CN104371987B - Method for separating yak stomach lysozyme - Google Patents
Method for separating yak stomach lysozyme Download PDFInfo
- Publication number
- CN104371987B CN104371987B CN201410603940.XA CN201410603940A CN104371987B CN 104371987 B CN104371987 B CN 104371987B CN 201410603940 A CN201410603940 A CN 201410603940A CN 104371987 B CN104371987 B CN 104371987B
- Authority
- CN
- China
- Prior art keywords
- yak
- lysozyme
- solution
- stomach
- concentration
- 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.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Polymers & Plastics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Animal Husbandry (AREA)
- Birds (AREA)
- General Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Gastroenterology & Hepatology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pharmacology & Pharmacy (AREA)
- Immunology (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for separating and purifying yak stomach lysozyme. The method comprises the following steps: (1) homogenizing to obtain homogenate; (2) carrying out coarse purification to obtain a coarse purified solution; (3) carrying out CM-Sepharose FF column chromatography to obtain a chromatographic solution; (4) carrying out Sephadex G-75 column chromatography to obtain a yak stomach lysozyme solution; and (5) and lyophilizing to obtain yak stomach lysozyme. By using the method disclosed by the invention, the pure yak stomach lysozyme can be separated from yak gastric mucosal tissue, has high enzyme activity, wide application range and antibacterial effect and can be used as an antibacterial agent. Meanwhile, the method is high in yield and simple and convenient to operate and has industrial application values.
Description
Technical field
The present invention relates to a kind of separation method of yak stomach lysozyme.
Background technology
Lysozyme is a kind of alkaline protein, and it is distributed widely in animal, plant and microorganism, in the blood of mammal
All have expressed in the histiocytes such as slurry, trachea, intestinal, stomach, kidney regulating liver-QI.Lysozyme passes through to destroy n- acetyl cell wall in Peptidoglycan
β-Isosorbide-5-Nitrae glycosidic bond between acid and n- n acetylglucosamine n, leads to cell wall structure to change, so that bacterolysises.Lysozyme
Due to having antibiotic property, and no toxicity, side effect, food, biological engineering, doctor can be applied to as potential Substitutes For Antibiotic
Treat and the fields such as animal and fowl fodder.Consider, lysozyme is used in as additive in feedstuff, can have from disease of domestic animals control aspect
Effect preventing and treating diarrhea in livestock, provides nutrition and intestinal protection for it simultaneously, thus the death that the diarrhoea reducing domestic animal leads to, improves it
The rate of animals delivered to the slaughter-house.
Yak, as the ruminant being distributed in extremely frigid zones, is the most important domestic animal in Qinghai-xizang Plateau Region.Due to its with
Difference between the animal living enviroment of hinterland, creates genetic specificity and the unique adaptability to environment of yak itself.
But so far, have no the document report with regard to isolating and purifying yak stomach lysozyme.
Content of the invention
In order to solve the above problems, the invention provides a kind of isolation and purification method of yak abomasum lysozyme.
A kind of isolation and purification method of present invention yak stomach lysozyme, comprises the steps:
(1) it is homogenized: take yak abomasum mucosa to organize, smash to pieces, add the Spirit of Mindererus. of mucosal tissue 2 times (v/w), even
Slurry, centrifugation, separate supernatant and precipitation, then add the Spirit of Mindererus. precipitating 2 times of weight (w/w) in precipitation, centrifugation, close
And supernatant, as homogenate；Wherein, the concentration of Spirit of Mindererus. is 10mm；
(2) slightly pure: to adjust the ph to 4 of homogenate, centrifugation, take supernatant, heat 2min in boiling water bath, treat that temperature is down to
Room temperature, then adjust ph to 5, centrifugation, obtain supernatant, as thick liquid；
(3) cm-sepharose ff column chromatography: using the Spirit of Mindererus. balance cm-sepharose for 10mm for the concentration
Ff post, takes the slightly pure liquid loading of step (2), the ammonium acetate eluting foreign protein being 10mm with concentration, then with concentration be 10mm～
The Spirit of Mindererus. linear gradient elution of 300mm, according to eluting collection of illustrative plates, the corresponding solution in second peak of collection, as chromatographic solution；
(4) sephadex g-75 column chromatography: sephadex g-75 is balanced for 0.2% (v/v) acetum using concentration
Post, takes the chromatographic solution loading of step (3), is the acetum eluting of 0.2% (v/v) with concentration, according to eluting collection of illustrative plates, collects
The corresponding solution in peak, obtains final product yak stomach lysozyme soln afterwards；
(5) lyophilizing, obtains final product yak stomach lysozyme.
In step (1) and step (2), the centrifugal force that described centrifugation adopts is 27000g, and centrifugation time is 15min.
In step (2), adjust ph to adopt glacial acetic acid for the first time, adjust for second ph to adopt ammonia.
In step (3), the speed of eluting is 1ml/min.
In step (3), during described linear gradient elution, the cumulative volume of eluent is 20cv.
Cv, represents column volume.
In step (4), the speed of eluting is 0.2ml/min.
In step (4), first on chromatographic column, Deca 1ml concentration is 0.2% acetum, then eluting.
Present invention also offers the yak stomach lysozyme of preceding method preparation, and its preparing feed additive or antimicrobial drug
Purposes in thing.
Can be separated from yak gastric mucosa tissue using the inventive method and obtain sterling yak abomasum lysozyme, its enzyme activity
Up to 29,268u/mg, all active in wider ph scope and ionic strength range, there is preferable thermal stability, to stomach
Protease is resistant, applied range, has commercial application value, also has bacteriostasis, can as Antibiogics usage,
It is also used as additive and is used in feedstuff, prevent and treat diarrhea in livestock.Meanwhile, the inventive method yield is high, easy and simple to handle, industry
Application prospect is good.
Brief description
Fig. 1 yak stomach lysozyme cm-sepharose ff ion-exchange chromatography；
Fig. 2 yak stomach lysozyme sephadex g-75 gel filtration；
Fig. 3 purified product sds-page electrophoretogram；
Fig. 4 purified product fungistatic effect；
The impact to yak stomach lysozyme activity for Fig. 5 ph；
The impact to enzymatic activity for Fig. 6 ionic strength；
The impact to enzymatic activity of Fig. 7 ionic strength and ph；
The impact to enzymatic activity for Fig. 8 temperature；
Fig. 9 yak stomach lysozyme activity is over time.
Specific embodiment
The specific embodiment of form by the following examples, makees further specifically to the above of the present invention
Bright.But this scope being interpreted as the above-mentioned theme of the present invention should not be only limitted to below example.All above-mentioned interior based on the present invention
Hold realized technology and belong to the scope of the present invention.
Experiment material and reagent:
Experiment material
Meiwa yak abomasum
Reagent
Instrument
Embodiment 1 isolation and purification method of the present invention
1st, acquired for materials
Meiwa yak abomasum is collected in the bluish white river in Chengdu slaughterhouse.After abomasum is taken out, with normal saline, it is carried out
Cleaning.It is subsequently placed into ice chest, takes back laboratory, be stored in -20 DEG C.
2nd, isolate and purify
2.1 prepare gastric mucosa homogenate
(1) yak abomasum is placed in 4 DEG C of environment and thaws.Then gastric mucosa is torn, weighs 7g yak abomasum gastric mucosa,
And it is added thereto to 10mm ammonium acetate (ph=6) 14ml.Finally carry out homogenized (remaining gastric mucosa preserves with tissue mashing machine
In -80 DEG C).
(2) homogenate is moved in the 50ml centrifuge tube after sterilizing, be centrifuged at 4 DEG C (27,000 × g, 15 points
Clock).Subsequently the supernatant after centrifugation is transferred in new 50ml centrifuge tube and (is stored in 4 DEG C).
(3) 10mm ammonium acetate (ph=6) is added to (10mm ammonium acetate: residual in the test tube containing gastric mucosa residue again
Slag=2:1, the proportioning of the two is volume mass ratio), it is centrifuged (27,000 × g, 15 minutes) at 4 DEG C.Again supernatant is proceeded to
(it is stored in 4 DEG C) in new centrifuge tube.
The thick purification of 2.2 samples
(1) to Deca glacial acetic acid in supernatant, adjust mixed liquor ph to 4.Then with the speed of 27,000 × g at 4 DEG C
Centrifugation 15 minutes.Subsequently supernatant is transferred in new centrifuge tube.
(2) supernatant after centrifugation is put in 100 DEG C of water, water-bath 2 minutes.Subsequently taking out makes its temperature be down to room temperature.
(3) ammonia is added dropwise in supernatant, adjusts ph to 5.Then with the centrifugation of 27,000 × g at 4 DEG C
15 minutes.Again supernatant is transferred in new centrifuge tube, and is stored in 4 DEG C.
2.3cm-sepharose ff column chromatography
(1) cm sepherose ff is installed on bio-rad protein purification instrument, and with balance liquid (10mm acetic acid
Ammonium) prepacked column is balanced.
(2) with 0.20 μm of filter, sample supernatant is filtered, and the 2ml supernatant after filtering is added chromatography system
In system.
(3) use balance liquid (the 10mm ammonium acetate of ph=5.8) with the flow velocity rinse-system of 1ml/min, eluting foreign protein.
(4) add eluent 10mm ammonium acetate in tomographic system, be stepped up 300mm ammonium acetate (ph=8), with
The flow velocity of 1ml/min carries out linear gradient elution (gradient: 0-100% elution buffer to cm-sepharose ff ion exchange column
Liquid, 20cv), and the solution containing stomach lysozyme is collected according to elution profile, and it is stored in 4 DEG C.
Elution profile is as shown in Figure 1:
Solution containing eluting peak is collected, and activity identification is carried out to it.Result shows, destination protein is present in
In the corresponding collecting pipe in second peak.
Therefore, the corresponding solution in second peak of collection in actual separation.
2.4sephadex g-75 column chromatography
(1) weigh 5～6g gel dry powder, after adding the eluent of 10 times of volumes, be placed in water-bath 2 in 100 DEG C of water-bath
Hour.Midway is slowly stirred manually.As found tiny gel pieces, after gel precipitation, remove supernatant, be repeated several times by until solidifying
No (it is continuously added appropriate eluent during water-bath, prevent gel slurry to be evaporated) till gel pieces in sol solution.
(2) by chromatographic column right angle setting indoors, and to it inject the eluent of 1/5 column volume, in order to exclude chromatographic column
The air at bottom.It is slowly added to gel slurry with tailing edge glass rod.Post bottom outlet is opened, simultaneously by chromatographic column and bio- after the completion of post to be installed
Rad protein purification instrument is connected, then with eluent, it is overnight balanced with (during dress post, whether the moment observes in chromatographic column has
Tomography is gentle bubble out existing, if there is bubble should be driven at once out of or re-starts dress post).
(3) 1ml lysozyme soln is slowly added into gel surface (to avoid impacting gel as far as possible, cause gel surface
Out-of-flatness, thus affect separating effect), then turn on chromatography column outlet.Enter after colloid after lysozyme soln, in gel surface
It is slowly added dropwise 1ml eluent (0.2% acetic acid), then with the eluent (0.2% acetic acid) of 0.2ml/min flow velocity, chromatographic column is entered
Row eluting.
(4) according to elution profile, collect the solution of lysozyme, and with freeze dryer, frozen dried is carried out to sample, obtain final product this
Bright lysozyme (sample preservation after process is in -20 DEG C).
The absorbance isolating and purifying rear protein solution is measured, as shown in Figure 2.Solution containing eluting peak is carried out
Collect, and its activity is measured.Result shows, destination protein is present in the corresponding collecting pipe in last peak.
Therefore, collect the corresponding solution in last peak in actual separation and be lysozyme soln of the present invention.
3rd, detect
3.1sds-page electrophoresis
(1) sds-page polyacrylamide gel preparation, is shown in Table 1:
Table 1sds-page polyacrylamide gel
table.1sds-page polyacrylamide gel
(2) draw 10 μ l enzymatic solution in pcr pipe, add 2 μ l 5 × sds-page loading buffer, and will mix
Close liquid to be placed in 100 DEG C of water, water-bath 15 minutes.
(3) gel preparing is put in electrophoresis tank, in gel, then add mixed liquor.To be mixed with 80v voltage
Liquid electrophoresis is to concentration glue and separation gel demarcation line, then adjusts voltage for 120v until electrophoresis terminates.
(4) running gel is moved in the vessel filling coomassie brilliant blue staining liquid, then be placed in shaking table slow concussion 4 hours
Preservative film closed vessel mouth (is used) in dyeing course in left and right.
(5) discard dye liquor, with water, gel is rinsed for several times, add Coomassie brilliant blue destaining solution, be placed in shaking table slow
Overnight, (using preservative film closed vessel mouth in decolorization) up to protein band in concussion.
3.2 Enzyme activity assay
Using bacteriolyze enzyme detection kit, the stomach lysozyme activity after isolating and purifying is measured, step is as follows:
(1) bacterium solution, standard protein solution and testing sample solution are placed in pre-temperature more than 5 minutes in 37 DEG C of calorstats.
(2) spectrophotometric wavelength is adjusted to 530nm, and with distilled water, the transmittance of UV detector is adjusted
Whole is 100%.
(3) will be added in cuvette and mix in the bacterium solution (2ml) of 37 DEG C of pre-temperatures and standard protein solution (0.2ml), and
Record the transmittance changing value in 2 minutes.
(4) will be added in cuvette in the bacterium solution (2ml) of 37 DEG C of pre-temperatures and testing sample solution (0.2ml), and repeat
(3) assay method in, obtains transmittance changing value.
(5) according to computing formula (testing sample activity (u/ml)=(treat test tube transmittance changing value ÷ standard protein printing opacity
Degree changing value) × standard protein activity (200u/ml)) calculate testing sample activity (u/ml).
3.3 enzymatic solution concentration mensurations
Using bradford quantification of protein test kit, the stomach lysozyme concentration after isolating and purifying is measured, step is such as
Under:
(1) 0,10,20,30,40,50,60 μ l bovine serum albumin titer (1mg/ml) are added separately in pcr pipe,
Add pbs and supply 150 μ l.
(2) titer in (1) is added in the cuvette of the Coomassie brilliant blue dye liquor containing 2.85ml and mixes, and in room
Temperature is lower to place 5 to 10 minutes.
(3) use at spectrophotometric determination 595nm, the light absorption value of the bovine serum albumin titer of different content, and record
Numerical value (using the absorbance value without bovine serum albumin titer as blank).
(4) according to the light absorption value obtaining and bovine serum albumin content, draw standard curve.
(5) 80 μ l lysozyme solns are added in pcr pipe, and supply 150 μ l with pbs.
(6) the lysozyme mixed solution in (5) is added in the cuvette of the dye liquor of Coomassie brilliant blue containing 2.85ml and mixes,
And place 5 to 10 minutes at room temperature.
(7) use at spectrophotometric determination 595nm, the light absorption value of lysozyme mixed solution, and record numerical value.
(8) according to standard curve, calculate the protein content under corresponding light absorption value, thus calculating molten after isolating and purifying
The protein concentration of bacterium enzymatic solution.
3.4 Resistence research
There is to gram positive bacteria using lysozyme the characteristic of bacteriolyze effect, checked using staphylococcus aureuses and separate
The anti-microbial property of purification gained lysozyme, step is as follows:
Lb solid medium (antibiotic-free) 8ml after (1) sterilizing to addition in the culture dish after sterilizing about, it is placed in
Horizontal plane, treats its solidification.
(2) take 0.5～1ml staphylococcus aureuses bacterium solution, pour in remaining lb solid medium, shake up.
(3) in the culture dish containing lb solid medium, be equidistantly placed 3 Oxford cups (10mm × 7.8mm ×
6.0mm).Subsequently the lb solid medium (8ml) containing staphylococcus aureuses bacterium solution is poured into culture dish, be positioned over level
Face, treats its solidification.
(4) it is separately added into the yak stomach lysozyme after 3.6ug and 2.6 μ g isolates and purifies in No. 1 and No. 2 Oxford cups, No. 3
For negative control (adding 100 μ l deionized waters).Subsequently put it in 37 DEG C of constant incubators and cultivate 24 hours, observe knot
Really.
3.4 peptide fingerprinting spectrums
Yak stomach lysozyme after purification carries out sds-page gel electrophoresiss, intercepts yak stomach lysozyme electrophoretic band part
Gel, send Shanghai Inst. of Life Science, CAS, using electrophoresis mass spectrograph (applied biosystem, maldi-
Tof/tof 4800 type), by 2010 editions two annex j mass spectrographys of Chinese Pharmacopoeia, carry out protein with peptide fingerprinting spectral technology
Identification.
4th, measurement result
4.1sds-page Electrophoretic
The present invention is isolated and purified obtained yak stomach lysozyme and carries out sds-page gel electrophoresiss, result such as Fig. 3.Warp
Electrophoresis detection finds, only shows a protein band, show that isolating and purifying obtained sample has obtained purification in gel.By with
Standard protein contrasts thus it is speculated that yak abomasum bacteriolyze enzyme molecular weight is about 15kda.
Experimental result illustrates, the present invention separates and obtained sterling yak abomasum lysozyme.
4.2 yak abomasum lysozyme soln purification result
The protein solution being obtained after yak gastric mucosa homogenate is isolated and purified each time carries out activity and concentration mensuration,
Its purification result is shown in Table 2.
Table 2 yak stomach bacteriolyze enzyme purification
Upper table shows, the separated yak stomach lysozyme after purification, finally obtaining is 29,268u/mg, purification than living
For 66.51 times, the response rate be 58.6%.
It is computed understanding, in 10g gastric mucosa, about contain 1.99mg lysozyme.Meanwhile, using the inventive method, Ke Yicong
1.117mg lysozyme is extracted, yield is 58.6% in 10g gastric mucosa.
Experimental result, the present invention separates the sterling yak abomasum lysozyme obtaining, and enzyme activity is high.The inventive method yield simultaneously
High.
4.3 identification of proteins results
Shanghai Inst. of Life Science, CAS qualification result shows, this albumen is cattle lysozyme 2 precursor
(lysozyme 2a precursor, partial, bos taurus).Show that the product that we isolate and purify is one and cattle
The similar lysozyme of lysozyme.
4.4 yak stomach lysozyme fungistatic effects
Identified, the yak stomach lysozyme isolating and purifying gained has obvious fungistatic effect to staphylococcus aureuses
(Fig. 4).Wherein, No. 1 inhibition zone is slightly big than No. 2 inhibition zones, shows the increase with stomach bacteriolyze enzyme dosage, its fungistatic effect also with
Enhancing.
Experimental result illustrates, yak abomasum lysozyme of the present invention has bacteriostatic activity, can be used for preparing antibacterials.
Experimental result illustrates, can separately obtain sterling yak abomasum lysozyme using the inventive method, and its enzyme activity is high, tool
There are bacteriostasis.The yield of the inventive method is high simultaneously, is suitable to commercial Application.
The enzymatic property of the yak abomasum lysozyme that embodiment 2 is isolated and purified using the inventive method
Take the yak abomasum lysozyme of the embodiment of the present invention 1 preparation, detect its enzymatic property:
1st, test method
1.1 buffer
Using buffer online calculation server (http://www.biomol.net/en/tools/
Buffercalculator.htm) buffer of different ph and ionic strength is calculated, and determine formula.
The impact to enzymatic activity for the 1.2ph
In room temperature environment, measure under same ion intensity (ionic strength=0.133), different ph are to enzymatic properties
Impact, step is as follows:
(1) prepare the buffer solution micrococcus luteuses (ultimate density is 0.25mg/ml) of different ph, and be incubated at 25 DEG C
1 hour.
(2) gained lysozyme soln (deionized water dissolving) will be isolated and purified and be positioned over 37 DEG C of insulations 1 hour.
(3) draw 150 μ l lysozyme solns (5 μ g lysozyme), be added in 2ml micrococcus luteuses solution, turn upside down
Mix.It is then placed in measurement changing value in 5 minutes for the light transmittance in ultraviolet spectrophotometer (wavelength is 530nm), and record
Result.
(4) using lysozyme ph=2 light transmittance changing value as 1, the light transmittance changing value under remaining ph is divided by ph=2
Actual light transmittance changing value, in this, as the change multiple of the light transmittance under this ph.
Different ph buffer:
1.10mm phosphate buffer (ph=2, ionic strength=0.133): weigh 0.245g phosphoric acid and 1.8755g nacl,
It is dissolved in 200ml deionized water, adjust ph to 2, be settled to 250ml.
2.10mm phosphate buffer (ph=3, ionic strength=0.133): weigh 0.245g phosphoric acid and 1.814g nacl,
It is dissolved in 200ml deionized water, adjust ph to 3, be settled to 250ml.
3.10mm acetate buffer solution (ph=4, ionic strength=0.133): weigh 0.15g acetic acid and 1.919gnacl, molten
Solution, in 200ml deionized water, adjusts ph to 4, is settled to 250ml.
4.10mm acetate buffer solution (ph=5, ionic strength=0.133): weigh 0.15g phosphoric acid and 1.8445g nacl,
It is dissolved in 200ml deionized water, adjust ph to 5, be settled to 250ml.
5.10mm phosphate buffer (ph=6.2, ionic strength=0.133): weigh 0.3g biphosphate and receive and 1.7445g
Nacl, is dissolved in 200ml deionized water, adjusts ph to 6.2, is settled to 250ml.
6.10mm phosphate buffer (ph=7, ionic strength=0.133): weigh 0.3g biphosphate and receive and 1.6255g
Nacl, is dissolved in 200ml deionized water, adjusts ph to 7, is settled to 250ml.
7.10mm phosphate buffer (ph=8, ionic strength=0.133): weigh 0.3g biphosphate and receive and 1.526g
Nacl, is dissolved in 200ml deionized water, adjusts ph to 8, is settled to 250ml.
8.10mm tris buffer (ph=9, ionic strength=0.133): weigh 0.3025g tris-base and
1.927g nacl, is dissolved in 200ml deionized water, adjusts ph to 9, is settled to 250ml.
The impact to enzymatic activity for 1.3 ionic strengths
In room temperature environment, measure under identical ph (ph=5), the impact to enzymatic properties for the different ionic strength, step
As follows:
(1) the buffer solution micrococcus luteuses (ultimate density be 0.25mg/ml) of different ionic strength are prepared, and 25
DEG C insulation 1 hour.
(2) gained lysozyme soln (deionized water dissolving) will be isolated and purified and be positioned over 37 DEG C of insulations 1 hour.
(3) draw 150 μ l lysozyme solns (5 μ g lysozyme), be added in 2ml micrococcus luteuses solution, turn upside down
Mix, be then placed in measurement changing value in 5 minutes for the light transmittance in ultraviolet spectrophotometer (wavelength is 530nm), and record
Result.
(4) using lysozyme ionic strength be 0.007 light transmittance changing value as 1, the printing opacity under remaining ionic strength
The actual light transmittance changing value that rate changing value is 0.007 divided by ionic strength, in this, as the light transmittance under this ionic strength
Change multiple.
The buffer of determination of activity under different ionic strength:
1.10mm acetate buffer solution (ph=5, ionic strength=7mm): weigh 0.15g acetic acid and 0.065g nacl, dissolving
In 200ml deionized water, adjust ph to 5, be settled to 250ml.
2.10mm acetate buffer solution (ph=5, ionic strength=20mm): weigh 0.15g acetic acid and 0.195g nacl, molten
Solution, in 200ml deionized water, adjusts ph to 5, is settled to 250ml.
3.10mm acetate buffer solution (ph=5, ionic strength=50mm): weigh 0.15g acetic acid and 0.632g nacl, molten
Solution, in 200ml deionized water, adjusts ph to 5, is settled to 250ml.
4.10mm acetate buffer solution (ph=5, ionic strength=125mm): weigh 0.15g acetic acid and 1.7275g nacl,
It is dissolved in 200ml deionized water, adjust ph to 5, be settled to 250ml.
5.10mm acetate buffer solution (ph=5, ionic strength=200mm): weigh 0.15g acetic acid and 2.824g nacl, molten
Solution, in 200ml deionized water, adjusts ph to 5, is settled to 250ml.
6.10mm acetate buffer solution (ph=5, ionic strength=300mm): weigh 0.15g acetic acid and 4.287g nacl, molten
Solution, in 200ml deionized water, adjusts ph to 5, is settled to 250ml.
7.10mm acetate buffer solution (ph=5, ionic strength=500mm): weigh 0.15g acetic acid and 7.2135g nacl,
It is dissolved in 200ml deionized water, adjust ph to 5, be settled to 250ml.
The impact to enzymatic activity for 1.4 temperature and the heat stability of enzyme
Lysozyme soln is respectively placed in 4 DEG C, 25 DEG C, 37 DEG C, 45 DEG C, 50 DEG C, 60 DEG C, 80 DEG C and 100 DEG C of water-baths.Water
Bath 15 minutes.Then draw 150 μ l lysozyme solns (5 μ g lysozyme), be added in 2ml micrococcus luteuses solution (25 DEG C of guarantors
Temperature 1 hour), change in 5 minutes for the light transmittance is measured by ultraviolet spectrophotometer (wavelength be 530nm), and by lysozyme
In 4 DEG C of light transmittance changing values as 1, the light transmittance changing value at a temperature of remaining divided by 4 DEG C of actual light transmittance changing value, with
This is as the change multiple of the light transmittance at a temperature of this.
The buffer of determination of activity under different temperatures: 10mm phosphate buffer (ph=6.2, ionic strength=50mm): claim
Take 0.3g biphosphate to receive and 1.7445g nacl, be dissolved in 200ml deionized water, adjust ph to 6, be settled to 250ml.
For studying the heat stability of albumen, measure light transmittance at optimum temperature and 100 DEG C relatively for the yak stomach lysozyme
Change, and the retention of enzymatic activity in 100 DEG C is calculated with this.
The impact to enzymatic activity of 1.5 ionic strengths and ph
Prepare the buffer solution micrococcus luteuses (ultimate density is 0.25mg/ml) of different ionic strength and ph, juxtaposition
It is incubated at 25 DEG C.Then draw lysozyme soln (5 μ g lysozyme) after 37 DEG C of insulations for the 150 μ l, be added to 2ml Resina garciniae micro-
Mix in coccus solution (25 DEG C are incubated 1 hour), light transmittance is measured at 5 points by ultraviolet spectrophotometer (wavelength is 530nm)
Change in clock.And respectively using lysozyme the light transmittance changing value of the ph=4 in different ionic strength as 1, remaining is different
In ionic strength, the light transmittance changing value of different ph, divided by the actual light transmittance changing value of ph=4 in this ionic strength, is made with this
Change multiple for the light transmittance under this ionic strength and ph.
The buffer of determination of activity under different ionic strength and ph:
1.10mm acetate buffer solution (ph=4, ionic strength=20mm): weigh 0.15g acetic acid and 0.268g nacl, molten
Solution, in 200ml deionized water, adjusts ph to 4, is settled to 250ml.
2.10mm phosphate buffer (ph=6.2, ionic strength=20mm): weigh 0.3g sodium dihydrogen phosphate and 0.1075g
Nacl, is dissolved in 200ml deionized water, adjusts ph to 6.2, is settled to 250ml.
3.10mm phosphate buffer (ph=7, ionic strength=20mm): weigh 0.3g sodium dihydrogen phosphate and 0.0025g
Nacl, is dissolved in 200ml deionized water, adjusts ph to 7, is settled to 250ml.
4.10mm tris buffer (ph=8, ionic strength=20mm): weigh 0.3g tris-base and 0.209g
Nacl, is dissolved in 200ml deionized water, adjusts ph to 8, is settled to 250ml.
5.10mm acetate buffer solution (ph=4, ionic strength=50mm): weigh 0.15g acetic acid and 0.706g nacl, molten
Solution, in 200ml deionized water, adjusts ph to 4, is settled to 250ml.
6.10mm phosphate buffer (ph=6.2, ionic strength=50mm): weigh 0.3g sodium dihydrogen phosphate and 0.5395g
Nacl, is dissolved in 200ml deionized water, adjusts ph to 6.2, is settled to 250ml.
7.10mm phosphate buffer (ph=7, ionic strength=50mm): weigh 0.3g sodium dihydrogen phosphate and 0.428g
Nacl, is dissolved in 200ml deionized water, adjusts ph to 7, is settled to 250ml.
8.10mm phosphate buffer (ph=8, ionic strength=50mm): weigh 0.3g sodium dihydrogen phosphate and 0.3155g
Nacl, is dissolved in 200ml deionized water, adjusts ph to 8, is settled to 250ml.
9.10mm acetate buffer solution (ph=4, ionic strength=133mm): weigh 0.15g acetic acid and 1.919g nacl, molten
Solution, in 200ml deionized water, adjusts ph to 4, is settled to 250ml.
10.10mm phosphate buffer (ph=6.2, ionic strength=133mm): weigh 0.3g biphosphate receive and
1.7445g nacl, is dissolved in 200ml deionized water, adjusts ph to 6, is settled to 250ml.
11.10mm phosphate buffer (ph=7, ionic strength=133mm): weigh 0.3g biphosphate and receive and 1.6255g
Nacl, is dissolved in 200ml deionized water, adjusts ph to 7, is settled to 250ml.
12.10mm phosphate buffer (ph=8, ionic strength=133mm): weigh 0.3g biphosphate and receive and 1.526g
Nacl, is dissolved in 200ml deionized water, adjusts ph to 8, is settled to 250ml.
13.10mm acetate buffer solution (ph=4, ionic strength=200mm): weigh 0.15g acetic acid and 2.899g nacl,
It is dissolved in 200ml deionized water, adjust ph to 4, be settled to 250ml.
14.10mm phosphate buffer (ph=6.2, ionic strength=200mm): weigh 0.3g sodium dihydrogen phosphate and
2.7209g nacl, is dissolved in 200ml deionized water, adjusts ph to 6.2, is settled to 250ml.
15.10mm phosphate buffer (ph=7, ionic strength=200mm): weigh 0.3g sodium dihydrogen phosphate and 2.599g
Nacl, is dissolved in 200ml deionized water, adjusts ph to 7, is settled to 250ml.
16.10mm phosphate buffer (ph=8, ionic strength=200mm): weigh 0.3g sodium dihydrogen phosphate and 2.505g
Nacl, is dissolved in 200ml deionized water, adjusts ph to 8, is settled to 250ml.
1.6 unconventional kinetic measurement
Configuration ph=5, the buffer solution micrococcus luteuses (ultimate density is 0.25mg/ml) of ionic strength=0.05,
It is placed in being incubated 1 hour in 25 DEG C.Then draw lysozyme soln (5 μ g lysozyme) after 37 DEG C of insulations for the 150 μ l, be added to
Mix in 2ml micrococcus luteuses solution.Measure the light transmittance number of different time by ultraviolet spectrophotometer (wavelength is 530nm)
Value (every 30 seconds records once).
Unconventional kinetic measurement buffer:
1.10mm acetate buffer solution (ph=5, ionic strength=50mm): weigh 0.15g acetic acid and 0.632g nacl, molten
Solution, in 200ml deionized water, adjusts ph to 5, is settled to 250ml.
2.0.066m sodium phosphate: weigh 1.7g sodium phosphate, be dissolved in 80ml deionized water, be settled to 100ml.
The impact to enzymatic activity for 1.7 pepsin
(1) 0.25mg yak stomach lysozyme is dissolved in the 0.01m hcl (nacl containing 0.1m) that 50ml ph is 2.
(2) 6mg pepsin is dissolved in 1ml 0.066m sodium phosphate (ph=6.2).
(3) prepare ph be 5, ionic strength be 0.133 buffer (attached 1 reagent configuration) dissolving micrococcus luteuses (final
Concentration is 0.25mg/ml), and it is incubated 1 hour at 25 DEG C.
(4) draw 9 μ l pepsin solutions and be added in 10ml lysozyme soln (stomach lysozyme: pepsin=1:1),
And adjust ph to 2.75.
(5) experimental group that will add pepsin (ph=2.75) and the matched group not adding pepsin (ph=2.75) are same
When be placed in 37 DEG C be incubated 1 hour, after taking-up all use 1m naoh adjustment ph to 6.5.
(6) draw lysozyme (experimental group and matched group) after 37 DEG C of insulations for the 150 μ l, be added to 2ml micrococcus luteuses
In solution, by ultraviolet spectrophotometer (wavelength is 530nm) detection mixed solution in 5 minutes iuuminting rate changing values.
2nd, result of the test
The impact to enzymatic activity for the 2.1ph
Stomach lysozyme activity, result such as Fig. 5 is measured in the buffer solution (ionic strength is 0.133) of different ph.Figure
In visible, stomach lysozyme of the present invention is all active in the range of ph2～9, wherein, ph be 5 weak acid environment in, activity
Highest.
The impact to enzymatic activity for 2.2 ionic strengths
The activity of stomach lysozyme, result such as Fig. 6 is measured in the buffer solution (ph is 5) of different ionic strength.
It can be seen that stomach lysozyme of the present invention is all active in the range of ionic strength is for 0.007～0.3, wherein,
In the buffer of ionic strength=0.05, the active highest of stomach lysozyme.
Meanwhile, hypersaline environment (ionic strength ＞ 0.05) is better than low-salt environment to the inhibition level of yak stomach lysozyme activity
(ionic strength ＜ 0.05).
The impact to enzymatic activity of 2.3 ionic strengths and ph
The activity of stomach lysozyme, result such as Fig. 7 is measured in the buffer solution of different ph values and ionic strength.Laterally observe
Find: the increase (0.02～0.2) with ionic strength, the optimum ph of yak stomach lysozyme progressively from close to 7 neutral value to
Ph is 5 meta-acid value movement, therefore different ionic strengths can affect the optimum ph value of yak stomach lysozyme；Longitudinal 2 observation finds:
Increase (0.02～0.2) with ionic strength, yak stomach lysozyme activity can decline (under the conditions of optimum ph), that is, in high salt
In environment, (ionic strength=0.2) yak stomach lysozyme activity can be suppressed, and compared with low-salt environment (ionic strength=
0.02), antibacterial effect at optimum ph for the stomach lysozyme declines 4 times about.
The impact to enzymatic activity for 2.4 temperature and the heat stability of enzyme
Yak stomach lysozyme activity, result such as Fig. 8 is measured in different temperatures environment.In figure shows: stomach bacteriolyze of the present invention
Enzyme is all active in the range of temperature is for 4～100 DEG C, and wherein, when temperature is 50 DEG C, its activity reaches highest.
Experimental result illustrates, relative to optimum temperature at 50 DEG C about, and it is unwise to temperature change for yak stomach lysozyme
Sense.
Measure the temperature stability of yak stomach lysozyme at high temperature, its result shows: yak stomach lysozyme is in 100 DEG C of water
Bath 15 minutes after, its activity still remain with this enzyme under optimum temperature activity 73% about.As can be seen here.Yak stomach bacteriolyze
Enzyme has preferable heat stability in high temperature.
2.5 unconventional kinetics
The activity measuring stomach lysozyme in 10mm acetate buffer solution (ph=5, ionic strength=0.05) changes over
Rule, result such as Fig. 9.It can be seen that prolongation over time, the activity of yak stomach lysozyme presents logarithm growth
Trend.
The impact to enzymatic activity for 2.6 pepsin
After adding the pepsin of equivalent in yak stomach lysozyme, adjust solution ph to 2.75, and little in 37 DEG C of incubations 1
When.Subsequently using 1m naoh, enzyme liquid ph is adjusted to 6.5, and measures its activity.Result showed, through pepsin digestion 1 hour
Stomach lysozyme (experimental group), its activity still remains with the 70% about of original stomach lysozyme (matched group) activity.
As can be seen here, yak stomach lysozyme is to pepsic resistance.
Experimental result illustrate, the inventive method be obtained sterling yak abomasum lysozyme have wider ph scope, from
All active in sub- strength range and temperature range, have preferable heat stability, to pepsin resistance, applied range.
To sum up, sterling yak abomasum lysozyme can be obtained using separation method of the present invention, its enzyme activity is high, in wider ph
All active in scope, ionic strength range and temperature range, there is preferable heat stability, to pepsic resistance, also
There are bacteriostasis, as Antibiogics usage, or can be used for as additive in feedstuff, prevent and treat diarrhea in livestock.With
When, the inventive method yield is high, easy and simple to handle, has industrial application value.
Claims (7)
1. a kind of isolation and purification method of yak stomach lysozyme it is characterised in that: comprise the steps:
(1) it is homogenized: take yak abomasum mucosa to organize, smash to pieces, add Spirit of Mindererus., the addition of Spirit of Mindererus. is mucosa group
2 times (v/w) knitting, homogenate, centrifugation, separate supernatant and precipitation, then add Spirit of Mindererus., Spirit of Mindererus. in precipitation
Addition be precipitation capacity 2 times (v/w), centrifugation, merge supernatant, as homogenate；Wherein, the concentration of Spirit of Mindererus. is
10mm；
(2) slightly pure: to adjust the ph to 4 of homogenate, centrifugation, take supernatant, heat 2min in boiling water bath, treat that temperature is down to room
Temperature, then adjust ph to 5, centrifugation, obtain supernatant, as slightly pure liquid；
(3) cm-sepharose ff column chromatography: using the Spirit of Mindererus. balance cm-sepharose ff post for 10mm for the concentration,
Take the slightly pure liquid loading of step (2), the ammonium acetate eluting foreign protein being 10mm with concentration, then be 10mm～300mm's with concentration
Spirit of Mindererus. linear gradient elution, according to eluting collection of illustrative plates, the corresponding solution in second peak of collection, as chromatographic solution；
(4) sephadex g-75 column chromatography: sephadex g-75 post is balanced for 0.2% (v/v) acetum using concentration, takes
The chromatographic solution loading of step (3), is the acetum eluting of 0.2% (v/v) with concentration, according to eluting collection of illustrative plates, collects last
The corresponding solution in individual peak, obtains final product yak stomach lysozyme soln；
(5) lyophilizing, obtains final product yak stomach lysozyme.
2. isolation and purification method according to claim 1 it is characterised in that: in step (1) and step (2), centrifugation adopt
Centrifugal force be 27000g, centrifugation time be 15min.
3. isolation and purification method according to claim 1 it is characterised in that: in step (2), adjust for the first time ph to adopt ice vinegar
Acid, adjusts for second ph to adopt ammonia.
4. isolation and purification method according to claim 1 it is characterised in that: in step (3), the speed of eluting is 1ml/
min.
5. isolation and purification method according to claim 1 it is characterised in that: in step (3), during linear gradient elution, wash
The cumulative volume of de- liquid is 20cv.
6. isolation and purification method according to claim 1 it is characterised in that: in step (4), the speed of eluting is 0.2ml/
min.
7. isolation and purification method according to claim 1 it is characterised in that: in step (4), first Deca on chromatographic column
1ml concentration is 0.2% acetum, then eluting.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410603940.XA CN104371987B (en) | 2014-10-31 | 2014-10-31 | Method for separating yak stomach lysozyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410603940.XA CN104371987B (en) | 2014-10-31 | 2014-10-31 | Method for separating yak stomach lysozyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104371987A CN104371987A (en) | 2015-02-25 |
| CN104371987B true CN104371987B (en) | 2017-01-25 |
Family
ID=52551184
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410603940.XA Active CN104371987B (en) | 2014-10-31 | 2014-10-31 | Method for separating yak stomach lysozyme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104371987B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101352673A (en) * | 2007-07-26 | 2009-01-28 | 复旦大学 | Affinity chromatography for separating lysozyme and preparation method thereof |
| CN103160482A (en) * | 2013-03-14 | 2013-06-19 | 江苏大学 | Method for preparing egg white lysozyme and active protein by adopting coseparation |
-
2014
- 2014-10-31 CN CN201410603940.XA patent/CN104371987B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101352673A (en) * | 2007-07-26 | 2009-01-28 | 复旦大学 | Affinity chromatography for separating lysozyme and preparation method thereof |
| CN103160482A (en) * | 2013-03-14 | 2013-06-19 | 江苏大学 | Method for preparing egg white lysozyme and active protein by adopting coseparation |
Non-Patent Citations (3)
| Title |
|---|
| Yak (Bos Grunniens) Stomach Lysozyme: Molecular Cloning, Expression and its Antibacterial Activities;Mingfeng Jiang et al.;《Animal Biotechnology》;20091217;第21卷(第1期);25-35 * |
| 反刍动物胃溶菌酶的研究进展;邓霄禹等;《中国畜牧兽医》;20121231;第39卷(第9期);22-25 * |
| 牦牛胃溶菌酶基因在毕赤酵母中的分泌表达及活性分析;任洪辉等;《中国遗传学会第九次全国会员代表大会暨学术研讨会论文摘要汇编（2009-2013）》;20130918;257 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104371987A (en) | 2015-02-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gunaratna et al. | Screening and evaluation of natural coagulants for water treatment | |
| CN103054902B (en) | Method for producing transfer factor in scale | |
| CN106008704A (en) | Method for producing lactoferrin and lactoperoxidase | |
| CN105648008A (en) | Feeding silk antibacterial peptide preparation and preparation method thereof | |
| CN102183653A (en) | Cryptosporidium parvum immune colloidal gold detection test paper strip and production method thereof | |
| CN103948632B (en) | Toad raises the medical usage of maggot antibacterial peptide | |
| CN110041425A (en) | A kind of high-purity sero-abluminous preparation method | |
| CN106011115A (en) | Preparation method and application of lamb abomasums effective component extracts | |
| CN104371987B (en) | Method for separating yak stomach lysozyme | |
| CN105131099B (en) | A method of sericin is prepared from tussah degumming of silk industrial wastewater | |
| CN105801689A (en) | Method for co-preparing ovalbumin and ovotransferrin | |
| Li et al. | The involvement of PDGF/VEGF related factor in regulation of immune and neuroendocrine in Chinese mitten crab Eriocheir sinensis | |
| CN109735513A (en) | A kind of purification process of Cryptosporidium protein kinase | |
| Kamiya et al. | Participation of the C-type hemolymph lectin in mineralization of the acorn barnacle Megabalanus rosa | |
| CN102200526A (en) | Qualitative detection method capable of distinguishing cow milk doped in human milk | |
| Rafiq et al. | Purification and partial characterization of a fructose-binding lectin from the leaves of Euphorbia helioscopia | |
| CN104587274B (en) | A kind of leaf of bamboo homo-orientin extract | |
| CN102028709A (en) | Process for extracting biologically active calcium ions by biotechnological wet method | |
| CN111826442B (en) | Target PLEKHN1 for preventing lung cancer and application thereof | |
| CN103864915B (en) | The method of purification of Recombinant human interferon-alpha-2 b and test kit | |
| CN103044542B (en) | Serpin and gene thereof and application in crucian ovum | |
| CN111825757A (en) | Method for removing heat source in recombinant IL-18 and product prepared by method | |
| CN113583084B (en) | Urechis unicinctus leaching solution protein extract and extraction method and application thereof | |
| CN104730261A (en) | Colloidal gold test strip for detecting chicken infectious anemia virus | |
| CN110172094B (en) | Method for extracting high-purity metallothionein from oratosquillas |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |