CN107084867A - 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
- Publication number
- CN107084867A CN107084867A CN201710261618.7A CN201710261618A CN107084867A CN 107084867 A CN107084867 A CN 107084867A CN 201710261618 A CN201710261618 A CN 201710261618A CN 107084867 A CN107084867 A CN 107084867A
- Authority
- CN
- China
- Prior art keywords
- solid fermentation
- filamentous fungi
- fermentation product
- total protein
- supernatant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of extracting method 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); to destroy the abundant tough and tensile cell membrane of filamentous fungi and discharge intracellular total protein, and extract under the protection of Extraction buffer (SII) and protease inhibitors high-quality trichoderma solid fermentation product total protein.Present invention employs a kind of method simple and easy to apply, the abundant 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 thing, high-quality total protein can be directly extracted from trichoderma solid fermentation product with reference to pellicle dialysis, the research for filamentous fungi proteomics in solid fermentation process provides technical guarantee.
Description
Technical field
The invention belongs to bio-science field, it is 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 are provided and ensured for intensive agriculture being changed into, but agricultural chemicals (agriculture
Medicine, chemical fertilizer etc.) a large amount of apply the problem of also bringing 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 perches substantial amounts of beneficial microbe, and they pass through not
Same mode promotes plant root growth and induces plant to produce defense reaction, strengthens the resistivity of environment to external world.Research
It was found that trichoderma is widely present in soil as a kind of beneficial microbe, colonize in after plant root symbiosis can be formed with plant
Body, the growth of plant is promoted by secreting secondary metabolite and plant growth regulator.Research finds that Trichoderma harzianum is biological
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 conventional biocontrol agent, while can promote the growth of plant, especially the growth-promoting to root system of plant has significantly
Effect.In recent years, prebiotic trichoderma because its is environment-friendly, safety non-toxic, parasitic plant pathogen the advantages of have as microorganism
Gradually it is widely used, is taken in terms of plant growth and prevention and control soil-borne disease is promoted in the agricultural production at home and abroad of machine fertilizer
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
Close, fermentation can be largely classified 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 that under the conditions of certain moisture content, microorganism is being adapted to support
The process of metabolite is grown and produced on the solid state substrate divided.Numerous studies show that solid-fermented technique is than liquid fermentation skill
Art has more advantages.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 the agricultural or industrial or agricultural accessory substance using low cost
As culture matrix, fund and running cost are greatly reduced；Finally, in solid fermentation process, mobility is relatively low, required equipment
Simply, meanwhile, reduce the cost that the downstream engineering of liquid fermentation is brought.
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 focus of world research, it is not only the expansion of genome times afterwards comprehensively research, the core of even more current life science research
It is intracardiac to hold.The technology that proteomics research is related to mainly includes electrophoretic techniques, tree species for bio-energy source, and both are 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 are sufficiently separated, this technology
Advantage be that it can study the posttranslational modification of albumen, but the reappearance of this technology is not high and be easily caused albumen and lose
Lose, have 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.It is biological
Mass-spectrometric technique is the mass-to-charge ratio (m/z) by determination sample to carry out composition and structural analysis, is the main skill of identification of proteins
Art, it has the advantages that sensitivity height, accuracy are good and easy to automate.
With scientific and technological progress and the development of society, research of the mankind to fungi is increasing, particularly in agricultural research
It is used for the trichoderma of plants probiotics in journey, there is important effect in terms of plant growth-promoting and biological and ecological methods to prevent plant disease, pests, and erosion.But, early stage it is a large amount of
Research is tight to rest on genome and transcript profile level, and the horizontal progress of protein groups is slow, and most important of which restriction factor is
It is difficult to obtain high-quality total protein.Compared with other species, the proteomics research of filamentous fungi is also in a starting
In the stage, it 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 invention aims to solve in protein science research process, the technical bottleneck of filamentous fungi total protein extraction
There is provided a kind of extracting method of filamentous fungi tunning total protein rapidly and efficiently for problem.
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 inwall, adds and shift to an earlier date precooling
Lysate SI and zirconia ball, are vortexed uniform；Add after DDT solution and be vortexed, then carry out ice bath, ice bath terminates mixed after freezing
Close and be ground on ball milling instrument；
(2) extract solution SII is added after grinding terminates, ice bath after solid fermentation product is well mixed with zirconium oxide bead；Ice
Bath is transferred on ball milling instrument after terminating and is ground；
(3) mixture ground in step (2) is transferred in centrifuge tube, centrifuged at 4 DEG C；, will be upper after centrifugation terminates
It is transferred to clearly in new centrifuge tube, and abandons precipitation；
(4) supernatant is carried out after ice bath, according to 1:4 (v/v) ratio adds 100%TCA solution and is well mixed, will be mixed
Close under the conditions of liquid is stored in -20 DEG C 2 hours, be then placed on ice until liquefaction；
(5) after after supernatant completely liquefaction, in 4 DEG C of centrifugations, supernatant is abandoned, centrifuge tube is upside down on blotting paper, removed many
Remaining liquid；The acetone for shifting to an earlier date precooling is added into precipitation, and is beaten with liquid-transfering gun come resorption by pellet resuspended among acetone,
After precipitation piping and druming is mixed, 4 DEG C of centrifugations 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, removing upper strata acetone, unnecessary acetone is volatilized clean, solid powder is filamentous fungi
Total protein in solid fermentation product, -20 DEG C are stored in by solid powder.
The preferred trichoderma of described filamentous fungi；Further preferred filamentous fungi is Guizhou trichoderma.Guizhou of the present invention
Trichoderma is applied to all Guizhou trichodermas, is particularly suitable for use in growth-promoting functions, the Guizhou trichoderma for producing bio-feritlizer, such as
Voluntarily separation and commercially available Trichoderma harziaum are applied to the inventive method；It is most preferably suited to Guizhou trichoderma NJAU4742
(Trichoderma guizhouense), bacterial strain preserving number is CGMCC No.12166.
Described 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 with solid fermentation matrix, obtains described filamentous fungi solid fermentation product；Described 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 1-2cm small fragment and 40 purposes is ground into pulverizer
Powder.The different stalk powders of 10g are weighed in 1L big triangular flask, are then prepared toward addition 23mL in triangular flask
Mandels salting liquids (initial aqueous rate about 70%), wait solid content and salting liquid fully mix after by culture medium at 121 DEG C it is high
Temperature sterilizing 30min is standby.
The compound method of SI solution described in step (1) is preferred：8M urea, 2M thiocarbamides, 1mM EDTA, 10mM PBS,
4 DEG C of refrigerators are preserved 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 compound methods described in step (1) are preferred：1.55gDTT is settled to 10mL, -20 DEG C with deionized water
Save backup；It is 10min that freezing mixing and ball milling instrument working procedure, which is set 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 comprises the following steps：
(1) cultured filamentous fungi solid fermentation product is weighed in the ball milling instrument zirconium oxide inwall jar cleaned up,
And add the Zirconia beads in the lysate SI for shifting to an earlier date precooling with 0.5mm；Tank body is placed on vortex instrument, with 1800-
Solid fermentation product is well mixed by 2200rpm/min with zirconium oxide bead；Open and enter DDT solution after tank body and be vortexed 2 minutes, will
Tank body ice bath 15min；Tank body is ground by ice bath after terminating on freezing mixing and ball milling instrument；Wherein filamentous fungi solid is sent out
Ferment product and lysate SI mass volume ratio 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 terminates, tank body is opened and adds extract solution SII, tank body is placed in whirlpool
Revolve on instrument, be well mixed solid fermentation product with zirconium oxide bead with middling speed, by tank body ice bath 30 minutes；Ice bath terminate after by tank
Body is transferred on ball milling instrument and is ground, and grinding temperature is set to 4 DEG C, and grinding net time is 10min, suspends 1 point within every 2 minutes
Clock；Filamentous fungi solid fermentation product and extract solution SII mass volume ratio are 1.8-2.2g：1mL；
(3) ground mixture is transferred in the centrifuge tube cleaned up, centrifuged under the conditions of 4 DEG C of 8000rpm
10min；After centrifugation terminates, supernatant is transferred in new centrifuge tube, and abandons precipitation, total protein is present in supernatant；
(4) after supernatant being carried out into ice bath 10 minutes, according to 1:4 (v/v) ratio adds 100%TCA solution and centrifuged
Pipe, which turns upside down, makes it be sufficiently mixed uniformly；2-4 hours under the conditions of mixed liquor is stored in into -20 DEG C；After cooling time has been arrived,
And be placed on ice until liquefaction；
(5) after after supernatant completely liquefaction, 10min is centrifuged under the conditions of 4 DEG C of 8000rpm；Centrifugation abandons supernatant after terminating, will
Centrifuge tube is upside down on blotting paper, removes unnecessary liquid；The acetone for shifting to an earlier date precooling is added into precipitation, and with liquid-transfering gun back and forth
Suction is beaten by pellet resuspended among acetone, and after precipitation piping and druming is mixed, 10min is centrifuged under the conditions of 4 DEG C of 8000rpm, is removed
The liquid on upper strata；Repeat the above steps 3 times, until after acetone color less depth, removing upper strata acetone, will be many 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 into -20 DEG C.
In the method for the invention, when removing supernatant after centrifugation, must trying one's best, it is clean to remove supernatant, otherwise holds
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；Added in ice-cold acetone
0.01% beta -mercaptoethanol, can protect thread true total protein not oxidized, but definitely can not excessively add, and can excessively make egg
White matter is denatured and is in unstable transition, accelerates the decomposition of protein.
Beneficial effect：
The present invention is matrix using agricultural wastes straw, carries out solid fermentation after being inoculated with trichoderma, and utilize mechanical breaking-wall method
And the method that suitable extractant phase is combined, high-quality trichoderma total protein, 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 be destroyed
The abundant tough and tensile cell membrane of filamentous fungi simultaneously discharges intracellular total protein, can greatly improve the extracted amount of albumen.
(2) agricultural crop straw inoculation trichoderma is carried out after solid fermentation, and substantial amounts of organic matter and humic acids are contained in matrix
Material, 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.
Brief description of the drawings
The total protein electrophoretogram for the Guizhou trichoderma NJAU4742 that Fig. 1 Different Extraction Methods are extracted.M is standard protein
marker；1 swimming lane is the protein electrophoresis result that conventional method one is extracted, and 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 that method used in the present invention is extracted.
The total protein electrophoretogram of Guizhou trichoderma NJAU4742 tunnings in Fig. 2 different substrates.M is standard protein
marker；1 swimming lane is that optimization method of the present invention extracts Guizhou trichoderma NJAU4742 rice straw tunning total proteins；2 be normal
Rule method three extracts Guizhou trichoderma NJAU4742 rice straw tunning total proteins；3 be that optimization method of the present invention extracts Guizhou
Trichoderma NJAU4742 wheat stalk tunning total proteins；4 be that conventional method three extracts Guizhou trichoderma NJAU4742 wheat stalks
Tunning total protein；5 be that optimization method of the present invention extracts Guizhou trichoderma NJAU4742 maize straw tunning total proteins；6
Guizhou trichoderma NJAU4742 maize straw tunning total proteins are extracted for conventional method three.
Fig. 3 Guizhou trichoderma NJAU4742 total protein two dimensional electrophoresis figure.(rice straw is fermentation substrate, present invention optimization
Method) Fig. 4 Guizhou trichoderma NJAU4742 total protein two dimensional electrophoresis figure.(rice straw is fermentation substrate, conventional method three)
Biomaterial preservation information
NJAU4742, Classification And Nomenclature Guizhou trichoderma (Trichoderma guizhouense), is preserved in China Microbiological bacterium
Preservation administration committee common micro-organisms center is planted, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese section
Institute of microbiology of institute, preservation date is on April 11st, 2016, bacterial strain preserving number CGMCC No.12166.
Embodiment：
Following examples illustrate this hair by taking Guizhou trichoderma NJAU4742 (Trichoderma guizhouense) as an example
Bright technical scheme, but therefore do not limit protection scope of the present invention.In fact, inventor is according to the inventive method, with many
Plant voluntarily separation and commercially available filamentous fungi carries out the extraction of solid fermentation product total protein, obtain 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 is preserved；Carrier ampholyte (Bio-Rad companies of the U.S.), Bio-Lyte 3/10Ampholyte, 40%, 10ml, 163-
1112,4 DEG C of refrigerators are preserved；Protein quantification kit Protein Assay Kit II.
The conidial preparations of Guizhou trichoderma NJAU4742 of embodiment 1 and solid fermentation
1. Guizhou trichoderma NJAU4742 brief introductions
Bacterial strain used in the present invention is 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, it 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 prepare
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. Guizhou trichoderma NJAU4742 solid fermentation
After rice straw is dried, it is cut into 1-2cm small fragment and the size of 40 mesh is ground into pulverizer, weigh 10g
Rice straw powder is in 1L big triangular flask, then toward the Mandels salting liquids [0.3g that addition 23mL is prepared in 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 standby at 121 DEG C after fully being mixed Deng solid content and salting liquid.
By the Guizhou trichoderma NJAU4742 spore suspensions prepared, according to 107Cfu/g dw inoculum concentration is inoculated into preparation
In good solid fermentation culture medium.After being inoculated with, triangular flask is placed in standing lucifuge culture in 28 DEG C of constant incubator, culture
Time is 5-7 days, and the solid fermentation product is by the extraction for follow-up total protein.
The distinct methods of embodiment 2 extract the total protein in the trichoderma NJAU4742 solid fermentation products of Guizhou
2.1 common fungus total protein extraction methods one
Quickly Guizhou trichoderma NJAU4742 solid fermentation products are ground to form uniformly using mortar under the conditions of liquid nitrogen frozen
Powder, the powder for taking 2g ground is transferred in clean 50mL centrifuge tube, adds (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, is during which mixed every 5min is reverse, 4 DEG C, take supernatant, supernatant after 24 000r/min centrifugations 30min
As shown in table 1, electrophoresis result is as shown in Figure 1 for protein concentration in the total protein as extracted, supernatant.
2.2 common fungus total protein extraction methods two
Quickly Guizhou trichoderma NJAU4742 solid fermentation products are ground to form uniformly using mortar under the conditions of liquid nitrogen frozen
Powder, the powder for taking 2g ground is transferred in clean 50mL centrifuge tube, adds (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；Ice bath 30min, is during which mixed once every 5min, and thalline is crushed in ice bath with ultrasonic cell disruption instrument
(750W, ultrasonic 6s are spaced 1s, ultrasound 20 times)；After ultrasound terminates, at 4 DEG C, 12000rpm pelleted by centrifugation 20min, centrifugation terminates
Afterwards, careful taking-up supernatant, supernatant be in total protein solution, supernatant protein concentration as shown in table 1, 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 to form uniformly using mortar under the conditions of liquid nitrogen frozen
Powder, the powder for taking 2g ground is transferred in clean 50mL centrifuge tube, adds the 10%TCA- acetone solns of precooling
(DTT containing 20mM), -20 DEG C stand overnight 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, 4 DEG C, 12000r/min centrifugation 25min abandon supernatant, repeated
2 times.Mud chamber's warm air is done, precipitation is dried to fine powder.The μ L of dielectrophoresis sample lysate 400 containing urea, thiocarbamide are added,
Room temperature extracts 2h, 4 DEG C of sample solution, 13 400r/min centrifugation 20min, supernatant be in total protein solution, supernatant albumen it is dense
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 to form uniformly using mortar under the conditions of liquid nitrogen frozen
Powder, the powder for taking 2g ground is transferred in clean 50mL centrifuge tube, is subsequently added 300mg glusulases, 100mg fine
The DDT100 μ L of the plain enzyme of dimension and 1M, add 25mM phosphate buffers (pH7.9) to cumulative 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 unnecessary enzyme after being repeated 2 times；Add lysate
(8M urea, 200mM sodium carbonate) 400 μ L, iceberg is incubated 20min after being well mixed, and 4 DEG C of sample, 12000r/min are centrifuged
25min, supernatant be in total protein solution, supernatant protein concentration as shown in table 1, 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 10mL shift to an earlier date in the lysate SI of precooling and 2g 0.5mm Zirconia beads；Tank body is placed in
On vortex instrument, solid matrix is well 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；Tank body is mounted in freezing mixing and ball milling instrument (German Lay is speeded, MM400) by ice bath after terminating
On be ground.Tank body is placed in 15min on ice by grinding after terminating, and is opened tank body and is added 1mL extract solution SII, tank body is put
In on vortex instrument, solid matrix is well mixed with zirconium oxide bead with middling speed, by tank body ice bath 30 minutes；Ice bath terminate after by tank
Body is transferred on ball milling instrument and is ground, and grinding temperature is set to 4 DEG C, and grinding net time is 10min, suspends 1 point within every 2 minutes
Clock.In the centrifuge tube that ground mixture is transferred to the 50mL cleaned up, 10min is centrifuged under the conditions of 4 DEG C of 8000rpm；
After centrifugation terminates, supernatant is transferred in new centrifuge tube, and abandon precipitation；After supernatant is carried out into ice bath 10 minutes, according to
1:4 (v/v) ratio adds 100%TCA solution (500g TCA are added in 227mL deionized waters, 4 DEG C of preservations) and centrifuge tube
Turn upside down well mixed, it is sufficiently mixed uniformly；4 hours under the conditions of mixed liquor is stored in into -20 DEG C；Cooling time is arrived
After, and be placed on ice until liquefaction, unsuitable artificial heating hydrotropy；After after supernatant completely liquefaction, at 4 DEG C
10min is centrifuged under the conditions of 8000rpm；Centrifugation abandons supernatant after terminating, and centrifuge tube is upside down on blotting paper, removes unnecessary liquid
Body；Toward precipitation in add 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 1mL liquid-transfering gun come resorption by pellet resuspended among acetone, try not with very big power
Gas so that mixed liquor is tried one's best uniformly.After precipitation piping and druming is mixed, 10min is centrifuged under the conditions of 4 DEG C of 8000rpm, upper strata is removed
Liquid (must not remove the precipitation of lower floor)；Repeat the above steps 3 times, until after acetone color less depth, in removal
Layer acetone, will be precipitated and dissolved among 400mL PBS (pH6.0) solution, supernatant is albumen in total protein solution, supernatant
As shown in table 1, electrophoresis result is as shown in Figure 1 for concentration.
The different protein extracting methods of table 1 extract total protein result in the trichoderma NJAU4742 solid fermentation products of Guizhou
* protein content is mgg-1Dw, expression is the total protein concentration extracted in every gram of dry fermentation product.
The Guizhou trichoderma NJAU4742 of embodiment 3 is in rice straw, wheat stalk and maize straw in solid fermentation product
The comparison of the extraction result of total protein
3.1 Guizhou trichoderma NJAU4742 solid fermentation
After different stalk (rice straw, wheat stalk and maize straw) drying, the small fragment for being cut into 1-2cm is used in combination
Pulverizer is crushed, and weighs the different solid fermentation matrix of 10g in 1L big triangular flask, is then prepared toward adding in triangular flask
Mandels salting liquids (compound method is shown in embodiment 1).By culture medium at 121 DEG C after fully being mixed Deng solid content and salting liquid
Lower high-temperature sterilization 30min is standby.
By the Guizhou trichoderma NJAU4742 spore suspensions prepared, according to 107Cfu/g dw inoculum concentration is inoculated into preparation
In good solid fermentation culture medium.After being inoculated with, triangular flask is placed in standing lucifuge culture in 28 DEG C of constant incubator, fermentation
Time is 5-7 days, and the solid fermentation product is by the extraction for follow-up total protein.
Protein extractions of the 3.2 Guizhou trichoderma NJAU4742 in different stalk top fermentation products
The pre-treatment of the solid fermentation product of Guizhou trichoderma NJAU4742 difference stalks is entered according to the method in embodiment 2
OK；After culture terminates, Guizhou trichoderma NJAU4742 is extracted in different stalk top fermentation things according to the inventive method 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 Guizhou trichoderma is extracted with common fungus total protein extraction method three in embodiment 2 and the inventive method
NJAU4742 fermentate total proteins, are respectively compared both extraction efficiencies, extract result as shown in table 2.Further use SDS-
PAGE method, intuitively to observe the extraction effect of albumen in different disposal, electrophoresis result as shown in Fig. 2 each being swum in figure
Road applied sample amount is all 10 μ L (not waiting Tot Prot in equal volume).Associated proteins extract solubility and SDS-PAGE electrophoresis results, from figure
In it is known that the present invention method extract Guizhou trichoderma NJAU4742 solid fermentation things total protein effect more preferably, 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 that this method is extracted is conventional method
2.16 times of three total proteins extracted, further illustrate that the total protein extraction amount after this method improvement 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 that the Different Extraction Method of embodiment 4 is extracted
Two-dimensional Electrophoresis Analysis
Due to total protein in the Guizhou trichoderma NJAU4742 wheat stalk solid fermentation products using the inventive method extraction
Quality highest, therefore compare the Guizhou trichoderma that the inventive method and conventional method three are extracted using the method for dielectrophoresis
Total protein in NJAU4742 wheat stalk tunnings, the detailed process of dielectrophoresis is as follows：
1st, first to isoelectric focusing
(1) the aquation sample-loading buffer (I) (DTT being free of, 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 each 2.5 μ L of 4-6,5-7 are added in tubule, it is fully mixed
400 μ L aquation sample-loading buffers are taken out after even from tubule, 100 μ L samples is added, fully mixes.
(2) the prefabricated adhesive tape of the IPG of -20 DEG C of freezen protectives (17cm pH 4-7) is taken from refrigerator, is placed 10 minutes in room temperature；
Along edge to the left side for focusing on disk or aquation disk bracket groove, the right side linearly adds sample；At groove two ends, each 1cm or so should not be loaded, in
Between sample liquid must link up.Note：Bubble should not be produced.Otherwise the distribution of protein in adhesive tape is had influence on；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) both positive and negative polarity of adhesive tape is distinguished, it is molten that IPG adhesive tape glue lightly is placed face down on into sample in focusing disk or aquation disk
On liquid so that the positive pole (indicating+) of adhesive tape corresponds to the positive pole for focusing on disk.Ensure that adhesive tape is contacted with electrode seal；Sample should not be 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 produces bubble；If having produced bubble, one end of adhesive tape is lightly lifted with tweezers, glue is moved up and down
Bar, until bubble is rushed to beyond adhesive tape.
(4) 2-3mL mineral oil is covered in every adhesive tape, the evaporation of liquid in adhesive tape hydration process is prevented.Need slow
Mineral oil is added, along adhesive tape, mineral oil is drop by drop slowly added in plastic support film, to good positive and negative electrode, closes 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.It is balanced immediately, second to SDS-PAGE electrophoresis, adhesive tape is otherwise placed in sample
In aquation disk, -20 DEG C of refrigerators are preserved.
2nd, second to SDS-PAGE
(1) acrylamide gel of preparation 10%
With 80mL gel solutions, per clotting glue 40mL, solution is injected separately into glass plate interlayer, 1cm sky is stayed on top
Between, with MilliQ water, ethanol 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.Satisfy after after gel sets, removing MilliQ water, ethanol or the water on separation gel surface
And n-butanol, rinsed with MilliQ water.The adhesive tape taken out from -20 DEG C of refrigerators, places 10 minutes prior to room temperature, dissolves it.
(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 dry thick filter paper upward.By another
Thick filter paper MilliQ water-soakeds, squeeze and remove excessive moisture, be then placed directly within 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 groove
Bar, adds 5mL adhesive tape level pads I in the groove for have adhesive tape.Sample hydration disk is placed on horizontal shaker and slowly rocks 15
Minute.
(3) adhesive tape level pad II is prepared
After balance terminates for the first time, the adhesive tape level pad I in sample hydration disk is thoroughly outwelled or sopped up.And use filter paper
The unnecessary equilibrium liquid of absorption (adhesive tape is erected on filter paper, in order to avoid loss albumen or damage gel surface).Add adhesive tape balance
Buffer solution II, continuation is slowly rocked 15 minutes on horizontal shaker.
Liquid unnecessary between glass plate above SDS-PAGE polyacrylamide gels is sucked with filter paper.By handle well second
Put on the table to gel, long glass plate under, short glass plate upward, the top of gel against oneself.By agarose sealing liquid
Dissolved by heating.By 10 × electrophoretic buffer, 10 times are diluted with graduated cylinder, into 1 × electrophoretic buffer.Rush buffer solution surface
Bubble.After second of balance terminates, the adhesive tape level pad II in sample hydration disk is thoroughly outwelled or sopped up.And inhaled with filter paper
Take unnecessary 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 in disk and remove, 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 is equally operated.
(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 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 produce 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, glue surface should not be encountered.Place 5 minutes, make 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 electrophoretic buffer, switch on power, 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, which reaches, can stop electrophoresis during bottom margin.After electrophoresis terminates, layer glass is gently pried open, gel is taken out, and
Corner cut is with marking (wearing gloves, prevent from polluting glue surface).
3rd, dielectrophoresis results contrast is analyzed
Total proteins and tradition of the Guizhou trichoderma NJAU4742 that the inventive method is extracted respectively in wheat stalk fermentate
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 protein sites, and hair method of the present invention is carried
More than 700 protein site of the 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 than extracting the difficulty of filamentous fungi total protein with pure culture condition under liquid fermentation condition, particularly with stalk
Class is solid fermentation matrix, and this kind of fermentation substrate is decomposed by filamentous fungi during the fermentation, is discharged a large amount of containing pigment
Polyphenols and humic acid material, even in having under protease inhibitors protective effect, also hold very much after Release of intracellular protein
Easily it is broken off, so can be on the low side compared with extracting liq ferments with the mycelia total protein under the conditions of pure culture.Due to solid hair
Ferment is closer to the actual metabolic process of filamentous fungi, in order to study the Key Metabolic mistake in filamentous fungi solid fermentation process
Journey, it is necessary to extract the total protein of high-quality.Meanwhile, the method that the present invention extracts filamentous fungi total protein is simple and easy to apply, it is not necessary to multiple
Miscellaneous instrument and equipment and expensive reagent, and it is also applied for carrying for other trichoderma filamentous fungi solid fermentation total proteins
Take, the protein science research belonged to for wooden enzyme in filamentous fungi solid fermentation process provides a kind of carrying for high-quality and efficient total protein
Take method.
Claims (8)
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 inwall, adds the cracking for shifting to an earlier date precooling
Liquid SI and zirconia ball, are vortexed uniform；Add after DDT solution and be vortexed, then carry out ice bath, ice bath terminates to mix ball after freezing
It is ground on mill instrument；
(2) extract solution SII is added after grinding terminates, ice bath after solid fermentation product is well mixed with zirconium oxide bead；Ice bath knot
It is transferred on ball milling instrument and is ground after beam；
(3) mixture ground in step (2) is transferred in centrifuge tube, centrifuged at 4 DEG C；After centrifugation terminates, supernatant is turned
Move on in new centrifuge tube, and abandon precipitation；
(4) supernatant is carried out after ice bath, according to 1:4 volume ratio adds 100%TCA solution and is well mixed, and mixed liquor is protected
In the presence of 2 hours under the conditions of -20 DEG C, then it is placed on ice until liquefaction；
(5) after after supernatant completely liquefaction, in 4 DEG C of centrifugations, supernatant is abandoned, centrifuge tube is upside down on blotting paper, it is unnecessary to remove
Liquid；The acetone for shifting to an earlier date precooling is added into precipitation, and is beaten with liquid-transfering gun come resorption by pellet resuspended among acetone, will be heavy
After piping and druming of forming sediment is mixed, 4 DEG C of centrifugations 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, unnecessary acetone is volatilized clean, solid powder is filamentous fungi solid
Total protein in tunning, -20 DEG C are stored in by solid powder.
2. extracting method according to claim 1, it is characterised in that described filamentous fungi is trichoderma.
3. extracting method according to claim 1, it is characterised in that described filamentous fungi is Guizhou trichoderma.
4. the extracting method according to any one of claim 1-3, it is characterised in that described filamentous fungi solid fermentation
Product is that filamentous fungi is inoculated in agricultural wastes straw to carry out solid fermentation, after mycelia is covered with solid fermentation matrix
Stop culture, obtain described filamentous fungi solid fermentation product；Described agricultural wastes straw is selected from rice straw, Wheat Straw
Any one in stalk or maize straw.
5. extracting method according to claim 1, it is characterised in that the compound method of the SI solution described in step (1)
For：4 DEG C of refrigerators are preserved after 8M urea, 2M thiocarbamides, 1mM EDTA, 10mM PBS, 0.1%Triton-X-100, fully dissolving, are made
With preceding addition Roche Protease inhibitor cocktail；Above-mentioned percent concentration is mass volume ratio concentration (w/v).
6. extracting method according to claim 1, it is characterised in that the DDT solution compound methods described in step (1) are：
1.55gDTT is settled to 10mL with deionized water, and -20 DEG C save backup；It is net that freezing mixing and ball milling instrument working procedure is set to grinding
Time is 10min, suspends 1 minute within every 2 minutes, grinding temperature is set to 4 DEG C.
7. extracting method according to claim 1, it is characterised in that the SII solution formulas described in step (2) are：3%
CHAPS, 1%SDS, 0.1% NaTDC, 0.2mg/mL BSA.
8. the extracting method according to any one of claim 1-7, it is characterised in that comprise the following steps：
(1) cultured filamentous fungi solid fermentation product is weighed in the ball milling instrument zirconium oxide inwall jar cleaned up, and is added
Enter the Zirconia beads with 0.5mm in the lysate SI of precooling in advance；Tank body is placed on vortex instrument, with 1800-2200rpm/
Solid fermentation product is well mixed by min with zirconium oxide bead；Open and enter DDT solution after tank body and be vortexed 2 minutes, by tank body ice bath
15min；Tank body is ground by ice bath after terminating on freezing mixing and ball milling instrument；Wherein filamentous fungi solid fermentation product with
Lysate SI mass volume ratio 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 15min on ice after grinding terminates, tank body is opened and adds extract solution SII, tank body is placed in vortex instrument
On, solid fermentation product is well mixed with zirconium oxide bead with middling speed, by tank body ice bath 30 minutes；Ice bath turns tank body after terminating
Move on to and be ground on ball milling instrument, grinding temperature is set to 4 DEG C, and grinding net time is 10min, suspends 1 minute within every 2 minutes；Silk
Shape fungus solids tunning and extract solution SII mass volume ratio are 1.8-2.2g：1mL；
(3) ground mixture is transferred in the centrifuge tube cleaned up, 10min is centrifuged under the conditions of 4 DEG C of 8000rpm；From
After hearty cord beam, supernatant is transferred in new centrifuge tube, and abandons precipitation, total protein is present in supernatant；
(4) after supernatant being carried out into ice bath 10 minutes, according to 1:4 (v/v) ratio is added on 100%TCA solution and centrifuge tube
Lower overturn makes it be sufficiently mixed uniformly；2-4 hours under the conditions of mixed liquor is stored in into -20 DEG C；After cooling time has been arrived, and will
It is placed on ice until liquefaction；
(5) after after supernatant completely liquefaction, 10min is centrifuged under the conditions of 4 DEG C of 8000rpm；Centrifugation abandons supernatant after terminating, and will centrifuge
Pipe is upside down on blotting paper, removes unnecessary liquid；The acetone for shifting to an earlier date precooling is added into precipitation, and is beaten with liquid-transfering gun come resorption
By pellet resuspended among acetone, after precipitation piping and druming is mixed, 10min is centrifuged under the conditions of 4 DEG C of 8000rpm, upper strata is removed
Liquid；Repeat the above steps 3 times, will be unnecessary using nitrogen evaporator until after acetone color less depth, removing upper strata acetone
Acetone volatilization is clean, and solid powder is the total protein in solid fermentation product, and solid powder is stored in into -20 DEG C.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710261618.7A CN107084867B (en) | 2017-04-20 | 2017-04-20 | A kind of extracting method of filamentous fungi solid fermentation product total protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710261618.7A CN107084867B (en) | 2017-04-20 | 2017-04-20 | A kind of extracting method of filamentous fungi solid fermentation product total protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107084867A true CN107084867A (en) | 2017-08-22 |
| CN107084867B CN107084867B (en) | 2018-05-25 |
Family
ID=59612951
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710261618.7A Active CN107084867B (en) | 2017-04-20 | 2017-04-20 | A kind of extracting method of filamentous fungi solid fermentation product total protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107084867B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108586589A (en) * | 2018-01-16 | 2018-09-28 | 南京农业大学 | A kind of preparation method and applications of the Swollenin albumen from Guizhou trichoderma |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433215A (en) * | 2011-09-22 | 2012-05-02 | 厦门汇盛生物有限公司 | Method of extracting grease from fungi or algae through physical wall-breaking |
| 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 |
-
2017
- 2017-04-20 CN CN201710261618.7A patent/CN107084867B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102433215A (en) * | 2011-09-22 | 2012-05-02 | 厦门汇盛生物有限公司 | Method of extracting grease from fungi or algae through physical wall-breaking |
| 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 |
Non-Patent Citations (4)
| Title |
|---|
| OLAF KNIEMEYER ET AL: "Optimisation of a 2-D gel electrophoresis protocol for the human-pathogenic fungus Aspergillus fumigatus", 《CURR GENET》 * |
| 孙宁宁等: "基于不同裂解液的蛋白质组样品制备方法的比较研究", 《分析化学》 * |
| 焦立新等: "一种念珠菌蛋白质的提取方法", 《中国组织工程研究与临床康复》 * |
| 胡彬彬等: "一种高效的真菌总蛋白质提取方法", 《中国生物工程杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108586589A (en) * | 2018-01-16 | 2018-09-28 | 南京农业大学 | A kind of preparation method and applications of the Swollenin albumen from Guizhou trichoderma |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107084867B (en) | 2018-05-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103952362B (en) | One strain is to the oranges and tangerines endogeny rayungus of various plants pathogenic bacteria tool bacteriostatic activity | |
| WO2016155567A1 (en) | Streptomyces and method for producing milbemycin a4 using same | |
| CN103614302A (en) | High-efficiency phosphate-solubilizing penicillium oxalicum with heavy metal tolerance characteristic | |
| CN103614304A (en) | High-efficiency phosphate-solubilizing aspergillus japonicus with heavy metal tolerance | |
| CN102286467A (en) | Method for extracting microbial total RNA in forest soil and litter | |
| CN108570433A (en) | One plant of acid proof defence pseudomonad CLP-6 and its application | |
| CN108220169A (en) | A kind of separation screening and its identification method of degraded polystyrene strain | |
| CN102212134A (en) | Polyclonal antibody against outer membrane protein of Candidatus liberobacter asiaticum, and preparation method and application thereof | |
| CN106434372A (en) | Application of coral-derived fungus aspergillus terreus strain C21-10 | |
| CN107418909B (en) | One plant of marine source bacillus licheniformis method biological reinforced for vinegar | |
| CN107084867B (en) | A kind of extracting method of filamentous fungi solid fermentation product total protein | |
| CN105648106A (en) | Exserohilum turcicum molecular detection primers and quick detection method | |
| CN103333911A (en) | Method for producing protein A by utilizing recombinant pichia pastoris | |
| CN102618517A (en) | Zearalenone (ZEN) toxin degrading enzyme for acinetobacter and coding gene and applications of ZEN toxin degrading enzyme | |
| CN102787089B (en) | Acinetobacterbeijerinckii and application of acinetobacterbeijerinckii | |
| CN108018216A (en) | Improve the method and application of sugar utilization and lemon acid yield in citric acid fermentation | |
| CN106631440A (en) | Compound microbial organic fertilizer for taxus chinensis as well as preparation method and application thereof | |
| CN103421697B (en) | Aspergillus oryzae LJ366 strain used for degrading aureomycin | |
| CN103421764B (en) | Kit for quickly extracting double-stranded RNA of mycovirus and application of kit | |
| CN105176846A (en) | FEDT-866 fungi strains for producing biological siderophore compounds and preparation method | |
| CN104593393A (en) | Formica sinensis AChE gene, formica sinensis AChE and preparation method thereof | |
| CN105648107A (en) | Helminthosporium maydis molecular detection primers and quick detection method | |
| CN103710363B (en) | Laccase gene Lac6 and expression protein and application thereof | |
| CN101805715A (en) | Active component extracted by using Bacillus licheniformis and application thereof | |
| CN105543138A (en) | Bacillus subtilis and application thereof to prevention and treatment of wheat sharp eyespot |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190121 Address after: 264000 No. 20 Huasheng Road, Zhifu District, Yantai City, Shandong Province Patentee after: Wuzhoufeng Agricultural Science & Technology Co., Ltd. Address before: 211225 Jiangsu Nanjing Lishui District Baima Town National Agricultural Science and Technology Park Nanjing Agricultural University base Patentee before: Nanjing Agricultural University |
|
| TR01 | Transfer of patent right |