CN107868762A - Produce bacillus cereus and its application of keratinase - Google Patents
Produce bacillus cereus and its application of keratinase Download PDFInfo
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- CN107868762A CN107868762A CN201711145653.9A CN201711145653A CN107868762A CN 107868762 A CN107868762 A CN 107868762A CN 201711145653 A CN201711145653 A CN 201711145653A CN 107868762 A CN107868762 A CN 107868762A
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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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/085—Bacillus cereus
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- 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
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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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
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention belongs to microorganism field, and in particular to the bacillus cereus of efficient degradation feather keratin（Bacillus cereus）The bacterial strains of Y 15, further relate to the application of bacterial strain.The bacillus cereus of the present invention（Bacillus cereus）Y 15, the bacterial strain were preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, culture presevation CGMCC NO.14221 on June 6th, 2017, and the colonial morphology in milk screening flat board is：Bacterium colony is white, and circular, surface wax is opaque, marginal wavy；Gram's staining is the positive, and thalline is in shaft-like.The bacillus cereus that the present invention is screened and cultivated（Bacillus cereus）The bacterial strain keratin degradation effects of Y 15 are excellent.
Description
Technical field
The invention belongs to microorganism field, and in particular to produce the bacillus cereus (Bacillus of keratinase
Cereus) Y-15 bacterial strains, the application of bacterial strain is further related to.
Background technology
Feather can cause the soil erosion, Heavy Metal Accumulation, disease as poultry farming, the accessory substance of butchery, phase stacking
Pathogenic microorganism amount reproduction, pernicious gas release, bring serious environmental problem.Meanwhile feather strucutre densification is complicated, is difficult to drop
Solution, difficulty is brought to offal treatment.The methods of traditional landfill, burning, alkali process, is handled feather waste, is turned
Change not only both expensive, cause secondary pollution, also creating several amino acids in conversion process is destroyed.Micro- life is utilized in recent years
Keratin discarded object progress harmless treatment becomes a kind of efficient, low energy consumption, ring in a mild condition for thing and keratinase
The method for transformation of guarantor.Exploitation can not only realize discarded object for the efficient keratinase preparation of animal waste processing
Harmless treatment, reduce environmental problem, while the higher value application of discarded object can also be realized, alleviate protein feed resource
The predicament of shortage.
Report including bacillus licheniformis, bacillus subtilis, Bacillus circulans, bacillus pumilus and waxy bud
More bacillus category bacteriums including spore bacillus (Bacillus cereus, B.cereus) etc. are all accredited as keratin drop
Solve bacterium (Critical Reviews in Biotechnology, 2014,34 (4):372-384.).In recent years about waxy bud
The report of spore bacillus keratin degraded has：B.cereus IZ-06b and B.cereus IZ-06r can make in wool/peptone
Grown in culture medium for sole carbon source/nitrogen source, the scarcity of peptide substrates can induce thalline to secrete extracellular clostridiopetidase A, bullet in culture medium
Property protease and keratinase (Journal of Applied Microbiology, 2009,107 (1):226–234.)；
B.cereus Wu2 growth and breeding and can secrete keratinase and two sulphur in feather is as sole carbon source and the culture medium of nitrogen source
Key reductase carrys out degradation of feather, and amino acid content rises (Journal of Bioscience and in hydrolysate
Bioengineering,2012,114(6): 640-647.)；B.cereus B5esz and B.cereus PCM 2849 are degradable
Bristles hair (the International Biodeterioration and treated in advance by sulphite
Biodegradation,2015,100:116-123； Waste and Biomass Valorization,2016,8(2):1-
11.).The country also reports degraded (patents of the B.cereus J2 for being isolated from plant's soil to feather
ZL201310716495.3) and be isolated from snake alimentary canal content B.cereus YSQ08 restructuring keratinase expression,
Zymologic property (modern food science and technology, 2016 (12):105-112.), B.cereus YSQ08 bacterium and enzyme collective effect are degraded
Research (feed wide-angle, 2016 (19) of feather:46-49.).
Although having screened a large amount of keratin degrading bacterias, comparatively strain enzyme-producing efficiency is not very high, protoenzyme
Yield far can not meet the needs of animal waste Efficient Conversion, need the angle egg of screening and identification with higher vigor badly
White enzyme resource.Only have BioResource-International.Inc. companies at presentWithAngle
Protease preparation is applied to the processing of keratin discarded object, creates more than 1,000 ten thousand dollars of income (Applied every year
Microbiology and Biotechnology,2013,97(23): 9931-9940.).Both enzyme preparations are by lichens
Bacillus PWD-1 keratinase KerA transformations (Applied＆Environmental Microbiology, 1995,
61(4):1469-74.).For the present Research, need badly and screen a kind of bacterial strain of high yield keratinase, and optimize its fermentation
Condition, further improve its yield of enzyme and enzyme activity, carry forward vigorously the research and development process of enzyme preparation, make its degraded keratin and
The significant effect in the material containing keratin of degrading.
The content of the invention
In order to solve above-mentioned technical problem, the present invention has screened one plant of production keratinase, keratin with stronger
Bacillus cereus (Bacillus cereus) Y-15 of degradation capability, it is micro- that the bacterial strain is preserved in China on June 6th, 2017
Biological inoculum preservation administration committee common micro-organisms center, culture presevation CGMCC NO.14221, bacillus cereus
Repeatedly still producing enzyme is stable for (Bacillus cereus) Y-15 continuous passages, and hydrolyzes circle/colony diameter>3.0, it is one plant of albumen
Enzyme superior strain；Colonial morphology in milk screening flat board is：Bacterium colony is white, and circular, surface wax is opaque, edge wave
Shape, Gram's staining are the positive, and thalline is in shaft-like.
Microbial inoculum containing above-mentioned bacillus cereus (Bacillus cereus) Y-15, and claimed by the present invention
Content.
It is bacillus cereus (Bacillus cereus) Y-15 answering in keratin of degrading that the present invention is also to be protected
With, and, applications of bacillus cereus (Bacillus cereus) Y-15 in the material containing keratin of degrading, contain
The material of keratin can be wool, camel hair, bristles, human hair, fowl plumage and animal hair.
Above-mentioned application, by bacillus cereus (Bacillus cereus) Y-15 strain fermentation supernatant crude enzyme liquids and angle
Albumen or the feather progress reaction treatment containing keratin.
Above-mentioned bacillus cereus (Bacillus cereus) Y-15, the fermentative medium formula that it optimizes is by weight
Percentage is：Corn flour 2.23%, wheat bran 2%, dregs of beans 0.88%, K2HPO40.1%, KH2PO40.1%, CaCl2
0.22%.
Keratin in bacillus cereus (Bacillus cereus) Y-15 strains for degrading feathers provided by the present invention
Method, including following step：
(1) by bacillus cereus (Bacillus cereus) Y-15 logarithmic phases bacterium solution by volume 2% ratio be inoculated with
Amount is inoculated in FM culture mediums；
FM culture mediums are obtained by following step：NaCl 0.05%, MgCl are taken by following percentage by weight20.01%,
CaCl20.006%, K2HPO40.1%, KH2PO40.04%, then in pH 7.5,1 × 105Sterilized under conditions of Pa
20min, add feather 1%；
Then at 30 DEG C, concussion and cultivate under 180r/min；
(2) metamorphosis after feather enzymolysis is observed, calculates degradation rate, while determine thalli growth and born of the same parents in FM culture mediums
During keratinase enzyme activity determination, light absorption value per minute under 660nm wavelength is raised with the variation relation of incubation time for outer enzyme activity
0.1 is defined as 1 enzyme-activity unit U.
The 16srDNA nucleotide sequences such as sequence table NO.1 of bacillus cereus (Bacillus cereus) Y-15 bacterial strains
It is shown.
Brief description of the drawings
Fig. 1 is for culture B.cereus Y-15 growth curve, producing enzyme curve of the invention in FM culture mediums and to feather
The degradation curve figure of keratin；
Fig. 2 is visually observation B.cereusY-15 degradation of feather effects；
Fig. 3 is the structure change after scanning electron microscopic observation feather enzymolysis；
Fig. 4 is the response surface design figure that different factors produce keratinase reciprocal effect to B.cereus Y-15；
Fig. 5 is B.cereus Y-15 crude enzyme liquid degradation of feather design sketch.
Embodiment
The present invention is further described with reference to the accompanying drawings and detailed description, so as to the technology of this area
Personnel know more about the present invention, but do not limit the present invention with this.
Bacillus cereus (Bacillus cereus) Y-15 bacterial strains be abbreviated as in this application Y-15 bacterium or
B.cereus Y-15；
Embodiment 1
Screening, purifying, identification and the preservation of bacterial strain
1.1 bacterial strain screenings and purifying
Mud sample is picked up from the mud that Shandong food factory of Ningyang sewage draining exit nearby submerges feather residue for a long time.Weigh 1 g silts
Mud sample product, be put into contain 10mL sterile salines and with bead triangular flask in, shaking makes mud sample be sufficiently mixed with water.Take
1mL suspensions are added in the test tube for filling 9mL sterile salines and fully mixed, and this is 10-1Dilution.10 are made by that analogy-2
~10-6The solution of several dilution factors.The solution example of above-mentioned different dilution factors is taken into 100 μ L coating milk screening flat board (10%
After 116 DEG C of skimmed milk power sterilizing 30min with beef-protein medium (beef extract 0.5%, peptone 1%, NaCl
0.5%, pH 7.5) according to 1:9 ratios are mixed, and plate is down flat after mixing), flat board is inverted in 30 DEG C of incubators and cultivates 24h.
Select grown fine on flat board, hydrolyzing circle, clearly bacterium colony continuous line in milk screening flat board purifies for 3 times to bacterial strain,
Purified bacterial strain microscopy be single bacterium colony after, glycerol tube is made and is stored in -80 DEG C.
25 plants of the bacterial strain of production protease must be stablized by being sieved altogether using the method for being coated with milk screening flat board, and bacterial strain Y-15 is continuous
Repeatedly still producing enzyme is stable for passage, and hydrolyzes circle/colony diameter>3.0, it is one plant of proteinase high-yield bacterial strain.Bacterial strain Y-15 is in milk
Colonial morphology in screening flat board is：Bacterium colony is white, and circular, surface wax is opaque, marginal wavy.Gram's staining is sun
Property, thalline is in shaft-like.
1.2 strain idenfication
The cultured Y-15 bacterium solutions of 1ml are taken, bacterium is extracted according to " hundred Tyke bacterial genomes extracts kits " specification
STb gene.Design 16S universal primers, using bacterial genomes as template, according to the PCR system in Taq archaeal dna polymerase specifications and
Program, PCR obtain DNA fragmentation.DNA fragmentation is reclaimed using DNA glue reclaims kit.Using Solution I ligases by DNA
Fragment is connected on carrier pMD19-T easy carriers, connection product Transformed E .coli DH5 α competent cells.Picking converts
Son, transformant sequencing are completed in Shanghai Bo Shang Bioisystech Co., Ltd.Using EzBioCloud 16S databases to 16S
RRNA sequencing results are analyzed.
16S rRNA sequence 1414bp are measured altogether through sequencing, and sequence is submitted to NCBI GenBank databases, sequence
Row number is MF797956, and NO.1 is shown in sequence table.Ezbiocloud database analysises show bacterial strain Y-15 16S rRNA sequences with
The type strains of B.cereus ATCC 14579 (No. GenBank：AE016877.1 16S rRNA sequences) are closest, similitude
For 99.72%, show that Y-15 is likely to one plant of B.cereus bacterial strain, be named as B.cereus Y-15.
1.3 culture presevation
Bacillus cereus (Bacillus cereus) Y-15 bacterial strains, preservation date are：On June 6th, 2017, preservation list
Position：China Committee for Culture Collection of Microorganisms's common micro-organisms center, culture presevation CGMCC NO.14221, preservation
Address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and bacillus cereus (Bacillus cereus) Y-15 of preservation connects
Resuming generation, repeatedly still producing enzyme is stable, and hydrolyzes circle/colony diameter>3.0, it is one plant of proteinase high-yield bacterial strain；It is flat in milk screening
Colonial morphology on plate is：Bacterium colony is white, and circular, surface wax is opaque, marginal wavy, and Gram's staining is positive, bacterium
Body is in shaft-like.
Embodiment 2
Crude enzyme liquid extracts and the degraded to feather
In culture medium after B.cereus Y-15 seed liquors are optimized by 2% inoculum concentration access, at 30 DEG C,
After 180r/min cultures 48h, centrifugation goes thalline to obtain crude enzyme liquid.1L culture mediums can obtain crude enzyme liquid total enzyme activity 11000U.Thick enzyme
Liquid handles feather at 45 DEG C under the conditions of 150r/min, the feather overall volume after visible enzymolysis is visually observed after 24h and is diminished, plumage
Gross weight is reduced, and solution is become cloudy by clarifying.Feather strucutre destroys, and pinna rachis curls, and the pinnule of most of feather is all degraded disappearance,
Nubbin pinna rachis (accompanying drawing 5).Feather waste degradation rate is 50% under the experiment condition, and degradation effect is notable, is shown thick
Enzyme liquid degradation of feather has good application prospect.
Embodiment 3
B.cereus Y-15 optimization culture conditions
With reference to Tiwary and Gupta method (Bioresource Technology, 2010,101 (15): 6103-
6110.) feather protein peptone culture medium (Feather peptone medium, FM1) (feather meal 0.5%, peptone is prepared
0.5%, glucose 1%, K2HPO40.3%, KH2PO40.1%, pH 7.5,1 × 105Pa sterilizings 20min).By B.cereus
Y-15 seed liquors are by 2% inoculum concentration access FM1 culture mediums, and at 30 DEG C, 180 r/min cultures 48h determines zymotic fluid keratin
Enzyme enzyme activity is 0.52U/mL.
Comprehensive utilization experiment of single factor, PB designs and center combination design to carry out B.cereus Y-15 condition of enzyme production
Optimization.
3.1 experiment of single factor
Select cultivation temperature (30 DEG C, 37 DEG C), medium pH (6.0~10.0,1 pH gradient of every 1.0 setting), inoculum concentration
(volume ratio) (2%, 4%, 6%, 8%), 9 kinds culture bacterium common carbon source (maltose, sucrose, fructose, galactolipin, starch,
Glycerine, mannitol, corn flour, wheat bran, 1% glucose in FM1 culture mediums is replaced respectively by 1% addition), 9 kinds culture bacteriums
Common nitrogen source (NH4Cl、(NH4)2SO4, urea, dusty yeast, beef extract, fructus hordei germinatus leaching powder, casein, dregs of beans, bean powder, by 0.5%
Addition replaces 0.5% peptone in FM1 culture mediums respectively) as medium exchange to be optimized, seed liquor is pressed these factors
2% inoculum concentration is respectively connected to culture medium, and 48h measure zymotic fluid keratinase enzyme activity is cultivated at 30 DEG C.
B.cereus Y-15 extracellular keratinase enzyme activity when cultivating 48h for 30 DEG C is 0.52U/mL, when cultivating for 37 DEG C
Enzyme activity is 0.26U/mL, and the former improves 1 times than the latter.Therefore 30 DEG C are more conducive to B.cereus Y-15 producing enzymes.
B.cereus Y-15 producing enzyme effects in pH 8.0 are best, and ectoenzyme enzyme activity is 0.65U/ml, bacterium during pH 7.0
Bacterial strain ectoenzyme enzyme activity is highest enzyme activity when strain ectoenzyme enzyme activity is 81.36%, the pH 9.0 and pH 10.0 of highest enzyme activity
Enzymatic activities are only the 13% of highest enzyme activity when 50%, pH 6.0.The above results show that B.cereus Y-15 are more suitable in neutrality
Producing enzyme under conditions of meta-alkalescence, too sour and too alkali environment are unfavorable for strain secretes keratinase.
With the increase of inoculum concentration, B.cereus Y-15 yield of enzyme successively decreases, the keratin of 4%, 6%, 8% inoculum concentration
Enzyme enzyme activity is followed successively by 87.76%, 71.43%, the 55.10% of 2% inoculum concentration.Therefore 2% inoculum concentration is selected in subsequent experimental.
Carbon source single factor experiment result shows that B.cereus Y-15 producing enzymes in the culture medium that fructose does carbon source are best, born of the same parents
Outer enzyme activity is 0.72U/mL, and the enzyme activity is defined as into highest enzyme activity 100%.Corn flour effect is close with fructose, is 0.70U/mL.
The order that remaining carbon source influences on producing enzyme is maltose (89.23%)>Wheat bran (80%)>Sucrose (67.69%)>Glucose
(66.15%)>Starch (63.08%)>Glycerine (55.38%)>Mannitol (53.85%)>Galactolipin (27.69%).Corn flour
More there is a price advantage than fructose, selecting corn flour to do carbon source can substantially reduce with enzyme cost, therefore subsequent experimental selection corn flour
As carbon source.
Nitrogen source single factor experiment result shows that B.cereus Y-15 producing enzymes in the culture medium that dregs of beans does nitrogen source are best, born of the same parents
Outer enzyme activity is 1.375U/mL, and the enzyme activity is defined as into highest enzyme activity 100%.The order that remaining nitrogen source influences on producing enzyme is beef
Cream (82.4%)>Urea (78.4%)>Bean powder (62.4%)>Casein (48.8%)>Dusty yeast (47.2%)>(NH4)2SO4
(46.4%)>Fructus hordei germinatus leaching powder (44.8%)>NH4Cl (44%)>Peptone (42.4%).From result, original FM1 cultures
Peptone, which does nitrogen source, in base can not promote the fine producing enzymes of B.cereus Y-15, and the bacterium yield of enzyme can be made by doing nitrogen source using dregs of beans
1.36 times are improved, and dregs of beans is cheap and easy to get, can substantially reduce with enzyme cost.Therefore selection dregs of beans does nitrogen source in subsequent experimental.
3.2PB design
Select corn flour, wheat bran, dregs of beans, feather meal, NaCl, CaCl2、K2HPO4This 7 factors, utilize Design
The PB design programs of expert 8.0.6 softwares, each factor of analysis and evaluation produce the influence of keratinase to bacterial strain Y-15.
Using the keratinase enzyme activity Input Software measured as response.Understood by software analysis, 7 influence factors
In, in experiment setting range of variables, corn flour, wheat bran, CaCl in culture medium2, NaCl additions to B.cereus Y-15 produce
Enzyme has positive role, wherein corn flour, wheat bran, CaCl2Positive role extremely significantly (p<0.01), NaCl positive roles do not show
Write (p>0.1)；Dregs of beans, feather meal and K2HPO4There are negative consequence, wherein dregs of beans and K to B.cereus Y-15 producing enzymes2HPO4's
Negative consequence extremely significantly (p<0.01), the not notable (p of feather meal negative consequence>0.1).
3.3 center combination design
Select corn flour, dregs of beans and CaCl23 significantly affect factor, are entered using Design expert 8.0.6 softwares
Row center combination design Optimum Experiment.Polynomial regression fit calculating and analysis are carried out using software, obtains formula：Y=+8.22
+0.30A-0.64B+0.40C-0.49AB+0.046AC-0.60BC-0.75A2-0.48B2-0.48 C2
Wherein Y represents the enzyme activity (U/mL) in zymotic fluid, and A represents corn flour (%), and B represents dregs of beans (%), and C is represented
CaCl2(%).The p value of the model be 0.0004 (<0.05) it is extremely significant, to show model；Wherein A corn flour p value 0.0542>
0.05, corn flour influences not notable, B dregs of beans p value 0.0010 and C CaCl to B.cereus Y-15 producing enzymes2P value 0.0165 is equal<
0.05, it is very notable to show that both influence on B.cereus Y-15 producing enzymes.It is 0.2307 that the model, which loses plan item p value, shows to lose and intends item
It is not notable.Coefficient of determination R2For 0.9098, represent that this model is capable of the 90.98% of explanatory variable response.AB (corn flour,
Dregs of beans) and BC (dregs of beans, CaCl2) interaction on B.cereus Y-15 producing enzymes influence it is very notable, p value is equal<0.05.AB and
BC corresponding surface figure is as shown in Figure 4.
By center combination design obtain B.cereus Y-15 producing enzymes optimum formula be：Corn flour 2.23%, wheat bran
2%, dregs of beans 0.88%, K2HPO40.1%, KH2PO40.1%, CaCl20.22%, pH 8.0.Formula production keratinase
Enzyme activity can reach 9.698U/mL in theory.Actually measured enzyme activity is 10.168 U/mL in experiment, is approached with predicted value, explanation
It is rationally reliable to optimize obtained model, being capable of prognostic experiment result exactly.By single factor experiment, PB designs and center combination
Design, the culture medium after optimization improve nearly 20 times than former FM1 culture mediums yield of enzyme.
Embodiment 4
Bacillus cereus (Bacillus cereus) Y-15 strains for degrading feather wastes
It is that Y-15 is (hereinafter abbreviated as bacillus cereus (Bacillus cereus) by B.cereus Y-15
B.cereus Y-15) logarithmic phase bacterium solution is inoculated in FM culture mediums (NaCl 0.05%, MgCl by 2% (volume ratio) inoculum concentration2
0.01%, CaCl20.006%, K2HPO40.1%, KH2PO40.04%, pH 7.5,1 × 105Added after Pa sterilizings 20min
Feather 1%), 30 DEG C, 180r/min concussion and cultivates, the metamorphosis after feather enzymolysis is visually observed, according to the side such as Benkiar A
Method calculates degradation rate (International Biodeterioration and Biodegradation, 2013,83 (6):
129-138.), while thalli growth and enzymatic activities are determined in FM culture mediums with the situation of change of incubation time.Keratinase enzyme
It is living according to measure the methods of Fang Z (Process Biochemistry, 2014,49:647-654.), will be every under 660nm wavelength
Minute light absorption value rise 0.1 is defined as 1 enzyme-activity unit (U).
The specific method of keratinase enzyme activity is shown in Fang Z, Zhang J, Liu BH, et al.Cloning,
heterologous expression and characterization of two keratinases from
Stenotrophomonas maltophilia BBE11-1[J].Process Biochemistry,2014,49: 647-
654。
B.cereus Y-15, which are inoculated in after FM culture mediums, to be grown rapid, and 6h enters exponential phase, 12h be afterwards into
Enter plateau；Extracellular keratinase enzyme activity reaches highest when cultivating 36h, and now the degradation rate to feather is about 40%.Afterwards
Enzyme activity is gradually reduced in culture medium, but with the growth of incubation time and increasing for bacterium amount, the degradation rate of feather is progressively being carried
It is high.Bacterial strain Y-15 is about 70% to the degradation rate of feather when cultivating 48h, cultivates to 96h degradation rates and has more reached 90% (accompanying drawing
1).Visually the feather after observation enzymolysis becomes imperfect, and volume diminishes, and pinnule shortens, and some pinnules come off from pinna rachis, have
Pinna rachis bar fractures (accompanying drawing 2).Scanning electron microscopic observation shows that the pinnule after enzymolysis attenuates, and becomes because surface scale is degraded
It is coarse, there is wire drawing；Plumage sprig quantity reduces, shortened；Pinna rachis attenuates, and surface texture is seriously damaged, and reels off raw silk from cocoons
Phenomenon (accompanying drawing 3).The above results show that B.cereus Y-15 bacterial strains can efficiently be digested to feather keratin.
Claims (9)
1. the bacillus cereus of one plant of production keratinase（Bacillus cereus）Y-15, the bacterial strain was on June 6th, 2017
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, culture presevation CGMCC NO.14221, wax
Sample bacillus（Bacillus cereus）Colonial morphologies of the Y-15 in milk screening flat board be：Bacterium colony is white, circular,
Surface wax is opaque, marginal wavy；Gram's staining is the positive, and thalline is in shaft-like.
2. contain bacillus cereus as claimed in claim 1（Bacillus cereus）Y-15 microbial inoculum.
3. bacillus cereus（Bacillus cereus）Applications of the Y-15 in keratin of degrading.
4. bacillus cereus（Bacillus cereus）Applications of the Y-15 in the material containing keratin of degrading.
5. the application described in claim 3 or 4, it is by bacillus cereus（Bacillus cereus）Y-15 bacterial strains are sent out
Material of the ferment supernatant crude enzyme liquid with keratin or containing keratin carries out reaction treatment.
6. application as claimed in claim 4, the material containing keratin include wool, camel hair, bristles, human hair, fowl plumage and beast
Hair.
7. bacillus cereus as claimed in claim 1（Bacillus cereus）Y-15, its fermented and cultured basigamy optimized
Side is by weight percentage：Corn flour 2.23%, wheat bran 2%, dregs of beans 0.88%, K2HPO40.1%, KH2PO40.1%, CaCl2
0.22%。
8. the bacillus cereus in claim 1（Bacillus cereus）Keratin in Y-15 strains for degrading feathers
Method, including following steps：
（1）By bacillus cereus（Bacillus cereus）Y-15 logarithmic phases bacterium solution by volume 2% ratio inoculum concentration connect
Kind is in FM culture mediums；
FM culture mediums are obtained by following step：NaCl 0.05%, MgCl are taken by following percentage by weight20.01%, CaCl2
0.006%, K2HPO40.1%, KH2PO40.04%, then in pH 7.5,1 × 105Sterilize 20 min under conditions of Pa, adds
Feather 1%；
Then the concussion and cultivate under 30 DEG C, 180 r/min；
（2）The metamorphosis after feather enzymolysis is observed, calculates degradation rate, while determine thalli growth and ectoenzyme in FM culture mediums
The variation relation with incubation time living, during keratinase enzyme activity determination, light absorption value rise 0.1 per minute under 660 nm wavelength is determined
Justice is 1 enzyme-activity unit U.
9. the bacillus cereus in claim 1（Bacillus cereus）The 16srDNA nucleotide sequences of Y-15 bacterial strains are such as
Shown in sequence table NO.1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711145653.9A CN107868762A (en) | 2017-11-17 | 2017-11-17 | Produce bacillus cereus and its application of keratinase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711145653.9A CN107868762A (en) | 2017-11-17 | 2017-11-17 | Produce bacillus cereus and its application of keratinase |
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| Publication Number | Publication Date |
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| CN108795814A (en) * | 2018-06-25 | 2018-11-13 | 信阳师范学院 | A kind of bacterial strain, screening technique and its application of degradable waste feathers |
| CN108823191A (en) * | 2018-06-13 | 2018-11-16 | 天津科技大学 | A kind of fermentation process for improving bacillus and producing protease |
| CN109055280A (en) * | 2018-09-25 | 2018-12-21 | 山东理工大学 | A kind of keratinase superior strain and its application |
| CN109797145A (en) * | 2019-03-28 | 2019-05-24 | 信阳师范学院 | A method of improving keratinase vigor |
| CN110218664A (en) * | 2019-05-10 | 2019-09-10 | 博益德（北京）生物科技有限公司 | The bacterial strain of one high-efficiency degradation feather and its application |
| CN110607261A (en) * | 2019-09-24 | 2019-12-24 | 中国农业科学院饲料研究所 | Bacillus cereus capable of efficiently degrading feathers and application thereof |
| CN111424026A (en) * | 2020-04-22 | 2020-07-17 | 江南大学 | Method for producing keratinase |
| CN112126536A (en) * | 2020-07-07 | 2020-12-25 | 山东省农业科学院生物技术研究中心 | Application and application method of bacillus cereus keratinase |
| CN113980832A (en) * | 2021-09-22 | 2022-01-28 | 山东大学 | Strain capable of efficiently degrading keratin and application thereof |
| CN114540252A (en) * | 2022-03-31 | 2022-05-27 | 山东省农业科学院 | Microbacterium P6 for converting livestock and poultry breeding waste and application |
| CN114540253A (en) * | 2022-03-31 | 2022-05-27 | 山东省农业科学院 | Application of bacillus pumilus P6 keratinase in detergent and application method |
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| CN108823191B (en) * | 2018-06-13 | 2021-01-22 | 天津科技大学 | Fermentation method for improving protease production of bacillus |
| CN108823191A (en) * | 2018-06-13 | 2018-11-16 | 天津科技大学 | A kind of fermentation process for improving bacillus and producing protease |
| CN108795814A (en) * | 2018-06-25 | 2018-11-13 | 信阳师范学院 | A kind of bacterial strain, screening technique and its application of degradable waste feathers |
| CN108795814B (en) * | 2018-06-25 | 2021-08-27 | 信阳师范学院 | Bacterial strain capable of degrading waste feather, screening method and application thereof |
| CN109055280A (en) * | 2018-09-25 | 2018-12-21 | 山东理工大学 | A kind of keratinase superior strain and its application |
| CN109055280B (en) * | 2018-09-25 | 2022-01-18 | 山东理工大学 | Keratinase high-yield strain and application thereof |
| CN109797145A (en) * | 2019-03-28 | 2019-05-24 | 信阳师范学院 | A method of improving keratinase vigor |
| CN110218664A (en) * | 2019-05-10 | 2019-09-10 | 博益德（北京）生物科技有限公司 | The bacterial strain of one high-efficiency degradation feather and its application |
| CN110607261A (en) * | 2019-09-24 | 2019-12-24 | 中国农业科学院饲料研究所 | Bacillus cereus capable of efficiently degrading feathers and application thereof |
| CN111424026A (en) * | 2020-04-22 | 2020-07-17 | 江南大学 | Method for producing keratinase |
| CN112126536A (en) * | 2020-07-07 | 2020-12-25 | 山东省农业科学院生物技术研究中心 | Application and application method of bacillus cereus keratinase |
| CN112126536B (en) * | 2020-07-07 | 2021-08-20 | 山东省农业科学院生物技术研究中心 | Application and application method of bacillus cereus keratinase |
| CN113980832A (en) * | 2021-09-22 | 2022-01-28 | 山东大学 | Strain capable of efficiently degrading keratin and application thereof |
| CN114540252A (en) * | 2022-03-31 | 2022-05-27 | 山东省农业科学院 | Microbacterium P6 for converting livestock and poultry breeding waste and application |
| CN114540253A (en) * | 2022-03-31 | 2022-05-27 | 山东省农业科学院 | Application of bacillus pumilus P6 keratinase in detergent and application method |
| 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 |
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