass plate above SDS-PAGE polyacrylamide gels is sucked with filter paper.Second will handled well
Put on the table to gel, long glass plate under, short glass plate upward, the top of gel against oneself.By agarose sealing liquid
It is dissolved by heating.By 10 × electrophoretic buffer, 10 times are diluted with graduated cylinder, into 1 × electrophoretic buffer.It rushes buffer solution surface
Bubble.After second balances, the adhesive tape level pad II in sample hydration disk is thoroughly outwelled or sopped up.And it is inhaled with filter paper
Take extra equilibrium liquid (adhesive tape is erected on filter paper, so as not to loss albumen or damage gel surface).By IPG adhesive tape from sample water
Change and removed in disk, one end of adhesive tape is clamped with tweezers makes glue surface soak end completely in 1 × electrophoretic buffer.Then by adhesive tape glue surface
It is placed on upward on the long glass plate of gel.Remaining adhesive tape equally operates.
(4) PAGE gel for being placed with adhesive tape is transferred on encapsulating frame, short glass plate one is facing to oneself.In gel
Top add in low melting-point agarose sealing liquid.With the syringe needle of tweezers, spatula or tack, lightly adhesive tape is pushed down on,
It is allowed to completely attach to polyacrylamide gel glue surface.It is careful not to generate any bubble below adhesive tape.With tweezers, pressure tongue
When plate or tack syringe needle push away adhesive tape, it should be noted that be the support membrane for promoting the gel back side, not encounter glue surface.It places 5 minutes, makes low
Melt agarose sealing liquid thoroughly solidifies.After low melting-point agarose sealing liquid completely solidification.Gel is transferred in electrophoresis tank.
After electrophoresis tank adds in electrophoretic buffer, power on, low current (5mA/gel/17cm) or low-voltage during starting treat sample
Product are walking out IPG adhesive tape completely, after concentration is into a line, then high current (or voltage) (20-30mA/gel/17cm), treat bromine
Phenol indigo plant indicator can stop electrophoresis when reaching bottom margin.After electrophoresis, layer glass is gently pried open, takes out gel, and
Corner cut is with marking (wearing gloves, prevent pollution glue surface).
3rd, dielectrophoresis results contrast is analyzed
Respectively by the method for the present invention extraction Guizhou trichoderma NJAU4742 wheat stalk fermentate total protein and tradition
The Guizhou trichoderma NJAU4742 that method three is extracted carries out dielectrophoresis detection in the total protein of wheat stalk fermentate, as a result as schemed
Shown in 3 and Fig. 4.The fungi total protein extracted from traditional method three is probably in more than 300 a protein sites, and hair method of the present invention carries
More than the 700 a protein site of fungi taken, and the effect of Protein Separation is more preferable, if the protein science with reference to present high-resolution is ground
Study carefully method (iTRAQ, SWATH etc.), the protein quantity of identification will be more.Total protein is extracted from solid fermentation product, itself
Difficulty is just bigger with the difficulty of pure culture condition extraction filamentous fungi total protein than under liquid fermentation condition, particularly with stalk
Class is solid fermentation matrix, and this kind of fermentation substrate is decomposed by filamentous fungi, released largely containing pigment during the fermentation
Even if polyphenols and humic acid material in the case where there is protease inhibitors protective effect, also hold very much after Release of intracellular protein
It is easily broken off, so can be on the low side compared with the mycelia total protein under the conditions of extracting liq fermentation and pure culture.Since solid is sent out
Ferment closer to filamentous fungi reality metabolic process, in order to study the Key Metabolic mistake in filamentous fungi solid fermentation process
Journey, it is necessary to extract good total protein.Meanwhile the method for present invention extraction filamentous fungi total protein is simple and practicable, need not answer
Miscellaneous instrument and equipment and expensive reagent, and it is also applied for carrying for other trichoderma filamentous fungi solid fermentation total proteins
It takes, a kind of carrying for high-quality and efficient total protein is provided for the protein science research in wooden enzyme category filamentous fungi solid fermentation process
Take method.
Claims (5)
1. a kind of extracting method of filamentous fungi solid fermentation product total protein, it is characterised in that comprise the following steps：
（1）Filamentous fungi solid fermentation product is placed in the ball milling instrument tank body of zirconium oxide inner wall, adds in the cracking for shifting to an earlier date precooling
Liquid SI and zirconia ball are vortexed uniform；It is vortexed after adding in DDT solution, then carries out ice bath, ice bath terminates to mix ball after freezing
It is ground on mill instrument；The preparation method of the SI solution is：8 M urea, 2 M thiocarbamides, 1 mM EDTA, 10 mM PBS,
4 DEG C of refrigerators preserve after 0.1%Triton-X-100, fully dissolving, use preceding addition Roche Protease inhibitor cocktail；On
It is mass volume ratio concentration to state percent concentration（w/v）;The DDT solution preparation methods are：1.55gDTT use deionization
Water is settled to 10 mL, and -20 DEG C save backup；Freezing mixing and ball milling instrument working procedure is arranged to grinding net time as 10min, and every 2
Minute pause 1 minute, grinding temperature is arranged to 4 DEG C;
（2）Extracting solution SII is added in after grinding, by solid fermentation product and zirconium oxide bead ice bath after mixing；Ice bath knot
It is transferred on ball milling instrument and is ground after beam；The SII solution formulas are：3% CHAPS, 1% SDS, 0.1% deoxycholic acid
Sodium, 0.2 mg/mL BSA;
（3）By step（2）In ground mixture be transferred in centrifuge tube, centrifuged at 4 DEG C；After centrifugation, supernatant is turned
It moves on in new centrifuge tube, and abandons precipitation；
（4）After supernatant is carried out ice bath, according to 1:4 volume ratio adds in 100% TCA solution and is uniformly mixed, and mixed liquor is protected
In the presence of under the conditions of -20 DEG C 2 it is small when, then place it on ice until liquefaction；
（5）After supernatant completely liquefaction, in 4 DEG C of centrifugations, supernatant is abandoned, centrifuge tube is upside down on blotting paper, it is extra to remove
Liquid；The acetone for shifting to an earlier date precooling is added in into precipitation, and is beaten with liquid-transfering gun come resorption by pellet resuspended among acetone, it will be heavy
After piping and druming mixing of forming sediment, 4 DEG C centrifuge, and remove the liquid on upper strata；Step 2-5 times that supernatant is abandoned in acetone resuspension-centrifugation-is repeated, until
After acetone color less depth, upper strata acetone is removed, extra acetone is volatilized clean, solid powder is filamentous fungi solid
Solid powder is stored in -20 DEG C by the total protein in tunning.
2. extracting method according to claim 1, it is characterised in that the filamentous fungi is trichoderma.
3. extracting method according to claim 1, it is characterised in that the filamentous fungi is Guizhou trichoderma.
4. extracting method according to any one of claim 1-3, it is characterised in that the filamentous fungi solid fermentation
Product is that filamentous fungi is inoculated in agricultural wastes straw to carry out solid fermentation, after mycelia is covered in solid fermentation matrix
Stop culture, obtain the filamentous fungi solid fermentation product；The agricultural wastes straw is selected from rice straw, Wheat Straw
Any one in stalk or maize straw.
5. extracting method according to claim 4, it is characterised in that include the following steps：
（1）Cultured filamentous fungi solid fermentation product is weighed in the ball milling instrument zirconium oxide inner wall jar cleaned up, and is added
Enter in the lysate SI of precooling in advance and the Zirconia beads of 0.5mm；Tank body is placed in vortex instrument, with 1800-2200 rpm/
Solid fermentation product is uniformly mixed by min with zirconium oxide bead；Enter DDT solution after opening tank body and be vortexed 2 minutes, by tank body ice bath
15min；Tank body is ground on freezing mixing and ball milling instrument after ice bath；Wherein filamentous fungi solid fermentation product with
The mass volume ratio of lysate SI is 1g：3-7mL；The mass ratio of filamentous fungi solid fermentation product and Zirconia beads is 1:
（0.8-1.2）；The mass volume ratio of filamentous fungi solid fermentation product and DDT solution is 1g：3-7μL；
（2）Tank body is placed in 15 min on ice after grinding, open tank body and adds in extracting solution SII, tank body is placed in vortex
On instrument, solid fermentation product is uniformly mixed with zirconium oxide bead with middling speed, by tank body ice bath 30 minutes；By tank body after ice bath
It is transferred on ball milling instrument and is ground, grinding temperature is arranged to 4 DEG C, and grinding net time is 10min, suspends 1 minute within every 2 minutes；
The mass volume ratio of filamentous fungi solid fermentation product and extracting solution SII are 1.8-2.2g：1mL；
（3）Ground mixture is transferred in the centrifuge tube cleaned up, 10 are centrifuged under the conditions of 4 DEG C of 8000 rpm
min；After centrifugation, supernatant is transferred in new centrifuge tube, and abandons precipitation, total protein is present in supernatant；
（4）Supernatant is subjected to ice bath after ten minutes, according to 1:4 volume ratio adds in 100% TCA solution and centrifuge tube is run up and down
It is made to be sufficiently mixed uniformly；When 2-4 is small under the conditions of mixed liquor is stored in -20 DEG C；After cooling time has arrived, and put
In on ice until liquefaction；
（5）After supernatant completely liquefaction, 10 min are centrifuged under the conditions of 4 DEG C of 8000 rpm；Supernatant is abandoned after centrifugation, it will
Centrifuge tube is upside down on blotting paper, removes extra liquid；The acetone for shifting to an earlier date precooling is added in into precipitation, and with liquid-transfering gun back and forth
Suction is beaten by pellet resuspended among acetone, and after precipitation is blown and beaten mixing, 10 min are centrifuged under the conditions of 4 DEG C of 8000 rpm,
Remove the liquid on upper strata；It repeats the above steps 3 times, after acetone color less depth, removes upper strata acetone, utilize nitrogen evaporator
Extra acetone volatilization is clean, and solid powder is the total protein in solid fermentation product, and solid powder is stored in -20
℃。
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| CN103436581A (en) * | 2013-09-06 | 2013-12-11 | 江苏丘陵地区镇江农业科学研究所 | Method for extracting rapeseed peptides through wet milling combined with enzymic method |
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| CN103436581A (en) * | 2013-09-06 | 2013-12-11 | 江苏丘陵地区镇江农业科学研究所 | Method for extracting rapeseed peptides through wet milling combined with enzymic method |
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