CN109055280A - A kind of keratinase superior strain and its application - Google Patents
A kind of keratinase superior strain and its application Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of keratinase superior strain and its application.The bacterial strain of high yield keratinase provided by the invention is specially molten bacillus (lysobacter sp.) YQ20, and deposit number is CGMCC No.16072.The strain enzyme-producing speed is exceedingly fast, after 12-48h ferments, keratinase yield can achieve 259-966U/mL fermentation liquid, it is that report can produce the molten bacillus of keratinase for the first time at present, it is also the bacterial strain that fermentation produces that keratinase is very fast, substrate specificity is wider, is easy to large-scale culture and industrial application.
Description
Technical field:
The invention belongs to field of biotechnology, and in particular to a kind of keratinase superior strain and its application.
Background technique:
In recent years, with the rapid development of large-scale farming industry, a large amount of animal wastes are produced, mainly with hair, plumage
The principal modes such as hair, angle, hoof exist.According to statistics, the annual feather Isoquant in the whole world reaches millions of tons.These wastes are except a small amount of
For down products and preparing outside amino acid, the overwhelming majority is not utilized rationally, a large amount of wasting of resources is not only caused, but also
Serious environmental pollution is caused, the main component for being primarily due to these wastes is the difficult keratin being degraded.
Keratin can be divided into alpha keratin and two kinds of β keratin, and alpha keratin is primarily present in the hair of mammal, plumage
In hair, beast angle, finger/toenail, claw and beast hoof.Harder β keratin is present in toenail, shell, squama, beak, the pawl of reptile
In son and bristle.Its crude protein content is 80% or more, and various total amino acid contents are 70% or more, simultaneously containing compared with how much amount member
Element, microelement and unknown growth factor are a kind of good forage protein and fertilizer source, have weight to its development and utilization
The application prospect wanted.
Traditional keratin treatment process mainly includes physical method, chemical method and bioanalysis.Either physically or chemically, exist
Energy consumption is high, environmental pollution is serious, the nutriment of product is easily destroyed the problems such as not easy to handle with the three wastes.Utilize microbial degradation
Etc. biotechnologys approach solve keratin and decompose using being increasingly taken seriously, but there are a large amount of disulfide bond, hydrogen bond for keratin
And hydrophobic effect, it is not easy to by general proteins enzyme hydrolysis.Keratinase (Keratinase) is that one kind can be with selective degradation angle
The enzyme of albumen has now been found that 30 multiple-microorganisms can secrete keratinase, and the research of domestic and international keratinase is mainly concentrated
Dermatophyte and candida albicans (Candida albicans) in fungi, the streptomycete in actinomyces
(Streptomyces) and the bacillus licheniformis (Bacillus in high temperature zygosaccharomyces (Thermonspora) and bacterium
) and bacillus subtilis (Bacillus subtilis) etc. licheniformis.
As Chinese invention patent ZL 201010574642.4 disclose a kind of efficient degradation feather keratin bacterial strain and its
Screening technique, the invention arrive efficient degradation feather keratin bacterial strain lichens gemma bar after primary dcreening operation, separation, enrichment, secondary screening
Bacterium (Bacillus licheniformis) F4 contains bacterial strain access in the fermented and cultured of complete feather, fermented and cultured 60h,
The small pinnule of feather is all sloughed, and remaining plumage stalk and the pinnule sloughed largely are degraded, bacterial strain depilation and degradation of feather angle
Albumen effect is obvious.
As Chinese invention patent ZL 200510048773.8 discloses producing strains and its preparation side of a kind of keratinase
Method, the invention screen to obtain strain excellent-bacillus subtilis (Bacillus subtilis) of one plant of production keratinase
The optimal pH of CGMCC No.1505, the produced keratinase crude enzyme liquid enzyme reaction of the bacterial strain are 7.5-8.0 or so, and optimum temperature is
50-60 DEG C, after keeping the temperature 1h under condition of different pH, in the range of pH6.4-10, enzymatic activity is kept at 80% or more.70
After keeping the temperature 30min in DEG C water-bath, there are also 90% or so for remaining enzyme activity；Heat preservation 120min after enzymatic activity remain within 70% with
On.In comparison, the keratinase than having registered has better heat-resistant quality and pH stability.
As Chinese invention patent ZL201310701537.6 discloses the streptomyces aureus of one plant of production keratinase and its answers
With method, which obtains one plant of streptomyces aureus (Streptomyces aureofaciens) K13, the bacterial strain fermented week
It after phase 64h, produces keratinase vigor and reaches 44.2U/ml, which is medium temperature basic keratins enzyme, optimum temperature
It is 50-60 DEG C, optimal pH 8.5, and enzyme enzyme activity in alkaline (pH7.0-10.0) environment is stablized.
The keratin such as feather are specifically digested using the keratinase of microorganism secretion in nature, keratin is direct
It is transformed into protein or compound amino acid, thus turning waste into treasure, environment can be protected to utilize resource again, this will also has very aobvious
The economic benefit of work and far-reaching social benefit.
Summary of the invention:
For the demand of the prior art, the present invention is intended to provide one plant can high yield keratinase molten bacillus and its fermentation side
Method and application.The strain enzyme-producing speed of high yield keratinase provided by the invention is exceedingly fast, and is that report can produce angle egg for the first time at present
The molten bacillus of white enzyme, and fermentation produce the bacterial strain that keratinase is very fast, substrate specificity is wider, are easy to large-scale culture and industrialization
Using.
One of technical solution provided by the present invention are as follows: a kind of molten bacillus of high yield keratinase, the molten bacillus are specific
For molten bacillus (lysobacter sp.) YQ20, which is preserved in Chinese microorganism strain preservation pipe on July 9th, 2018
Reason committee common micro-organisms center, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences's microbe research
Institute, postcode 100101, deposit number are CGMCC No.16072.
The physicochemical property of the molten bacillus YQ20 is as follows: Gram-negative bacteria, and bacterium colony is in the shape of a rod on LB culture medium.Bacterium colony figure
Piece is shown in Fig. 1.
The two of technical solution provided by the present invention are as follows: application of the molten bacillus YQ20 in degradation of feather.
The three of technical solution provided by the present invention are as follows: application of the molten bacillus YQ20 in production keratinase.
A kind of the four of technical solution provided by the present invention are as follows: production using molten bacillus YQ20 fermenting and producing keratinase
Method, specific as follows:
(1) seed culture
Condition of culture: speed of agitator 160-220rpm, temperature are 30-37 DEG C, and ferment 12-24h；
Seed culture medium (g/L): beef extract powder 3, peptone 10；Sodium chloride 5；Remaining is water, pH7.4；
(2) fermented and cultured
Fermentation condition: inoculum concentration 2-10%, speed of agitator 160-220rpm, temperature are 30-40 DEG C, and ferment 12-48h；
Fermentation medium (g/L): NaCl 5.0, KH2PO4 0.5、K2HPO4 1.0、MgSO4·7H2O 1.0, wool, plumage
Hair or hoof tips 10.0, remaining is water, and pH is natural；
After 12-48h ferments, keratinase yield can achieve 259-966U/mL fermentation liquid.
The utility model has the advantages that
Molten bacillus CGMCC No.16072 provided by the invention can using wool, feather or hoof tips as sole carbon source and
Nitrogen source quickly carries out the accumulation of cell culture and keratinase, compared with prior art, using molten bacillus CGMCC No.16072,
Added degradation of substrates can be visually observed only surplus shorter slag wool, reach the different types of wool of degradation, birds by fermentation for 24 hours
The effect of keratin in feather and hoof tips has extensive prospects for commercial application.
Detailed description of the invention:
Fig. 1: the microscope photo of molten bacillus CGMCC No.16072；
Fig. 2: bacterial strain screening-hydrolytic circle screening；
Fig. 3: bacterial strain screening-chicken feather degradation test；
Fig. 4: molten bacillus CGMCC No.16072 16S rRNA；
Fig. 5: molten bacillus CGMCC No.16072 coarse wool degradation effect figure；
Wherein, figure A is the control group for not being inoculated with CGMCC No.16072；Figure B, C are the reality for being inoculated with CGMCC No.16072
Test group；
Fig. 6: molten bacillus CGMCC No.16072 maos of native ferment effect figures；
Wherein, figure A is the control group for not being inoculated with CGMCC No.16072；Figure B is the experiment for being inoculated with CGMCC No.16072
Group；
Fig. 7: amino acid classes and concentration in chicken feather fermentation liquid；
Fig. 8: amino acid classes and concentration in goose feather fermentation liquid；
Fig. 9: amino acid classes and concentration in drake feather fermentation liquid.
Specific embodiment:
In order to which the objects, technical solutions and advantages of this patent are more clearly understood, below in conjunction with specific embodiment, to this
Patent is further elaborated.It should be appreciated that specific embodiment described herein is only to explain this patent, and do not have to
It is of the invention in limiting.
Culture medium used in the present invention is as follows:
Seed culture medium (g/L): beef extract powder 3, peptone 10；Sodium chloride 5；Remaining is water, pH7.4；
Fermentation medium (g/L): NaCl 5.0, KH2PO4 0.5、K2HPO4 1.0、MgSO4·7H2O 1.0, wool, plumage
Hair or hoof tips 10.0, remaining is water, and pH is natural.
The present invention measures method used in keratinase enzyme activity and condition is specific as follows: fermentation liquid passes through centrifuging and taking supernatant
Crude enzyme liquid is done, production feather meal does substrate, and by termination reaction method, the amount with amino acid caused by keratinase degradation substrate is
According to detecting 280nm absorbance value data and measure enzyme activity.It is specific as follows:
0.5mL crude enzyme liquid is taken, is added 0.5mL 0.1mol/L phosphate buffer (pH7.0), 10mg feather meal is then added,
10min is reacted in 37 DEG C of isothermal vibration reactors, the trichloroacetic acid (TCA) that 2mL 10% is added terminates reaction.6000rpm from
Heart 20min, takes supernatant in OD280nmLower measurement light absorption value.
Keratinase enzyme activity (U) is defined as: absorbance value OD280nmIncreasing by 0.01 per hour is an enzyme-activity unit U.
The screening of 1 YQ20 bacterial strain of embodiment
Collect sheepfold, cattle pen, birds waste aheap, feather aheap, slaughterhouse choosing etc. ground acquisition soil sample, respectively
The wool that 5g soil sample is weighed with 1% is put into conical flask, and the sterile water that 45mL is then added obtains enriched medium, by taper
Bottle is placed on 37 DEG C, cultivates one week in 220rpm shaking table, is then coated on the bacterium solution in enriched medium after sterile water dilutes
Screening and culturing medium (casein 10.0g/L, MgSO4·7H2O 5.0g/L, NaCl 5.0g/L, agar 15.0g/L, add dilute alkali heating
Dissolution), it is placed in 37 DEG C of constant incubators and cultivates 48 hours.The diameter (such as Fig. 2) for measuring periphery of bacterial colonies hydrolysis circle, selects diameter
Biggish single colonie carries out strain feather degradation test, and (the single colonie toothpick picking for obtaining screening, is then vertically put into kind
Sub- inoculation of medium obtains seed liquor in 220rpm shaking table culture 24 hours then at 37 DEG C.Chicken feather fermentation medium is prepared,
Cultured seed liquor is inoculated into chicken feather fermentation medium with 4% inoculum concentration and is marked, puts 37 DEG C, 220rpm shakes
Bed culture 24 hours, observes the degradation situation of chicken feather), as shown in figure 3, chicken feather is degradable after fermentation 24 hours.As a result,
One plant of pure culture is finally obtained, number is YQ20 bacterial strain.
It is 687U/mL that the keratinase enzyme activity in fermentation liquid is measured at 12 hours；
The chicken feather fermentation medium group becomes (g/L): NaCl 5.0, KH2PO4 0.5、K2HPO4 1.0、MgSO4·
7H2O 1.0, chicken feather 10.0, remaining is water, and pH is natural.
The identification of 2 YQ20 bacterial strain of embodiment
The YQ20 bacterial strain that embodiment 1 is obtained carried out 16SrRNA (Fig. 4) identification, by blast analysis (https: //
Blast.ncbi.nlm.nih.gov/Blast.cgi) determine that YQ20 is molten bacillus.Molten bacillus YQ20 was protected on July 6th, 2018
It is hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.16072.
The common and braid wool of the molten bacillus CGMCC No.16072 of embodiment 3 ferments
The molten bacillus CGMCC No.16072 that embodiment 2 is obtained is inoculated with coarse wool fermented and cultured.
Seed culture condition: the time for 24 hours, 35 DEG C, 200rpm；
Seed culture medium (g/L): beef extract powder 3, peptone 10, sodium chloride 5, remaining is water, pH7.4；
Fermentation culture conditions: seed liquor is inoculated into fermentation medium with 5% inoculum concentration, and 35 DEG C, 200rpm, ferment 48h；
Fermentation medium (g/L): NaCl 5.0, KH2PO4 0.5、K2HPO4 1.0、MgSO4·7H2O 1.0, coarse wool
10.0；
Coarse wool generates after the techniques such as multistep cashmere extraction carry out wool utilization from wool processing enterprise of Shandong Province
Waste wool.Degradation effect is shown in Fig. 5.Scheming A is the coarse wool fermentation medium for not being inoculated with YQ20, still be can see after fermented clear
Clear hair ball.Figure B&C is the coarse wool fermentation medium for being inoculated with YQ20, and after fermentation, hair ball disappears, and culture medium is by clear and transparent change
For the liquid of brown viscous；
It is 259U/mL that the keratinase enzyme activity in fermentation liquid is measured at 36 hours.
The hair soil of the molten bacillus CGMCC No.16072 of embodiment 4 ferments
The molten bacillus CGMCC No.16072 that embodiment 2 is obtained carries out seed culture and fermented and cultured.
220rpm, 30 DEG C of seed cultures for 24 hours after, be inoculated into hair soil fermentation medium with 4% inoculum concentration, when 30 DEG C of fermentations
Between 48h.
The native fermentation medium (50mL) of hair: the hair soil for taking the scouring of wool of wool processing factory to generate is appropriate, includes wool, soil and sheep
Excrement adds originally water sedimentation sandstone, extracts the moisture in wool, weighs weight in wet base 2g, adds muddy water 50mL.Ferment effect such as Fig. 6.Scheme A
For the hair soil fermentation medium for not being inoculated with YQ20, clearly hair ball still can see after fermented.Figure B is inoculation YQ20
The native fermentation medium of hair, after fermentation, hair ball disappears.
Analysis of amino acids in the substrate specificity catalytic test and fermentation liquid of 5 enzyme of embodiment
The molten bacillus CGMCC No.16072 that embodiment 2 is obtained is at 30 DEG C, under the conditions of 160rpm after seed culture 12h,
It is inoculated into the fermentation medium containing different poultry feathers respectively by 10% inoculum concentration, 160rpm, 30 DEG C of culture 48h pass through
Degradation rate of the remaining insoluble solid content verifying bacterium of detection to different substrates.
Fermentation medium (g/L): NaCl 5.0, KH2PO4 0.5、K2HPO4 1.0、MgSO4·7H2O 1.0, chicken feather, duck
Hair, goose feather or hoof tips 10.0, remaining is water, and pH is natural.
When chicken feather culture medium fermentation 12h, measuring the keratinase enzyme activity in fermentation liquid is 720U/mL；
When drake feather culture medium fermentation 12h, measuring the keratinase enzyme activity in fermentation liquid is 672U/mL；
When goose feather culture medium fermentation 12h, measuring the keratinase enzyme activity in fermentation liquid is 872U/mL；
When hoof tips culture medium fermentation 48h, measuring the keratinase enzyme activity in fermentation liquid is 966U/mL；
Degradation rate method for measuring is that the complete fermentation liquid that will ferment first is filtered with vacuum pump, is not degraded
Residue has been left on filter paper, then with residue, weighing, while the blank that also to weigh on high-temperature blast drying oven drying filter paper
Sample finally calculates degradation rate using the weight for the weight and blank fermented.
Above-mentioned filtered filter liquor is carried out solid content filtering to remove, is analyzed using automatic amino acid analyzer.
By detection, it is found that the crude enzyme liquid that the strain fermentation generates has higher degradation to various feathers and hoof tips, and send out
Difference is presented depending on raw material in rich amino acids in zymotic fluid, content.The above results have the industrial application of the enzyme good
Good directive significance, degradation rate the results are shown in Table 1, and analysis of amino acids is shown in that (control group is not to be inoculated with CGMCC to Fig. 7-9
No.16072, experimental group are inoculated with CGMCC No.16072):
Table 1: the degradation rate of different substrates
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
The limitation to the scope of the patents therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art,
Under the premise of not departing from this patent design, the respective embodiments described above can also make several deformations, combination and improve, these all belong to
In the protection scope of this patent.Therefore, the protection scope of this patent should be subject to the claims.
Claims (4)
1. a kind of molten bacillus of high yield keratinase, which is characterized in that the molten bacillus is specially molten bacillus (lysobacter
Sp.) YQ20, deposit number are CGMCC No.16072.
2. application of the molten bacillus YQ20 described in claim 1 in degradation of feather.
3. application of the molten bacillus YQ20 described in claim 1 in production keratinase.
4. application of the YQ20 as claimed in claim 3 in production keratinase, which is characterized in that use YQ20 fermenting and producing angle
The method of protease is specific as follows:
(1) seed culture
Condition of culture: speed of agitator 160-220rpm, temperature are 30-37 DEG C, and ferment 12-24h；
Seed culture medium is based on g/L: beef extract powder 3, peptone 10；Sodium chloride 5；Remaining is water, pH7.4；
(2) fermented and cultured
Fermentation condition: inoculum concentration 2-10%, speed of agitator 160-220rpm, temperature are 30-40 DEG C, and ferment 12-48h；
Fermentation medium is based on g/L: NaCl 5.0, KH2PO4 0.5、K2HPO4 1.0、MgSO4·7H2O 1.0, wool, feather
Or hoof tips 10.0, remaining is water, and pH is natural.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113881655A (en) * | 2021-11-01 | 2022-01-04 | 山东理工大学 | Keratinase and application thereof |
| CN114717218A (en) * | 2022-05-07 | 2022-07-08 | 加来(济南)生活科技有限公司 | Method for efficiently producing keratinase |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107868762A (en) * | 2017-11-17 | 2018-04-03 | 山东省农业科学院生物技术研究中心 | Produce bacillus cereus and its application of keratinase |
-
2018
- 2018-09-25 CN CN201811118989.0A patent/CN109055280B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107868762A (en) * | 2017-11-17 | 2018-04-03 | 山东省农业科学院生物技术研究中心 | Produce bacillus cereus and its application of keratinase |
Non-Patent Citations (2)
| Title |
|---|
| J.D. ALLPRESS等: "Production, purification and characterization of an extracellular keratinase from Lysobacter NCIMB 9497", 《LETTERS IN APPLIED MICROBIOLOGY》 * |
| JAMILE QUEIROZ PEREIRA等: "Isolation of three novel Antarctic psychrotolerant feather-degrading bacteria and partial purification of keratinolytic enzyme from Lysobacter sp. A03", 《INTERNATIONAL BIODETERIORATION & BIODEGRADATION》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113881655A (en) * | 2021-11-01 | 2022-01-04 | 山东理工大学 | Keratinase and application thereof |
| CN114717218A (en) * | 2022-05-07 | 2022-07-08 | 加来(济南)生活科技有限公司 | Method for efficiently producing keratinase |
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