CN105821023B - A kind of isolation and purification method of keratinase and application - Google Patents
A kind of isolation and purification method of keratinase and application Download PDFInfo
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- CN105821023B CN105821023B CN201610289190.2A CN201610289190A CN105821023B CN 105821023 B CN105821023 B CN 105821023B CN 201610289190 A CN201610289190 A CN 201610289190A CN 105821023 B CN105821023 B CN 105821023B
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- 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)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
Abstract
Isolation and purification method and application the present invention relates to a kind of keratinase, step are：After crude enzyme liquid is precipitated with ammonium sulphate gradient, precipitation, dialysis is resuspended with buffer solution；With buffer solution ion balance exchange column, loading, enzyme activity peak is across in peak；Collecting has the component of enzyme activity, is dialysed with buffer solution；With buffer solution ion balance exchange column, loading with NaCl gradient elutions, obtains keratinase after purification.1L zymotic fluids can harvest the pure enzyme 16.59mg of keratinase Ker FJ, total enzyme activity 2989U in the present invention.And the keratinase Ker FJ obtained have extraordinary effect in terms of animal waste degradation, depilation and as detergent additives.
Description
Technical field
Isolation and purification method and application the present invention relates to a kind of keratinase, belong to biotechnology.
Background technology
Poultry feather, wool, animal skin, hoof tips main component be keratin.Keratin is rigid albumen, and structure causes
Close complexity it is difficult to by such as hydrolysis such as trypsase, pepsin and papain of common protease, can only be referred to as " angle
The protease of protease " is hydrolyzed.Due to keratinase can degrade common proteases be difficult to the feather rich in keratin degraded,
Wool, hoof nail, hair are therefore widely used in the processing of curriery, textile industry and keratin waste.Existing commercially available angle
Protease enzyme preparation is for exampleIt is that (Gupta R are transformed by keratinase KerA Deng all
et al.,Appl Microbiol Biotechnol.2013,97:9931–9940)。
In global range livestock culture industry, butchery, curriery, fur manufacturing industry bring a large amount of poultry feather and pig,
These protein wastes of the fur hoof tips of ox, sheep etc., these wastes are rich in keratin, discharge chlorine vulcanization under anaerobic
Object and ammonia bring serious environmental problem, while are difficult to degrade again, and difficulty is brought to waste processing.Microorganism and secretion
Can degrade ectoenzyme (mainly keratinase) these keratin wastes.Microorganism and keratinase are in mild reaction condition
Lower that keratin difficult to degrade is converted into polypeptide and amino acid, the hydrolysate rich in nitrogen can be used as feed feeding poultry,
The nutritive value and animal for improving feed digest and assimilate efficiency；It can serve as the nutritious fertilizer of soil；For foliage dressing；May be used also
Produce the bioenergies such as methane and biological hydrogen.How the harmless treatment of animal waste is solved, recycling becomes China
Aquaculture important topic urgently to be resolved hurrily.
Fur is impregnated, is lost hair or feathers in traditional curriery, softening and pelts tanning process need to use a large amount of alkali, sulfide, salt, You Jirong
The intractable chemical substance such as agent, lime, not only generates S2-Pollution, and COD, BOD value in waste water are increased, waste water is in strong
Alkalinity and with foul smell, causes serious environmental pollution.Traditional chemical reagent is replaced to lose hair or feathers using enzyme unhairing, can be kept away
Exempt from using vulcanized sodium and other noxious materials, so as to reduce the generation of waste, reduce discharge of industrial wastes, environmental protection is to realize
One of effective way of cleaner leather-making production.Commercialization protease preparation
It has been widely used in the programs such as cleaning, the depilation of fur in curriery (Gupta et al., Appl with Clarizyme
Microbiol Biotechnol.2013,97:9931–9940).But replace chemical reagent of high cost using protease completely, urgently
More efficient enzyme preparation need to be developed.Keratinase energy efficient degradation common proteases are difficult to the keratin degraded, including being rich in angle
Feather, wool, hoof nail, the hair of albumen, therefore in leather industry, keratinase just become than common proteases more have it is excellent
The substitute of gesture.There is no collagenase activity, the keratinase for having faint elastase activity to can be used for the depilation of curriery, this
A little enzyme relaxation keratin tissues, do not destroy the elasticity of leather and extract complete fur, keratin hydrolyzate can also act as retanning agent
Full leather quality, improve fur quality (Gupta et al., Appl Microbiol Biotechnol.2013,97:
9931–9940).More document reports bacillus Bacillus subtilis S14 of production keratinase
(Macedo AJ et al.,Appl Environ Microbiol.2005,71:594–596)、B.pumilus(Kumar AG
et al.,J Appl Microbiol.2008,104:411–420)、B.licheniformis ER-15(Tiwary E,
Gupta R et al.,Bioresour Technol.2010,101:6103-6110)、B.halodurans PPKS-2
(Prakash P et al.,Appl Biochem Biotechnol.2011,160:1909–1920)、B.halodurans
JB99(Shrinivas D,Naik GR et al.,Int Biodeter Biodeger.2011,65:29-35) extracellular thick enzyme
Liquid is to the depilation of different furs；The country is mainly around the screening for the strain resource for having depilation ability and crude enzyme liquid hair removal effect
Valency etc. comment roughly to study the hair removal effect of bacterial strain (Xie Fei etc., microorganism journal, 2010,50 (4)：537-541；Liu Yan etc.,
Chinese leather, 2013,42 (23)：15-18).It has been reported that from Brevibacillus brevis Brevibacillus brevis
Depilation (the Jaouadi NZ of rabbit, goat skin, sheepskin, ox-hide can be completed in the keratinase KERUS independent roles of US575
et al.,Plos One,2013,8(10):e76722)。
Compared with common proteases, keratinase can be easier to wash off the keratinization waste that clothing surface speckles with, and can be used as
The additive of detergent.The alkali resistance of the application requirement protease is good, holding high activity is remained within the scope of wider temperature, this
Outside will also to protein stain have good washing effect, the ionic/nonionic surface active agent being resistant in detergent,
The other compositions such as oxidant, bleaching agent, enzyme and stabilization (Vojcic L the et al., New for keeping activity itself
Biotechnol,2015,32(6):342–348).Although the report that protease can be applied to detergent additives has more,
The application study of keratin enzyme in this regard is less.From Brevibacillus brevis Brevibacillus sp.AS-S10-II's
Keratinase is in EDTA, SDS, Triton X-100, Tween 20, H2O2In still high enzyme is kept to live, but with variety classes wash
After agent common insulation enzyme activity have in various degree reduce (Rai KR, Mukherjee AK.Biochem Eng are J.2011,54:
47-56)；Keratinase optimal pH from B.pumilus KS12 is 10, most suitable enzyme activity temperature 60 C, detergent saponin(e,
Sodium taurocholate, SDS, Triton X, Tween-80, oxidant H2O2, enzyme activity has different journeys in sodium hypochlorite and various brands detergent
Degree improves, but have no to the report of spot washing effect (Rajput R et al., Enzyme Research, 2010,2010:
7)。
So far, screen, excavate, research have keratinase production capacity microbial strains and continual exploitation this
A little bacterial strains productions have potential industry, business application potentiality keratinase resource, be still in keratinase research it is important, treat
It solves the problems, such as.
Invention content
Isolation and purification method and application the purpose of the present invention is to provide a kind of keratinase pass through purifying side of the invention
Method, 1L B.subtilis FJ-3-16 zymotic fluids can harvest the pure enzyme 16.59mg of keratinase Ker FJ, total enzyme activity 2989U.
Technical scheme is as follows：
A kind of isolation and purification method of keratinase, is as follows：
By bacillus subtilis Bacillus subtilis FJ-3-16 fermented and cultureds, crude enzyme liquid is obtained, uses mass fraction
After 40~70% ammonium sulphate gradient precipitation crude enzyme liquid, with pH 10.0,50mM Tris-HCl buffer solutions, which are resuspended, to be precipitated, thoroughly
Analysis；With pH 10.0,50mM Tris-HCl buffer solutions balance Q Sepharose fast-flow ion exchange columns, loading, enzyme activity
Peak is across in peak；Collecting has the component of enzyme activity, with pH 7.0, the dialysis of 50mM Tris-HCl buffer solutions；With pH 7.0,50mM
Tris-HCl buffer solutions balance SP Sepharose fast-flow ion exchange columns, loading, with 0~1M NaCl gradient elutions,
The component of enzyme activity is eluted at 0.25M NaCl to get keratinase Ker FJ after purification.
The purity (Fig. 1) for the keratinase Ker FJ that each purification step obtains is detected using SDS-PAGE.Through ammonium persulfate
Gradient precipitates and two-step solution chromatography, and we obtain electrophoretically pure keratinase Ker FJ.
In the present invention, bacillus subtilis Bacillus subtilis FJ-3-16 were preserved on 01 20th, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center, culture presevation CGMCC NO.12086, preservation address
For Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Classification And Nomenclature is Bacillus subtilis.
In the present invention, the fermentation medium of the crude enzyme liquid is：
FM3 culture mediums (gL-1)：Corn flour 20, soy meal 20, wheat bran 10, KH2PO41, K2HPO43, CaCl21, pH
7.5。
The condition of fermented and cultured is：Temperature is 30 DEG C, rotating speed 200rpm, cultivates 72h, and inoculum concentration is 6% (volume ratio).
After fermented and cultured, the method that obtains crude enzyme liquid：
With the dregs after filtered through gauze fermented and cultured, 11000rpm centrifugation 15min, it is thick enzyme to collect fermented liquid supernatant
Liquid.
In the present invention keratinase Ker FJ isolate and purify the specific steps are：
(1) bacillus subtilis Bacillus subtilis FJ-3-16 single bacterium colonies are seeded to LB liquid with transfer needle
In culture medium, 30 DEG C, 200rpm cultures 18h；
LB fluid nutrient mediums (gL-1)：Peptone 10, dusty yeast 5, NaCl 10, pH 7.5；
(2) the logarithmic phase seed liquor in LB fluid nutrient mediums is inoculated in FM3 culture mediums by 6% (volume ratio) inoculum concentration
In, 30 DEG C, 200rpm cultures 72h；
FM3 culture mediums (gL-1)：Corn flour 20, soy meal 20, wheat bran 10, KH2PO41, K2HPO43, CaCl21, pH
7.5；
(3) fall the culture medium dregs after step (2) culture with filtered through gauze, 11000rpm centrifugations 15min collects zymotic fluid
Supernatant is crude enzyme liquid；
(4) by bacillus subtilis Bacillus subtilis FJ-3-16 fermented and cultureds, crude enzyme liquid is obtained, uses quality
After the ammonium sulphate gradient that score is 40~70% precipitates crude enzyme liquid, with pH 10.0,50mM Tris-HCl buffer solutions be resuspended precipitation,
Dialysis；With pH 10.0,50mM Tris-HCl buffer solutions balance Q Sepharose fast-flow ion exchange columns, loading, enzyme
Peak living is across in peak；Collecting has the component of enzyme activity, with pH 7.0, the dialysis of 50mM Tris-HCl buffer solutions；With pH 7.0,
50mM Tris-HCl buffer solutions balance SP Sepharose fast-flow ion exchange columns, loading, with 0~1M NaCl gradients
Elution, elutes the component of enzyme activity to get keratinase Ker FJ after purification at 0.25M NaCl.
Compared with the prior art, the present invention has the following advantages：
In the prior art, the protease of various bacteria secretion is mostly to be combined by ion-exchange chromatography with sieve chromatography
Reach purifying purpose.Compared with sieve chromatography, ion-exchange chromatography is with applied sample amount is big, flow velocity is fast, it is short to take, operation letter
Just the advantages that sample concentration, collected is high.The present invention removes the colour component of crude enzyme liquid first with Q anion columns, recycles
SP post separation other impurities components and obtain pure enzyme, purification step is easy to operate, the rate of recovery is high, purification effect is good.By this hair
Bright purification process, 1L B.subtilis FJ-3-16 zymotic fluids can harvest the pure enzyme 16.59mg of keratinase Ker FJ, total enzyme activity
2989U.And the keratinase Ker FJ obtained animal waste degradation, depilation and as having in terms of detergent additives it is non-
Often good effect.
Description of the drawings
Fig. 1 is the SDS-PAGE detection figures for isolating and purifying keratinase Ker FJ；
Fig. 2 is the evolution location drawing of phylogenetic analysis bacterial strain B.subtilis FJ-3-16；
Fig. 3 is that keratinase Ker FJ scheme the expansion of natural keratin substrate feather and wool and degradation；
Fig. 4 is depilation figures of the keratinase Ker FJ to sheepskin and donkey hide；
Fig. 5 surveys remnant enzyme activity after keeping the temperature 1h, 6h, 12h in various brands detergent for keratinase Ker FJ；
Fig. 6 is keratinase Ker FJ as detergent additives, (catsup, ginger powder, sweet fermented flour sauce, peppery to different spots
Sauce.Bloodstain) clean effect figure.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But embodiment is only exemplary, does not form any restrictions to the scope of the present invention.Those skilled in the art should
It should be appreciated that the details and form of technical solution of the present invention can be repaiied without departing from the spirit and scope of the invention
Change or replace, but these modifications and replacement are each fallen in protection scope of the present invention.
The isolation and purification method of 1 keratinase of embodiment, is as follows：
(1) bacillus subtilis Bacillus subtilis FJ-3-16 single bacterium colonies are seeded to LB liquid with transfer needle
In culture medium, 30 DEG C, 200rpm cultures 18h；
LB fluid nutrient mediums (gL-1)：Peptone 10, dusty yeast 5, NaCl 10, pH 7.5；
(2) the logarithmic phase seed liquor in LB fluid nutrient mediums is inoculated in FM3 culture mediums by 6% (volume ratio) inoculum concentration
In, 30 DEG C, 200rpm cultures 72h；
FM3 culture mediums (gL-1)：Corn flour 20, soy meal 20, wheat bran 10, KH2PO41, K2HPO43, CaCl21, pH
7.5；
(3) fall the culture medium dregs after step (2) culture with filtered through gauze, 11000rpm centrifugations 15min collects crude enzyme liquid；
(4) after precipitating crude enzyme liquid with the ammonium sulphate gradient that mass fraction is 40~70%, with pH 10.0,50mM Tris-
Precipitation, dialysis is resuspended in HCl buffer solutions；With pH 10.0,50mM Tris-HCl buffer solutions balance Q Sepharose fast-flow
Ion exchange column, loading, enzyme activity peak is across in peak；Collecting has the component of enzyme activity, is buffered with pH 7.0,50mM Tris-HCl
Liquid is dialysed；With pH 7.0,50mM Tris-HCl buffer solutions balance SP Sepharose fast-flow ion exchange columns, loading,
With 0~1M NaCl gradient elutions, the component of enzyme activity is eluted at 0.25M NaCl to get keratinase Ker after purification
FJ。
Using the purity of SDS-PAGE detection keratinase Ker FJ, as shown in Figure 1, obtaining electrophoretically pure keratinase
Ker FJ。
Bacillus subtilis Bacillus subtilis FJ-3-16 stack feather waste for a long time from poultry dressing farm
It screens and obtains in soil, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on 01 20th, 2016
The heart, culture presevation CGMCC NO.12086, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and Classification And Nomenclature is
Bacillus subtilis。
Embodiment 2B.subtilis FJ-3-16 strain idenfications
LB fluid nutrient mediums (g/L)：Peptone 10.0, dusty yeast 5.0, NaCl 10.0, pH 7.5.Such as it is used for transformant
It screens, 50 μ g/mL ampicillins (Amp) of final concentration is added in culture medium.
LB solid mediums (g/L)：Peptone 10.0, dusty yeast 5.0, NaCl 10.0, agar 15.0, pH 7.5.
FJ-3-16 bacterial strain single bacterium colonies are inoculated in 5mL LB liquid mediums, 37 DEG C of overnight incubations.Take 1mL cultured
Bacterium solution extracts bacteria total DNA according to " hundred Tyke bacterial genomes extracts kits " specification.
Design 16S universal primers 27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5 '-
ACGGCTACCTTGTTACGACTT-3’).Using bacterial genomes as template, according to the PCR bodies in Taq archaeal dna polymerase specifications
System and program, PCR obtain DNA fragmentation.
PCR system is as shown in table 1, and PCR programs are as shown in table 2：
Table 1
Table 2
DNA fragmentation is recycled using DNA plastic recovery kits (Omega), operation is carried out by kit specification.Recycling
PCR product is connected to carrier after agarose gel electrophoresis detects, using Solution I ligases (the precious biology in Dalian)
On pMD19-T easy (the precious biology in Dalian) carrier.10 μ l of linked system：4 μ l, pMD19-T easy carriers of DNA fragmentation, 1 μ l,
Solution I 5μl.Condition of contact：16 DEG C, 8h.Connection product Transformed E .coli DH5 а competent cells, method for transformation are pressed
According to《Molecular Cloning:A Laboratory guide》(third edition) (Pehanorm Brooker, Russell write, 2002) illustrates to carry out.According to pMD19-T
Easy carrier sequences design primer M13F：CAGGAAACAGCTATGAC, M13R：GTTTTCCCAGTCACGA utilizes bacterium colony PCR
Method screens transformant, and PCR system and program are the same as above-mentioned.Transformant sequencing is complete in Bo Shang biotechnologys (Shanghai) Co., Ltd.
Into.
Sequencing measures 16S rRNA sequences 1514bp altogether.By sequence, BLAST compares analysis in ncbi database
(http://blast.ncbi.nlm.nih.gov/Blast.cgiPROGRAM=blastn＆PAGE_TYPE= BlastSearch＆LINK_LOC=blasthome), sequence identity it is higher be bacillus subtilis Bacillus
Subtilis, identity 99%.Utilize the 16S of the Clustalx X1.83 softwares bacterial strain higher to sequence identity
RRNA carry out multiple alignment, using 6.0 softwares of MEGA adjacent method (Neighbor-Joining Method) draw sequence into
Change tree, as shown in Figure 2.
The enzyme activity determination of 3 keratinase Ker FJ of embodiment
The crude enzyme liquid that 1ml has diluted is taken to add in the keratin substrates (TCI companies, K0044) that 1ml mass fractions are 2%,
2ml 0.4M TCA termination reactions are added in after reacting 1h at 55 DEG C, supernatant 1ml is taken after 11000rpm centrifugations 2min, adds in 5ml
0.4M Na2CO3With 1ml Folin reagents, after 40 DEG C of 20min that develop the color, in 660nm colorimetrics, in 660nm light absorption value liters per minute
High 0.1 is defined as 1 enzyme-activity unit (U).
Determination of protein concentration
(green skies biotechnology) is operated according to " BCA determination of protein concentration kit " specification, is obtained in the present invention
B.subtilis FJ-3-16 zymotic fluids, keratinase total enzyme activity 73820.16U in 1L.Utilize purification process of the present invention, 1L
B.subtilis FJ-3-16 zymotic fluids can harvest the pure enzyme 16.59mg of keratinase Ker FJ, total enzyme activity 2989U.
The application of keratinase Ker FJ that 3 present invention of embodiment obtains
1st, animal waste is degraded
Keratinase Ker FJ substrates are extensive, the casein (casein) that can degrade, soluble keratin (reference
Wawrzkiewi methods are made by oneself, Wawrzkiewicz K et al., J Med Vet Mycol, and 1987,25:261-268), plumage
Mao Fen, wool powder, reddish black keratin (Keratin azure, Sigma, K8500), keratin (TCI companies, K0044) etc..
Degradations of the keratinase Ker FJ to natural feather, wool：
Enzymatic hydrolysis condition：45 DEG C, 150rpm enzymolysis 48h.Unpasteurized wool, feather 2.5mg/ml, enzyme concentration 25U.Addition
β-ME mass concentrations are 0.1%.
Digest result：Keratinase Ker FJ have the feather under native state and wool very strong degradation, enzymolysis
The pinnule part of Shi Yumao is dissociated from pinna rachis, the fracture of hard pinna rachis, and wool ball becomes loose.In reducing agent β-ME (0.1%)
In the presence of degrade more thoroughly, degradation rate is increased to 80% by 30%, as shown in Figure 3.
2nd, depilation
Experiment condition：45 DEG C, 150rpm is digested for 24 hours.Donkey hide, sheepskin are cut into 5cm × 5cm fritters purchased from slaughterhouse；Enzyme is dense
Spend 45U.
Experimental result：Keratinase Ker FJ are notable to the hair removal effect of black donkey hide and Aries skin, and the enzyme does not have collagen
Enzymatic activity has faint elastase activity；The visual hide lines observed after losing hair or feathers is complete, pore is clear, the collagen of hide
Layer is not damaged, as shown in Figure 4.
3rd, as detergent additives
Keratinase Ker FJ are catalyzed the optimal pH 9.0~9.5 of keratin hydrolysis；PH has good stability, pH 6.0~
Enzyme activity is stablized in the range of 11.5, and when pH12.0 still keeps 90% enzyme activity.Most suitable 55 DEG C of the enzyme activity temperature of the enzyme；Good thermal stability,
Enzyme activity half-life period about 10.6h at 40 DEG C.The enzyme belongs to medium temperature basic keratins enzyme.Metal ions M n2+、K+、Na+、Si2+、Li2+、
Mg2+、Ca2+、Ba2+On enzyme activity without influence；5mM reducing agent DTT, DTNB, β-ME make enzyme activity improve 10%；Mass fraction is 1%
Detergent saponin(e, Triton X-100, Tween-80 cannot not only reduce enzyme activity, moreover it is possible to enzyme activity be made to improve 104~32.56%
Differ；Mass fraction is 1~3% oxidant H2O2Enzyme activity is acted on without reduction.It is above-mentioned statistics indicate that keratinase Ker FJ
Anti-adversity is excellent.
(1) experiment condition：Keratinase Ker FJ are diluted to suitable concentration, with the detergent 1 that mass fraction is 2%:1 is mixed
It closes, enzyme concentration 40U/ml after mixing, after being incubated at room temperature 1h, 6h, 12h, remnant enzyme activity is surveyed using Folin- phenol method.With not with
Enzyme activity during the keratinase Ker FJ heat preservation 1h of detergent mixing is as 100%.
Experimental result：Keratinase Ker FJ commercial liquid detergent it is profound, it is vertical it is white, open rice, green wave, blue moon, peace
In profit, 1~12h of incubation at room temperature enzyme activity does not reduce not only, is increased instead；1h is kept the temperature in super, white cat to remain to remain
More than 95% enzyme activity；Enzyme activity reduces in carving board, Tide, as shown in Figure 5.
(2) experiment condition：Green wave detergent concentration 1%, enzyme concentration 9.6U/ml, wash conditions：30 DEG C, 150rpm washings
1h。
Experimental result：Compared with not adding the green wave that keratinase Ker FJ mass fractions are 1%, it is added to the washing of enzyme
When washing catsup, ginger powder, sweet fermented flour sauce, thick chilli sauce, bloodstain when spots, washing effect is more preferable, as shown in Figure 6 for agent.
The above results show that keratinase Ker FJ have application potential of the exploitation into detergent additives.
Claims (8)
1. a kind of isolation and purification method of keratinase, step are as follows：
By bacillus subtilis Bacillus subtilis FJ-3-16 fermented and cultureds, crude enzyme liquid is obtained, is 40 with mass fraction
After~70% ammonium sulphate gradient precipitation crude enzyme liquid, with pH 10.0, precipitation, dialysis is resuspended in 50mM Tris-HCl buffer solutions；With
PH 10.0,50mM Tris-HCl buffer solutions balance Q Sepharose fast-flow ion exchange columns, and loading, enzyme activity peak exists
Across in peak；Collecting has the component of enzyme activity, with pH 7.0, the dialysis of 50mM Tris-HCl buffer solutions；With pH 7.0,50mM
Tris-HCl buffer solutions balance SP Sepharose fast-flow ion exchange columns, loading, with 0~1M NaCl gradient elutions,
The component of enzyme activity is eluted at 0.25M NaCl to get keratinase Ker FJ after purification；
The bacillus subtilis Bacillus subtilis FJ-3-16 were preserved in China Microbiological on 01 20th, 2016
Culture presevation administration committee common micro-organisms center, culture presevation CGMCC NO.12086, preservation address are court of Beijing
The positive institute 3 of area's North Star West Road 1, Classification And Nomenclature are Bacillus subtilis；
The fermentation medium of the crude enzyme liquid is：
FM3 culture mediums (gL-1)：Corn flour 20, soy meal 20, wheat bran 10, KH2PO41, K2HPO43, CaCl21, pH 7.5.
2. according to the method described in claim 1, it is characterized in that, the condition of crude enzyme liquid fermented and cultured is：Temperature is 30 DEG C, is turned
Speed is 200rpm, cultivates 72h.
3. according to the method described in claim 2, it is characterized in that, inoculum concentration is 6% (volume ratio) during crude enzyme liquid fermented and cultured.
4. the according to the method described in claim 3, it is characterized in that, preparation method of the crude enzyme liquid：
With the dregs after filtered through gauze fermented and cultured, 11000rpm centrifugation 15min, it is crude enzyme liquid to collect fermented liquid supernatant.
5. according to the method described in claim 1, it is characterized in that, the keratinase isolate and purify the specific steps are：
(1) bacillus subtilis Bacillus subtilis FJ-3-16 single bacterium colonies are seeded to LB Liquid Cultures with transfer needle
In base, 30 DEG C, 200rpm cultures 18h；
LB fluid nutrient mediums (gL-1)：Peptone 10, dusty yeast 5, NaCl 10, pH 7.5；
(2) the logarithmic phase seed liquor in LB fluid nutrient mediums is inoculated in by 6% (volume ratio) inoculum concentration in FM3 culture mediums, 30
DEG C, 200rpm cultures 72h；
FM3 culture mediums (gL-1)：Corn flour 20, soy meal 20, wheat bran 10, KH2PO41, K2HPO43, CaCl21, pH 7.5；
(3) fall the culture medium dregs after step (2) culture with filtered through gauze, 11000rpm centrifugations 15min collects fermented liquid supernatant
As crude enzyme liquid；
(4) bacillus subtilis Bacillus subtilis FJ-3-16 fermented and cultureds are obtained into crude enzyme liquid, is with mass fraction
After 40~70% ammonium sulphate gradient precipitation, with pH 10.0, precipitation, dialysis is resuspended in 50mM Tris-HCl buffer solutions；Use pH
10.0,50mM Tris-HCl buffer solutions balance Q Sepharose fast-flow ion exchange columns, loading, enzyme activity peak across
In peak；Collecting has the component of enzyme activity, with pH 7.0, the dialysis of 50mM Tris-HCl buffer solutions；With pH 7.0,50mM Tris-HCl
Buffer solution balances SP Sepharose fast-flow ion exchange columns, loading, with 0~1M NaCl gradient elutions, in 0.25M
The component of enzyme activity is eluted at NaCl to get keratinase Ker FJ after purification.
6. application of the keratinase that such as prepared by any one of claim 1-5 the methods in terms of animal waste degradation.
7. application of the keratinase that such as prepared by any one of claim 1-5 the methods in terms of depilation.
8. application of the keratinase that such as prepared by any one of claim 1-5 the methods in terms of as detergent additives.
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| CN113341135A (en) * | 2018-07-30 | 2021-09-03 | 杭州莱和生物技术有限公司 | Kit for detecting trace amount of poison in hair by fluorescence immunoassay, and image detection method and system thereof |
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| CN114540252B (en) * | 2022-03-31 | 2022-09-13 | 山东省农业科学院 | Microbacterium P6 for converting livestock and poultry breeding waste and application |
| CN114540253B (en) * | 2022-03-31 | 2022-10-11 | 山东省农业科学院 | Application of bacillus pumilus P6 keratinase in detergent and application method |
| CN114574399B (en) * | 2022-04-01 | 2022-08-19 | 山东省农业科学院 | Application of Empedobacter brevis LY-5 keratinase in washing products |
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