CN109797145A - A method of improving keratinase vigor - Google Patents
A method of improving keratinase vigor Download PDFInfo
- Publication number
- CN109797145A CN109797145A CN201910246031.8A CN201910246031A CN109797145A CN 109797145 A CN109797145 A CN 109797145A CN 201910246031 A CN201910246031 A CN 201910246031A CN 109797145 A CN109797145 A CN 109797145A
- Authority
- CN
- China
- Prior art keywords
- keratinase
- added
- solution
- manganese ion
- feather
- 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.)
- Pending
Links
- 108010059345 keratinase Proteins 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 21
- 210000003746 feather Anatomy 0.000 claims abstract description 40
- 241000894006 Bacteria Species 0.000 claims abstract description 35
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 claims abstract description 27
- 229910001437 manganese ion Inorganic materials 0.000 claims abstract description 27
- 239000007788 liquid Substances 0.000 claims abstract description 17
- 238000000855 fermentation Methods 0.000 claims abstract description 15
- 230000004151 fermentation Effects 0.000 claims abstract description 15
- 230000015556 catabolic process Effects 0.000 claims abstract description 13
- 238000006731 degradation reaction Methods 0.000 claims abstract description 13
- 230000001580 bacterial effect Effects 0.000 claims abstract description 11
- 241000193755 Bacillus cereus Species 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 56
- 239000002609 medium Substances 0.000 claims description 31
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 27
- 108090000790 Enzymes Proteins 0.000 claims description 14
- 102000004190 Enzymes Human genes 0.000 claims description 14
- 230000000694 effects Effects 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 14
- 239000007787 solid Substances 0.000 claims description 12
- 239000001963 growth medium Substances 0.000 claims description 11
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 8
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 claims description 8
- 238000005119 centrifugation Methods 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 7
- 235000018102 proteins Nutrition 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 239000005018 casein Substances 0.000 claims description 6
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 6
- 235000021240 caseins Nutrition 0.000 claims description 6
- 102000011782 Keratins Human genes 0.000 claims description 5
- 108010076876 Keratins Proteins 0.000 claims description 5
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 claims description 4
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 229910052751 metal Inorganic materials 0.000 claims description 4
- 239000002184 metal Substances 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 4
- 239000007853 buffer solution Substances 0.000 claims description 3
- 238000000354 decomposition reaction Methods 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- 239000002699 waste material Substances 0.000 claims description 3
- 238000002835 absorbance Methods 0.000 claims description 2
- 239000011572 manganese Substances 0.000 claims description 2
- 230000001376 precipitating effect Effects 0.000 claims description 2
- 235000017550 sodium carbonate Nutrition 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 238000010561 standard procedure Methods 0.000 claims description 2
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims 1
- 239000011565 manganese chloride Substances 0.000 claims 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims 1
- 244000144977 poultry Species 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 238000004140 cleaning Methods 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 235000011187 glycerol Nutrition 0.000 description 8
- 229940099596 manganese sulfate Drugs 0.000 description 7
- 239000011702 manganese sulphate Substances 0.000 description 7
- 235000007079 manganese sulphate Nutrition 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 230000001954 sterilising effect Effects 0.000 description 7
- 102000029797 Prion Human genes 0.000 description 6
- 108091000054 Prion Proteins 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 239000003814 drug Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 244000153158 Ammi visnaga Species 0.000 description 3
- 235000010585 Ammi visnaga Nutrition 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000008676 import Effects 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 239000007836 KH2PO4 Substances 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 238000004090 dissolution Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000010985 leather Substances 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 101710200191 Feather keratin Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000005660 chlorination reaction Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 229910052748 manganese Inorganic materials 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007483 microbial process Effects 0.000 description 1
- 230000000116 mitigating effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000003895 organic fertilizer Substances 0.000 description 1
- 235000013348 organic food Nutrition 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000009941 weaving Methods 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
Abstract
The invention discloses a kind of methods for improving keratinase vigor, including following aspect: (1) manganese ion are added in the medium to cultivate the bacterial strain Bacillus cereus FDB-18B of production keratinase to improve the vigor of its produced keratinase in fermentation liquid；(2) manganese ion is added in feather fermentation medium, to improve the bacterium in the degradation capability of feather fermentation mesoptile；(3) manganese ion is added when measuring keratinase vigor, to significantly improve the vigor of keratinase.
Description
Technical field
The present invention relates to a kind of methods for improving keratinase vigor using manganese ion, belong to microorganisms technical field.
Technical background
With the raising of ratio carnivorous in our people's diet structure, the waste feathers quantity that butchering fowl generates is also continuous
Increase (mainly chicken feather).It is estimated that every year by 11,000,000 tons of generation of scale.Although the main component of feather is keratin,
But its special construction keeps its highly stable, it is difficult to be digested utilization；This not only results in waste of resources, and gives environmental protection
Bring pressure.Therefore the removal of keratin must rely on chemistry or microbial process.Chemical method needs higher energy to disappear
Consumption, and certain microorganisms that can produce keratinase can accelerate this process, and whole process can be made more green
Environmental protection.The biologic enzymolysis method of keratin is a kind of microorganism decomposition keratin by can produce keratinase, to eliminate environment
The method of harm has great development prospect.Keratinase is protease that can be special with one kind of degradation of feather keratin,
Other than it can act on other soluble protein substrates, it may also act on insoluble protein.Keratinase has
Extensive development prospect can be used for handling the feather that relevant industries (agricultural, stock keeping animal husbandry) generate, and can be used for washing
It washs, weave and the keratinase preparation in the fields such as leather processing.Keratinase is added in cleaning product can reduce dosage of scour
And improve cleaning efficiency.In addition to this it is possible to improve external preparation for skin drug for cosmetics, industry and medicine aspect
Curative effect has important economical and environmentally friendly meaning.
It is prion-infected to have become a clinically important problem.The non-disposable medical instrument used or equipment are (such as
The various various sensor probes and operation tool that needs to go deep into inside of human body or can be contacted with tissue), above may be used
Prion can be contained, be an important channel for causing it to propagate between patients.Since prion is to traditional ablation method
(such as prion needs the immersion a few hours in 1M NaOH that can make its protein denaturation to tolerance；Or in longer high temperature
Can be inactivated by thermal denaturation under high pressure), in addition certain medical articles itself can not receive sterilization condition violent in this way,
Become difficult cleaning.Existing research shows that the keratinase for capableing of degradation of feather can pass through cleaning under mild conditions
Effectively remove prion.It is of course also possible to remove other blood constituents.Thus, keratinase can be used for medical instrument cleaning solution
Enzyme preparation ingredient.But for a long time, the cleaning of these articles all relies on the cleaning solution of import.Therefore, exploitation can have
Effect removes the protease of prion, or improves its vigor, it will provides important support for the solution of problem.
Summary of the invention
It is an object of the invention to solve the deficiencies in the prior art, a kind of easy to operate, effective raising angle is provided
The innovative approach of prolease activity will can be used for the cleaning solution of medical treatment cleaning and provide important support to develop, alleviate clinically this
A little products are only capable of relying on the situation of import.
Technical solution
A method of improving keratinase vigor, which is characterized in that by addition manganese ion to improve keratinase
Vigor.
1. manganese ion is added in the culture medium of Bacillus cereus FDB-18B bacterial strain, to improve its produced angle egg
The vigor of white enzyme, the specific steps are as follows:
(1) Bacillus cereus FDB-18B bacterial strain glycerol stocks bacterium solution is taken, streak inoculation is in LB solid medium
On, it is then cultivated at 37 DEG C for 24 hours, there is bacterium colony generation on LB solid medium；
(2) picking colony is inoculated in equipped in 10ml LB liquid medium, 37 DEG C, cultivate 16h under 150rpm, obtains bacterium
Liquid；
(3) 1ml bacterium solution is taken to be inoculated in 50ml LB liquid medium, manganese ion to concentration is added into culture medium is
Then 0.1-2mM cultivates 32h at 37 DEG C, 150rpm, obtains bacterium solution；
(4) bacterium solution for obtaining step (3) carries out centrifuging and taking supernatant, with standard method using casein as substrate, measurement
660nm absorbance simultaneously calculates prolease activity.
Further, in step (1), the composition of every liter of LB solid medium are as follows: peptone 10.0g, yeast powder 5.0g, chlorination
Sodium 10.0g, 50% glycerine water solution 8ml, agar 20.0g, pH7.0；102 DEG C of sterilizing 5min.
Further, in step (2) and (3), the component of the LB liquid medium are as follows: peptone 10.0g/L, yeast powder
5.0g/L, sodium chloride 10.0g/L, with NaOH solution tune pH7.0；121 DEG C of sterilizing 20min.
2. manganese ion is added in feather fermentation medium, to improve keratinase to the capacity of decomposition of feather, specific step
It is rapid as follows:
(1) Bacillus cereus FDB-18B bacterial strain glycerol stocks bacterium solution is taken, streak inoculation is in LB solid medium
On, it is then cultivated at 37 DEG C for 24 hours, there is bacterium colony generation on LB solid medium；
(2) picking colony is inoculated in equipped in 10ml LB liquid medium, 37 DEG C, cultivate 16h under 150rpm, obtains bacterium
Liquid；
(3) it takes 1ml bacterium solution to be inoculated in 40ml feather fermentation medium, while manganese ion is added to concentration into culture medium
For 0.1-2mM, then cultivated for 24 hours at 37 DEG C, 150rpm；
(4) the remaining feather that will degrade after culture pulls rinsing drying out, claims remaining feather dry weight, decomposes according to feather is calculated
The formula of rate: 111.8%* (1-W residual/W is initial) calculates feather degradation rate.
Further, in step (3), the preparation method (g/L) of feather fermentation medium: NaCl 0.5, K2HPO41.4
KH2PO40.7, MgCl20.5, yeast powder 0.2, feather 30 is spare by 102 DEG C of high pressure sterilization 5min.
3. manganese ion is added when measuring keratinase vigor, to improve the vigor of keratinase, following step is specifically included
It is rapid:
1) three 1.5ml centrifuge tubes are taken, 0.65% casein solution, 500 μ l, 10Mm NaOOCCH3-5mMCa is added
(OOCCH3) 2 buffer solution, 50 μ l, 33 μ l, 0.1M manganese ion solution of ultrapure water, 12 μ l；
2) an above-mentioned centrifuge tube is added 500 μ l of 0.11M solution of trichloroacetic acid, adds the angle egg that 5 μ l freeze as CK
White enzyme shakes up spare；
3) two above-mentioned centrifuge tubes are placed on 50 DEG C of metal dry bath device as experimental group and preheat 5min, 5 μ l are then added
The keratinase frozen shakes up and puts back to dry bath device reaction 10min, and 500 μ l of 0.11M solution of trichloroacetic acid is added immediately after and shakes
It is even；
4) three centrifuge tubes are put into 30 DEG C of environment and react 30min, after 12000g centrifugation 5min protein isolate it is heavy
It forms sediment；
5) it takes the 200 above-mentioned centrifuged supernatants of μ l in new 1.5ml centrifuge tube, 500 μ l of 0.5M Na2CO3 solution is added,
100 μ l of 0.5M forint phenol, is put into 30 DEG C of environment after shaking up and reacts 30min；
6) 12000g is centrifuged 5min, and CK makees the control of school zero, supernatant is taken to survey experimental group OD660 value；
7) by the OD660 value measured compared with standard curve, practical enzyme activity is obtained.
The utility model has the advantages that
1. the present invention provides a kind of easy to operate, the effective side that keratinase vigor is improved using manganese ion
Method keeps the utilization of keratinase more efficient.
2. the degradation to feather can be improved in the method that the present invention improves keratinase vigor, the product after degradation can be used
Make feed additive, strengthen its proteinaceous nutrient ingredient, play a significant role in the cultivation of domestic birds and animals, generates economic effect
Benefit.The mixed liquor that feather fermentation obtains, nutriment rich in can be used as organic fertilizer in agricultural production, be used for
The production of green food and organic food reduces the dependence to chemical fertilizer.Meanwhile also containing viable bacteria in these fermentation liquids simultaneously, it is right
In improving soil, also helpful, its resource utilization is realized in pollution of the mitigation feather to environment.
3. the method provided by the invention for improving keratinase vigor can be used for relevant industries (washing, weaving and leather
Processing industry) etc. fields keratinase preparation, keratinase is added in cleaning product can reduce dosage of scour and improve cleaning
Efficiency.In addition to this, it is expected to the enzyme preparation ingredient for medical instrument cleaning solution, realizes the removal to prion, is delayed simultaneously
Clinically these products are only capable of relying on the situation of import solution.The curative effect of external preparation for skin drug can be improved in terms of medicine, have good
Good development prospect.
Detailed description of the invention
Fig. 1 is the keratinase vigor test result of embodiment 1；
Fig. 2 is the keratinase of embodiment 2 to feather degradation capability test result；
Fig. 3 is that the vigor test result after manganese ion is added when keratinase vigor measures.
Specific embodiment
Embodiment 1: the vigor that manganese ion improves the bacterium produced keratinase in culture is added in the medium
(1) Bacillus cereus FDB-18B bacterial strain glycerol stocks bacterium solution is taken with oese, streak inoculation is in LB solid
On culture medium, cultivated for 24 hours at 37 DEG C；
(2) it is inoculated in the test tube equipped with 10ml LB liquid medium with sterile toothpick picking colony, is cultivated at 37 DEG C
16h(150rpm)；
(3) it takes 1ml bacterium solution to be inoculated in 50ml LB liquid medium, while manganese sulfate being added in the medium to concentration
For 2mM, 32h (150rpm) is cultivated at 37 DEG C；
(4) 500 μ l are taken to cultivate the bacterium solution after 32h, it is spare that 12000g is centrifuged 5min；
(5) three 1.5ml centrifuge tubes are taken, 0.65% (mass concentration) casein solution 500 μ l, 10Mm is added
NaOOCCH3-5mMCa(OOCCH3)2Buffer molten 50 μ l；
(6) using an above-mentioned centrifuge tube as CK, 500 μ l of 0.11M solution of trichloroacetic acid is added, after adding 50 μ l centrifugation
Supernatant of bacteria solution liquid, shake up spare；
(7) it using two above-mentioned centrifuge tubes as experimental group, is placed on 50 DEG C of metal dry bath device and preheats 5min, be then added
Supernatant of bacteria solution liquid after 50 μ l centrifugation shakes up and puts back to dry bath device reaction 10min, 0.11M solution of trichloroacetic acid is added immediately after
500 μ l shake up；
(8) three centrifuge tubes are put into 30 DEG C of environment and react 30min, after 12000g centrifugation 5min it is spare；
(9) it takes the 200 above-mentioned centrifuged supernatants of μ l in new 1.5ml centrifuge tube, 0.5M Na is added2CO3500 μ l of solution,
100 μ l of 0.5M forint phenol shakes up to be put into 30 DEG C of environment and reacts 30min；
(10) 12000g is centrifuged 5min, and CK makees the control of school zero, supernatant is taken to survey experimental group OD660Value；
(11) OD that will be measured660Value obtains practical enzyme activity value compared with standard curve.
(12) it is compared with control, the addition of manganese sulfate improves enzyme activity 11% (Fig. 1) in fermentation liquid.
Embodiment 2: manganese sulfate is added in feather fermentation medium and improves the produced keratinase of the bacterium to feather degradation energy
Power
(1) Bacillus cereus FDB-18B bacterial strain glycerol stocks bacterium solution is taken with oese, streak inoculation is in LB solid
On culture medium, cultivated for 24 hours at 37 DEG C；
(2) it is inoculated in the test tube equipped with 10ml LB liquid medium with sterile toothpick picking colony, is cultivated at 37 DEG C
16h(150rpm)；
(3) 1ml bacterium solution is taken to be inoculated in 40ml feather fermentation medium (containing dry feather 30g/L), while in the medium
It is 0.2mM that manganese sulfate to concentration, which is added, and (150rpm) for 24 hours is cultivated at 37 DEG C；
Feather fermentation medium (g/L): NaCl 0.5, K2HPO41.4, KH2PO40.7, MgCl20.5, yeast powder 0.2,
Feather 30.It is spare by 102 DEG C of high pressure sterilization 5min.
(4) it pulls the remaining feather of degradation out rinsing drying (65 DEG C, 2.5h), claims the dry weight of remaining feather；
(5) according to feather degradation rate calculation formula: 111.8%* (1-W residual/W is initial) calculates feather palliating degradation degree.
(6) it is compared with control, the addition of manganese sulfate improves feather degradation rate 16% (Fig. 2).
Embodiment 3: manganese ion is added when keratinase vigor measures and improves keratinase vigor
(1) three 1.5ml centrifuge tubes are taken, 0.65% casein solution (mass concentration) 500 μ l, 10Mm is added
NaOOCCH3-5mMCa(OOCCH3)250 μ l of buffer solution, 33 μ l, 0.1M manganese sulfate solution of ultrapure water, 12 μ l (into solution manganese from
Sub- concentration is 2mM)；
(2) above-mentioned centrifuge tubes are added 500 μ l of 0.11M solution of trichloroacetic acid, add the angle that 5 μ l freeze as CK
Protease shakes up spare；
(3) it takes other two above-mentioned centrifuge tubes as experimental group, is placed on 50 DEG C of metal dry bath device and preheats 5min, then
The keratinase that 5 μ l freeze is added, shakes up and puts back to the reaction of dry bath device, 500 μ of 0.11M solution of trichloroacetic acid is added immediately after 10min
L shakes up；
(4) three centrifuge tubes are put into 30 DEG C of environment and react 30min, after 12000g centrifugation 5min it is spare；
(5) it takes the 200 above-mentioned centrifuged supernatants of μ l in new 1.5ml centrifuge tube, 0.5M Na is added2CO3500 μ l of solution,
100 μ l of 0.5M forint phenol shakes up to be put into 30 DEG C of environment and reacts 30min；
(6) 12000g is centrifuged 5min at room temperature, and CK makees the control of school zero, supernatant is taken to survey experimental group OD660Value；
(7) OD that will be measured660Value obtains practical enzyme activity value compared with standard curve.
(8) it is compared with control, manganese sulfate 2mM is added, enzyme activity 64% (Fig. 3) can be improved.
Embodiment 4: keratinase freezes preservation
(1) the Bacillus cereus FDB-18B bacterial strain fermentation liquor 40ml of culture 32h is taken to be placed in 50ml centrifuge tube,
10min is centrifuged under the conditions of 4 DEG C and 12000g；
(2) supernatant is transferred in two new 50ml centrifuge tubes (about 40ml), 8g (NH4) is added to each centrifuge tube
2SO4；It is put into 4 DEG C of environment after completely dissolution and stands 12h；
(3) after being centrifuged 5min (4 DEG C) under the conditions of 14000g, incline supernatant；
(4) buffer (the 10Mm NaOOCCH for containing 50% glycerol is added into centrifuge tube3-5mMCa(OOCCH3)2) dissolution
The protein of precipitating is placed in sterile centrifugation tube and saves for use at -20 DEG C to get the enzyme solutions of the keratinase of the bacterium.
Embodiment 5: the culture of bacterial strain is decomposed to feather, freezes and recovers
Culture: it in gnotobasis, with the single bacterium colony on the toothpick picking culture medium after sterilizing, is put into equipped with 10ml/
In the test tube of branch LB culture medium, 12h (150rpm) is cultivated at 37 DEG C；
LB culture medium (g/L): peptone 10；Yeast powder 5；NaCl 10；50% glycerine water solution 8ml；By 121 DEG C
High pressure sterilization 20min is spare；
Freeze: in gnotobasis, take culture 12h after bacterium solution with 50% glycerine water solution according to 7:3 volume ratio
Example mixing, loaded in culture presevation pipe and being deposited under conditions of -80 DEG C.
Recovery: it culture presevation pipe and is put under conditions of taking-up is stored in -80 DEG C and prevents from thawing on ice, with aseptic inoculation ring
It dips appropriate bacterium solution scribing line or is coated on LB solid medium；Single colonie after culture for 24 hours on picking plate does subsequent experimental.
Claims (7)
1. a kind of method for improving keratinase vigor, which is characterized in that improve the work of keratinase by addition manganese ion
Power.
2. the method as described in claim 1, which is characterized in that other than can be by the protein substrate of routine protein enzyme effect,
The enzyme can also decompose the keratoprotein that poultry waste feathers etc. are difficult to degrade.
3. the method as described in claim 1, which is characterized in that the addition manganese ion refers in Bacillus cereus
Manganese ion is added in the culture medium of FDB-18B bacterial strain, to improve the vigor of its produced keratinase, the specific steps are as follows:
(1) Bacillus cereus FDB-18B bacterial strain glycerol stocks bacterium solution is taken, streak inoculation is on LB solid medium, so
It is cultivated at 37 DEG C afterwards for 24 hours, there is bacterium colony generation on LB solid medium；
(2) picking colony is inoculated in equipped in 10ml LB liquid medium, 37 DEG C, cultivate 16h under 150rpm, obtains bacterium solution；
(3) 1ml bacterium solution is taken to be inoculated in 50ml LB liquid medium, it is 0.1- that manganese ion to concentration is added into culture medium
Then 2mM cultivates 32h at 37 DEG C, 150rpm, obtains bacterium solution；
(4) bacterium solution for obtaining step (3) carries out centrifuging and taking supernatant, with standard method using casein as substrate, measures 660nm
Absorbance simultaneously calculates prolease activity.
4. the method as described in claim 1, which is characterized in that the addition manganese ion refers to and adds in feather fermentation medium
Enter manganese ion, to improve keratinase to the capacity of decomposition of feather, the specific steps are as follows:
(1) Bacillus cereus FDB-18B bacterial strain glycerol stocks bacterium solution is taken, streak inoculation is on LB solid medium, so
It is cultivated at 37 DEG C afterwards for 24 hours, there is bacterium colony generation on LB solid medium；
(2) picking colony is inoculated in equipped in 10ml LB liquid medium, 37 DEG C, cultivate 16h under 150rpm, obtains bacterium solution；
(3) it takes 1ml bacterium solution to be inoculated in 40ml feather fermentation medium, while manganese ion to concentration is added into culture medium and is
Then 0.1-2mM is cultivated for 24 hours at 37 DEG C, 150rpm；
(4) feather for the remnants that will degrade after culture pulls rinsing drying out, claims remaining feather dry weight, according to calculating feather resolution ratio
Formula: 111.8%* (1-W residual/W is initial) calculates feather degradation rate.
5. the method as described in claim 1, which is characterized in that the addition manganese ion refers to when measuring keratinase vigor
Manganese ion is added to specifically comprise the following steps: to improve the vigor of keratinase
1) three 1.5ml centrifuge tubes are taken, 0.65% casein solution, 500 μ l, 10Mm NaOOCCH3-5mMCa (OOCCH3) is added
2 buffer solution, 50 μ l, 33 μ l, 0.1M manganese ion solution of ultrapure water, 12 μ l；
2) an above-mentioned centrifuge tube is added 500 μ l of 0.11M solution of trichloroacetic acid, adds the keratin that 5 μ l freeze as CK
Enzyme shakes up spare；
3) two above-mentioned centrifuge tubes are placed on 50 DEG C of metal dry bath device as experimental group and preheat 5min, 5 μ l are then added and freeze
Keratinase, shake up put back to dry bath device reaction 10min, immediately after be added 500 μ l of 0.11M solution of trichloroacetic acid shake up；
4) three centrifuge tubes are put into 30 DEG C of environment and react 30min, after 12000g centrifugation 5min protein isolate precipitating；
5) it takes the 200 above-mentioned centrifuged supernatants of μ l in new 1.5ml centrifuge tube, 500 μ l, 0.5M good fortune of 0.5M Na2CO3 solution is added
100 μ l of woods phenol, is put into 30 DEG C of environment after shaking up and reacts 30min；
6) 12000g is centrifuged 5min, and CK makees the control of school zero, supernatant is taken to survey experimental group OD660 value；
7) by the OD660 value measured compared with standard curve, practical enzyme activity is obtained.
6. the method as described in claim 1, which is characterized in that the manganese ion is MnSO4、MnCl2Or Mn (NO3)2Solution.
7. the method as described in claim 1, it is characterised in that manganese ion concentration used is 0.1-5mM；Preferably,
Its concentration is 2mM in live body system, 0.2mM in cultivating system.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910246031.8A CN109797145A (en) | 2019-03-28 | 2019-03-28 | A method of improving keratinase vigor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910246031.8A CN109797145A (en) | 2019-03-28 | 2019-03-28 | A method of improving keratinase vigor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109797145A true CN109797145A (en) | 2019-05-24 |
Family
ID=66564254
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910246031.8A Pending CN109797145A (en) | 2019-03-28 | 2019-03-28 | A method of improving keratinase vigor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109797145A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN114262676A (en) * | 2021-12-21 | 2022-04-01 | 江苏丘陵地区南京农业科学研究所 | Method for producing amino acid by efficiently degrading feather keratin with bacillus |
| 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 (3)
| 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 |
| CN108548746A (en) * | 2018-03-22 | 2018-09-18 | 东北农业大学 | A method of measuring feather degradation rate |
| 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 |
-
2019
- 2019-03-28 CN CN201910246031.8A patent/CN109797145A/en active Pending
Patent Citations (3)
| 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 |
| CN108548746A (en) * | 2018-03-22 | 2018-09-18 | 东北农业大学 | A method of measuring feather degradation rate |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| ALEXANDRE JOSE MACEDO ET AL.: "Properties of a non collagen-degrading Bacillus subtilis keratinase", 《CANADIAN JOURNAL OF MICROBIOLOGY》 * |
| S. AROKIYARAJ ET AL.: "Optimizing the fermentation conditions and enhanced production of keratinase from Bacillus cereus isolated from halophilic environment", 《SAUDI JOURNAL OF BIOLOGICAL SCIENCES》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN114262676A (en) * | 2021-12-21 | 2022-04-01 | 江苏丘陵地区南京农业科学研究所 | Method for producing amino acid by efficiently degrading feather keratin with bacillus |
| CN114262676B (en) * | 2021-12-21 | 2024-01-26 | 江苏丘陵地区南京农业科学研究所 | Method for efficiently degrading feather keratin to produce amino acid by using bacillus |
| 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 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109797145A (en) | A method of improving keratinase vigor | |
| CN100375637C (en) | Biological deodorization and purification agent, and its use | |
| CN103468613B (en) | Bacillus megatherium, method for preparing microbial inoculum through solid fermentation of bacillus megatherium and application of microbial inoculum | |
| CN102304483A (en) | Enterococcus faecium for feeding and applications thereof | |
| CN101838166A (en) | Animal carrion and static compost hazard-free treatment method of products thereof | |
| CN104548175B (en) | A kind of composite biological deodorant and the preparation method and application thereof | |
| CN104974962B (en) | One plant of acinetobacter phosphorus decomposing growth-promoting bacterium Y40 and its application | |
| CN108795814A (en) | A kind of bacterial strain, screening technique and its application of degradable waste feathers | |
| CN104328066B (en) | Bacillus subtilis H4, decomposed inoculant prepared therefrom and application of the decomposed inoculant | |
| CN103215208B (en) | Haemophilus parasuis culture medium | |
| CN1800358A (en) | Keratinase-proudicng bacterium and its preparation method | |
| CN109294937A (en) | Novel thermophilic alkaline-resisting amphimicrobian bacterial strain and the microorganism agent comprising above-mentioned bacterial strains for carcass of degrading | |
| CN104277994A (en) | Sinorhizobium SCAUs65 and application thereof | |
| CN110125149A (en) | A kind of processing method of life wet refuse | |
| KR20150136624A (en) | A composition of soil treatment and a process for the decontamination of soil using the same | |
| CN105272395B (en) | A kind of complex microorganism preparations and the preparation method and application thereof for manioc waste fast degradation | |
| KR100733135B1 (en) | Two novel bacteria of Bacillus sp. CK-10 having a decomposing capacity of nitrogen and/or phosphorus in water and a method of decomposing of nitrogen and/or phosphorus by the said bacteria | |
| CN106350558A (en) | Method for jointly degrading feathers by aid of enzyme bacteria | |
| CN109400392A (en) | A kind of astaxanthin fertilizer and preparation method thereof containing microorganism | |
| CN108976045A (en) | A method of biological organic fertilizer is prepared using dead livestock and poultry | |
| CN105457989A (en) | Resourceful treatment method for waste bodies of dead livestock and poultry animals | |
| CN107267414A (en) | One plant of plant azotobacteria DJG211 and its application in soil and plant vigor is improved | |
| CN103173396B (en) | Lactobacillus murinus QT-0304 strain and strain fermentation liquor thereof, and applications of strain fermentation liquor in preventing plant pathogen | |
| JP2021045085A (en) | Method for producing sterilized meat lump | |
| CN106244491A (en) | A kind of banana stem leaves residuum decomposing microbial inoculum suppressing droop and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190524 |
|
| RJ01 | Rejection of invention patent application after publication |