CN108048350A - Fit cold cellulase producing bacteria - Google Patents
Fit cold cellulase producing bacteria Download PDFInfo
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- CN108048350A CN108048350A CN201711368415.4A CN201711368415A CN108048350A CN 108048350 A CN108048350 A CN 108048350A CN 201711368415 A CN201711368415 A CN 201711368415A CN 108048350 A CN108048350 A CN 108048350A
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- Prior art keywords
- cold
- cellulase
- producing bacteria
- cellulase producing
- enzyme
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- Granted
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- 108010059892 Cellulase Proteins 0.000 title claims abstract description 64
- 229940106157 cellulase Drugs 0.000 title claims abstract description 63
- 241000894006 Bacteria Species 0.000 title claims abstract description 56
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 239000002253 acid Substances 0.000 abstract description 5
- 238000000034 method Methods 0.000 abstract description 5
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 238000009629 microbiological culture Methods 0.000 abstract description 3
- 108090000790 Enzymes Proteins 0.000 description 32
- 102000004190 Enzymes Human genes 0.000 description 32
- 229940088598 enzyme Drugs 0.000 description 32
- 239000000243 solution Substances 0.000 description 27
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 18
- 230000000694 effects Effects 0.000 description 15
- 238000000855 fermentation Methods 0.000 description 15
- 230000004151 fermentation Effects 0.000 description 15
- 230000001580 bacterial effect Effects 0.000 description 11
- 239000012153 distilled water Substances 0.000 description 11
- 229920002678 cellulose Polymers 0.000 description 10
- 239000001913 cellulose Substances 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 10
- 239000011780 sodium chloride Substances 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 8
- 239000001888 Peptone Substances 0.000 description 8
- 108010080698 Peptones Proteins 0.000 description 8
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 8
- 239000008103 glucose Substances 0.000 description 8
- 235000019319 peptone Nutrition 0.000 description 8
- 238000001514 detection method Methods 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 239000006228 supernatant Substances 0.000 description 6
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 5
- 239000002054 inoculum Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 239000001768 carboxy methyl cellulose Substances 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 239000000835 fiber Substances 0.000 description 4
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 4
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- 241000100580 Zhihengliuella halotolerans Species 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 229910021645 metal ion Inorganic materials 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 239000002689 soil Substances 0.000 description 3
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 108010047754 beta-Glucosidase Proteins 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 244000153158 Ammi visnaga Species 0.000 description 1
- 235000010585 Ammi visnaga Nutrition 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 229920002299 Cellodextrin Polymers 0.000 description 1
- 108010001682 Dextranase Proteins 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 101710112457 Exoglucanase Proteins 0.000 description 1
- 229920001503 Glucan Polymers 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
- 239000007836 KH2PO4 Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 102000006995 beta-Glucosidase Human genes 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 229920003123 carboxymethyl cellulose sodium Polymers 0.000 description 1
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 description 1
- 229940063834 carboxymethylcellulose sodium Drugs 0.000 description 1
- FYGDTMLNYKFZSV-ZWSAEMDYSA-N cellotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](O[C@@H]2[C@H](OC(O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-ZWSAEMDYSA-N 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 108010091371 endoglucanase 1 Proteins 0.000 description 1
- 108010091384 endoglucanase 2 Proteins 0.000 description 1
- 108010092450 endoglucanase Z Proteins 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000009941 weaving Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
-
- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract
The invention discloses one kind to fit cold cellulase producing bacteria, and it is 14938 that this, which fits cold cellulase producing bacteria in the deposit number of China General Microbiological culture presevation administrative center (CGMCC), and preservation date is on November 20th, 2017.The ability fitted cold cellulase producing bacteria and there is production cellulase of the present invention, and the method that the application suitable cold cellulase producing bacteria prepares suitable cold cellulase is simple, easy to implement, which has suitable to cold, the characteristic of acid and alkali-resistance, is with a wide range of applications.
Description
Technical field
The present invention relates to cellulase producing bacterias, and in particular, to fits cold cellulase producing bacteria with fitting cold cellulase
And preparation method thereof.
Background technology
Cellulase includes endoglucanase (EC 3.2.1.4), exoglucanase (EC 3.2.1.91) and β-Portugal
Cellulose complete hydrolysis can be glucose by synergistic effect by polyglycoside (EC 3.2.1.21) three types.Wherein inscribe
Dextranase mainly acts on the noncrystalline domain of cellulose, cuts β-Isosorbide-5-Nitrae-glycosidic bond, hydrocellulose at random.Circumscribed glucan
Enzyme hydrolyzes β-Isosorbide-5-Nitrae-glycosidic bond in unformed cellulose, forms soluble cellodextrin and cellobiose.Beta-glucosidase
Enzyme hydrolyzable non-reducing end hydrolyzation of glucose residue, glucose is resolved by cellobiose and cellulose dextrin.
At present, the bacterial strain of most of research cellulase-producing is normally taken from conventional environment, is usually that nutrition is relatively abundant, ecology
The more complicated place of structure, and often tolerance is not high for the bacterium producing multi enzyme preparation obtained, easily degenerates.The extreme ecological environment of some uniquenesses
Area tends to breed the bacterial strain with unique physiological activity, if it is possible to is screened wherein with special nature fiber
The bacterial strain of plain enzyme, this is beneficial to substantially reduce cost, saves resource, while will also be more suitably applied to industrial production.
China Qinghai-xizang Plateau Region belongs to plateau temperate zone, height above sea level, very cold drying, average annual precipitation 140mm, Nian Ping
Equal 1 DEG C of temperature, day and night the temperature difference is larger.The climatic environment of this area is more extreme, but the mountain peak of its internal distribution, glacier, high mountain
Abundant and peculiar living resources are still bred in all kinds of habitats such as lake and alt wetland, and this area is increasingly becoming section in recent years
Learn the famous scenic spot investigated with ecological Studies.Qinghai-Tibet unique geographical environment and weather conditions create the group of this area microorganism
Falling structure and physiological property and other areas has huge difference, thus is screened for the cellulase producing strain of this area, has
Important ecological benefits, economic benefit and social benefit, the novel strain obtained will have in fields such as food, feed and pharmacy
Potential application prospect.
The content of the invention
The object of the present invention is to provide one kind to fit cold cellulase producing bacteria with fitting cold cellulase and preparation method thereof, should
The cellulase of suitable cold cellulase producing bacteria production has the characteristics that suitable low temperature, resistance to acid and alkali, is expected to be applied to biological energy source
Multiple industrial circles such as source, laundry weaving, food fermentation, feed.
To achieve these goals, the present invention provides one kind to fit cold cellulase producing bacteria, described to fit cold cellulase
Producing strains are 14938 in the deposit number of China General Microbiological culture presevation administrative center (CGMCC), preservation date 2017
On November 20, in.
The present invention also provides a kind of preparation method for fitting cold cellulase, the preparation method comprises the following steps：(1) will
Suitable cold cellulase producing bacteria described in claim 1 is inoculated in culture in liquid LB (Luria-Bertani)；It (2) will culture
Gained bacterium solution, which is forwarded in fermentation medium, ferments；(3) bacterium solution after fermentation is centrifuged, takes supernatant.
The present invention also provides a kind of cold cellulase is fitted according to what previously described preparation method was prepared.
Through the above technical solutions, cold cellulase producing bacteria its Classification And Nomenclature provided by the invention of fitting is salt tolerant Liu Zhi perseverance
Bacterium (Zhihengliuella halotolerans) La12, the 16S rDNA gene orders of bacterial strain are protected as described in SEQ ID NO1
For China General Microbiological culture presevation administrative center, (abbreviation CGMCC, address are Tibetan unit：BeiChen West Road, Chaoyang District, BeiJing City 1
Number institute 3, Institute of Microorganism, Academia Sinica), preservation date is on November 20th, 2017, and deposit number is：14938.This
The invention ability fitted cold cellulase producing bacteria and have production cellulase, the cellulase have suitable to cold, acidproof
The characteristic of alkali, is with a wide range of applications.
Other features and advantages of the present invention will be described in detail in subsequent specific embodiment part.
Description of the drawings
Attached drawing is for providing a further understanding of the present invention, and a part for constitution instruction, with following tool
Body embodiment is together for explaining the present invention, but be not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the hydrocellulose photo provided by the invention for fitting cold cellulase producing bacteria；
Fig. 2 is influence of the temperature to cellulase activity provided by the invention in detection example 2；
Fig. 3 is influences of the pH to cellulase activity provided by the invention in detection example 3；
Fig. 4 is influence of the metal ion to cellulase activity provided by the invention in detection example 4.
Specific embodiment
The specific embodiment of the present invention is described in detail below.It is it should be appreciated that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to limit the invention.
The endpoint of disclosed scope and any value are not limited to the accurate scope or value herein, these scopes or
Value should be understood to comprising the value close to these scopes or value.For numberical range, between the endpoint value of each scope, respectively
It between the endpoint value of a scope and individual point value and can be individually combined with each other between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
The present invention provides one kind to fit cold cellulase producing bacteria, described to fit cold cellulase producing bacteria Chinese common micro-
The deposit number of biological inoculum preservation administrative center (CGMCC) is 14938, and preservation date is on November 20th, 2017.
Cold cellulase producing bacteria its Classification And Nomenclature provided by the invention of fitting is salt tolerant Liu Zhi perseverance bacterium (Zhihengliuella
Halotolerans) La12, for the 16S rDNA gene orders of bacterial strain as described in SEQ ID NO1, depositary institution is common micro- for China
(abbreviation CGMCC, address are biological inoculum preservation administrative center：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology), preservation date is on November 20th, 2017, and deposit number is：14938.
This is fitted cold cellulase producing bacteria and screens to obtain by the following method：(1) 3000-5000 meters of Qinghai-Tibet Platean sea is chosen
3-20 centimeters soil below place's vegetation earth's surface is pulled out, through water dissolution, dilution；(2) soil liquid after dilution is coated on fiber
It is cultivated on plain enriched medium, to grow bacterium colony point；(3) picking colony point on cellulose screening and culturing medium culture with length
Go out bacterium colony；(4) the cellulose screening and culturing medium for growing bacterium colony dyed, decolourized, check transparent circle；(5) filtering out has
Hydrolyze the strain on the cellulose screening and culturing medium of circle.Will specifically illustrate later.
The present invention also provides a kind of preparation method for fitting cold cellulase, the preparation method comprises the following steps：(1) will
Suitable cold cellulase producing bacteria described in claim 1 is inoculated in culture in liquid LB (Luria-Bertani)；It (2) will culture
Gained bacterium solution, which is forwarded in fermentation medium, ferments；(3) bacterium solution after fermentation is centrifuged, takes supernatant.
The ability of the present invention fitted cold cellulase producing bacteria and have production cellulase, which has suitable
Cold property, the characteristic of acid and alkali-resistance can be made according to the previously described suitable cold cellulase producing bacteria of above-mentioned technical proposal cultivation and fermentation
Standby to obtain fitting cold cellulase, preparation method is simple, easy to implement.
In a kind of preferred embodiment of the present invention, in order to improve the yield of suitable cold cellulase producing bacteria, it is preferable that
The formula of liquid LB is：Compared with the water of 1000mL, dusty yeast 4-6g, sodium chloride 8-12g, peptone 8-11g, pH 7.2-
7.6。
In a kind of preferred embodiment of the present invention, in order to improve the yield of suitable cold cellulase producing bacteria, it is preferable that
Condition of culture is in step (1)：When culture 18-36 is small under conditions of 24-30 DEG C of rotating speed is 100-300rpm.
In a kind of preferred embodiment of the present invention, in order to improve the yield of suitable cold cellulase producing bacteria, hair is improved
Ferment efficiency, it is preferable that bacterium solution and the volume ratio of fermentation medium are 2-3 in step (2):100.
In a kind of preferred embodiment of the present invention, in order to improve the yield of suitable cold cellulase producing bacteria, hair is improved
Ferment efficiency, it is preferable that the formula of fermentation medium is：Compared with the water of 1000mL, dusty yeast 4-6g, sodium chloride 8-12g, albumen
Peptone 8-11g, sodium carboxymethylcellulose 0.4-0.6g, pH 7.2-7.6.
In a kind of preferred embodiment of the present invention, in order to improve the yield of suitable cold cellulase producing bacteria, hair is improved
Ferment efficiency, it is preferable that fermentation condition is in step (2)：40-50 is cultivated under conditions of 24-30 DEG C of rotating speed is 100-300rpm
Hour.
In a kind of preferred embodiment of the present invention, in order to improve the recovery rate of suitable cold cellulase, it is preferable that step
(3) condition of centrifugation includes in：20-30min is centrifuged with 3000-5000rmp rotating speeds under the conditions of 2-6 DEG C.
The present invention also provides a kind of cold cellulase is fitted according to what previously described preparation method was prepared.
This, which fits cold cellulase, has suitable to cold, the characteristic of acid and alkali-resistance, is with a wide range of applications.
The present invention will be described in detail by way of examples below.
1st, the screening of cold cellulase producing bacteria is fitted
Sampling point around vegetation, gathers 5 to 10 centimeters soil below earth's surface at the Qinghai-Tibet 4000 meters of height above sea level of selection.It weighs
Pedotheque 1g, be added to a sterile water for filling 99mL and with bead triangular flask in, pedotheque is abundant
Dissolving, mixing, from into 10-2Then dilution adds sterile water by 10 times of dilution methods again and is diluted to 10 successively-3、10-4、10-5、
10-6Dilution.Take 10-4、10-5、10-6Each 0.1mL of dilution of three dilution factors is coated on cellulose enrichment conditions base tablet
On, it inserts and culture 2 days is inverted in 25 DEG C of constant incubators.Cellulose enrichment conditions base：CaCl20.1g, MgSO40.1g,
(NH4)2SO42.0g, KH2PO40.5g, K2HPO42.0g, NaCl 6g, agar 1.5%, sodium carboxymethylcellulose 0.5% steam
Distilled water 1000mL, pH is adjusted to 7.0, sterilize in 121 DEG C, under 0.11Mpa 30 minutes it is spare.
The well-grown bacterium colony point of sterilizing toothpick picking is placed in 25 DEG C of constant temperature incubations on cellulose screening and culturing medium tablet
It is cultivated 3 days in case.After bacterium colony is grown, the Congo red dye liquor of appropriate 1mg/mL is added in into tablet, dyes 10 minutes, goes to contaminate
Liquid adds the NaCl solution of appropriate 1mol/L, impregnates 5 minutes and carries out decolorization, whether there is with size to sentence according to transparent circle
Disconnected and selection, the result is shown in Figure 1.
2nd, the Molecular Identification of cold cellulase producing bacteria is fitted
Bacterial gene is extracted using the TIANamp Bacteria DNA Kit of TIANGEN Biotech (Beijing) Co., Ltd.
Group DNA, takes 5mL bacterium solutions, thalline were collected by centrifugation under 10000rpm, supernatant discarding.By 200 μ L buffer solutions GA, 20 μ L
Proteinase K solution, 220 μ L buffer solutions GB, 500 μ L buffer solutions GD, 600 μ L rinsing liquids PW, 50 μ L distilled water according to will
It asks and sequentially adds, obtain bacterial strain La1 genomic DNAs.
Using the genomic DNA of strain to be tested as template, carrying out 16S rDNA amplifications, (94 DEG C are denatured 3 minutes, 94 DEG C of unwindings 30
Second, 55 DEG C are annealed 30 seconds, and 72 DEG C extend 1.5 minutes, repeat 30 Xun Huans of 2-4 step reactions, 72 DEG C extend 8 minutes.Reaction knot
Shu Hou is detected into row agarose gel electrophoresis, is recycled PCR product using TIANGEN Ago-Gel DNA QIAquick Gel Extraction Kits, is sent
Sequencing identification is carried out to Jin Sirui bio tech ltd.Blast program is submitted to exist the 16SrDNA sequences of acquisition
Sequence alignment is carried out in GenBank, analyzes the molecular biology classification status of the bacterial strain.It turns out that the 16SrDNA bases of bacterial strain
Because sequence is as described in SEQ ID NO.1, it is salt tolerant Liu Zhi perseverance bacterium that this, which fits cold cellulase producing bacteria Classification And Nomenclature,
(Zhihengliuella halotolerans)La1。
Embodiment 1
La1 bacterial strains (of the invention fits cold cellulase producing bacteria) are inoculated in 20mL liquid LB (yeast using oese
Powder 5g, sodium chloride 10g, peptone 10g, distilled water 1L, pH are adjusted in 7.4), and it is small that 24 are cultivated in 28 DEG C of 180rpm shaking tables
When.Gained bacterium solution is forwarded to by 2% inoculum concentration equipped with fermentation medium (dusty yeast 5g, sodium chloride 10g, peptone 10g, carboxylic
Sodium carboxymethylcellulose pyce 0.5g, distilled water 1L, pH are adjusted into triangular flask 7.4), and it is small that 48 are cultivated in 28 DEG C of 180rpm shaking tables
When.After fermentation, zymotic fluid 4000rmp rotating speeds under the conditions of 4 DEG C is taken to centrifuge 30 minutes, it is spare as enzyme solution to collect supernatant.
Embodiment 2
La1 bacterial strains (of the invention fits cold cellulase producing bacteria) are inoculated in 20mL liquid LB (yeast using oese
Powder 4g, sodium chloride 8g, peptone 8g, distilled water 1L, pH adjust in 7.2), cultivated in 30 DEG C of 300rpm shaking tables 36 it is small when.
Gained bacterium solution is forwarded to by 3% inoculum concentration equipped with fermentation medium (dusty yeast 4g, sodium chloride 8g, peptone 8g, carboxymethyl fibre
The plain sodium 0.4g of dimension, distilled water 1L, pH is adjusted into triangular flask 7.6), when culture 50 is small in 30 DEG C of 300rpm shaking tables.Fermentation
After, zymotic fluid 5000rmp rotating speeds under the conditions of 6 DEG C is taken to centrifuge 20 minutes, it is spare as enzyme solution to collect supernatant.
Embodiment 3
La1 bacterial strains (of the invention fits cold cellulase producing bacteria) are inoculated in 20mL liquid LB (yeast using oese
Powder 6g, sodium chloride 12g, peptone 11g, distilled water 1L, pH are adjusted in 7.6), and it is small that 18 are cultivated in 24 DEG C of 100rpm shaking tables
When.Gained bacterium solution is forwarded to by 2% inoculum concentration equipped with fermentation medium (dusty yeast 6g, sodium chloride 12g, peptone 11g, carboxylic
Sodium carboxymethylcellulose pyce 0.6g, distilled water 1L, pH are adjusted into triangular flask 7.6), and it is small that 40 are cultivated in 24 DEG C of 100rpm shaking tables
When.After fermentation, zymotic fluid 3000rmp rotating speeds under the conditions of 2 DEG C is taken to centrifuge 25 minutes, it is spare as enzyme solution to collect supernatant.
Detect example 1
Fit the determination of activity of cold cellulase
1st, the formulation of concentration of glucose standard curve
The glucose solution of the 1mg/mL of different volumes is taken respectively, and it is 1mL to add in distilled water to total volume, is obtained a series of
The glucose solution of various concentration.1mL DNS solution is dispensed into each pipe, mixing, boil 5min and be placed in cold water several points
Clock adds in distilled water into each tube reaction liquid to 10mL.Light absorption value is surveyed under 540nm.Using the data obtained, glucose mark is drawn
Directrix curve.
2nd, standard conditions enzyme activity determination
The enzyme solution obtained in 1.0% carboxymethylcellulose sodium solution of 0.5mLpH 5.0 and embodiment 1-3 is taken respectively,
30 DEG C of water-bath 60min.1mL DNS are added in after reaction.It is cooled down after boiling water bath 5min, adds 8mL distilled water
Dilute reaction solution measures light absorption value in 540nm.Control group is not added with enzyme solution, adds in 0.5mL enzyme solutions and 100mL after reaction
DNS reagents, afterwards repeat before the step of.Standard solution is made with glucose, with the enzyme needed for 1 μm of ol glucose of generation per minute
Amount is as an enzyme-activity unit, then calculates the enzyme activity of the cellulase.
As a result the strain enzyme-producing vigor is 0.3U/mL, it is seen that cold cellulase of fitting of the invention has certain hydrolysis fibre
The plain vigor of dimension.
Detect example 2
With reference to detection example 1 in enzyme activity determination method measure enzyme optimum temperature, respectively 0 DEG C, 10 DEG C, 20 DEG C, 30 DEG C, 40
DEG C, 50 DEG C, 60 DEG C, 70 DEG C, add enzyme solution under the conditions of 80 DEG C and measure enzyme activity, calculate each reaction system enzyme activity.The result is shown in
Fig. 2, from Figure 2 it can be seen that the optimal reactive temperature of the enzyme is 35 DEG C.
Detect example 3
The optimal pH of enzyme is measured with reference to 1 enzyme activity determination method of detection example, dilutes enzyme solution with the different buffer solutions of pH2-11, and
Its vigor is measured, calculates each reaction system enzyme activity, the result is shown in Fig. 3.As seen from Figure 3, the optimal reaction pH of the enzyme is
5.0。
Detect example 4
Influence of each metal ion species to enzyme activity is measured with reference to 1 enzyme activity determination method of detection example, is added respectively into enzyme solution
Enter different ions, including K+、Li+、NH4 +、Cu2+、Co2+、Rb2+、Mg2+、Ca2+、Zn2+、Mn2+, SDS, T-100, EDTA, make it
Final concentration of 1mmol/L, while using untreated enzyme solution as control, under the conditions of the optimum temperature and optimum pH of enzyme reaction
The influence of each ions enzyme vigor is measured, calculates each reaction system enzyme activity, the result is shown in Fig. 4.From fig. 4, it can be seen that the enzyme pair
Most of metal ion and chemical reagent have tolerance.
Above-mentioned testing result show the present invention fit the cellulase that is produced of cold cellulase producing bacteria have suitable to cold,
The characteristic of acid and alkali-resistance.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail, within the scope of the technical concept of the present invention, a variety of simple variants can be carried out to technical scheme, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that the specific technical features described in the above specific embodiments, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Sequence table
<110>Anhui Normal University
<120>Fit cold cellulase producing bacteria
<130> 05760
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1512
<212> DNA
<400> 1
agagtttgat cctggctcag gatgaacgct ggcggcgtgc ttaacacatg caagtcgaac 60
gatgaagccc agcttgctgg gtggattagt ggcgaacggg tgagtaacac gtgagtaacc 120
tgccctcgac tccaggataa gcccgggaaa ctgggtctaa tactggatat tcaatttcta 180
ccgcatggtg gtttttggaa aggattctgg tcgaggaggg actcgcggcc tatcagcttg 240
ttggtgaggt aatggctcac caaggcgacg acgggtagcc ggcctgagag ggtgaccggc 300
cacactggga ctgagacacg gcccagactc ctacgggagg cagcagtggg gaatattgca 360
caatgggcga aagcctgatg cagcgacgcc gcgtgaggga tgacggcctt cgggttgtaa 420
acctctttca gtagggaaga agcgaaagtg acggtacctg cagaagaagc gccggctaac 480
tacgtgccag cagccgcggt aatacgtagg gcgcaagcgt tatccggaat tattgggcgt 540
aaagagctcg taggcggttt gtcgcgtctg ccgtgaaagt ccggggctta actccggatc 600
tgcggtgggt acgggcagac tagagtgctg taggggagac tggaattcct ggtgtagcgg 660
tgaaatgcgc agatatcagg aggaacaccg atggcgaagg caggtctctg ggcagtaact 720
gacgctgagg agcgaaagca tggggagcga acaggattag ataccctggt agtccatgcc 780
gtaaacgttg ggcactagat gtgggggaca ttccacgttt tccgcgtcgt agctaacgca 840
ttaagtgccc cgcctgggga gtacggccgc aaggctaaaa ctcaaaggaa ttgacggggg 900
cccgcacaag cggcggagca tgcggattaa ttcgatgcaa cgcgaagaac cttaccaagg 960
cttgacatgg accggatcgg gctagaaata gtctttccct tcggggctgg ttcacaggtg 1020
gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1080
accctcgttc tatgttgcca gcacgtgatg gtggggactc ataggagact gccggggtca 1140
actcggagga aggtggggac gacgtcaaat catcatgccc cttatgtctt gggcttcacg 1200
catgctacaa tggccggtac aatgggttgc gatactgtga ggtggagcta atcccaaaaa 1260
gccggtctca gttcggattg gggtctgcaa ctcgacccca tgaagtcgga gtcgctagta 1320
atcgcagatc agcaacgctg cggtgaatac gttcccgggc cttgtacaca ccgcccgtca 1380
agtcacgaaa gttggtaaca cccgaagctg gtggcctaac cccttgtggg aaggagctgt 1440
cgaaggtggg accggcgatt gggactaagt cgtaacaagg tagccgtacc ggaaggtgcg 1500
gctggatcac ct 1512
Claims (1)
1. one kind fits cold cellulase producing bacteria, which is characterized in that described to fit cold cellulase producing bacteria in Chinese common micro- life
The deposit number of object culture presevation administrative center (CGMCC) is 14938, and preservation date is on November 20th, 2017.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110564631A (en) * | 2019-08-09 | 2019-12-13 | 辽宁省农业科学院 | yeast for producing cellulase at low temperature and screening method thereof |
| CN111879741A (en) * | 2020-07-15 | 2020-11-03 | 安徽师范大学 | Method for detecting activity of alpha-glucosidase |
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| CN102533563A (en) * | 2011-11-08 | 2012-07-04 | 中国科学院微生物研究所 | Celluase producing bacterium and application thereof |
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| CN102533563A (en) * | 2011-11-08 | 2012-07-04 | 中国科学院微生物研究所 | Celluase producing bacterium and application thereof |
| CN105039195A (en) * | 2015-05-22 | 2015-11-11 | 中国科学院水生生物研究所 | Cellulase producing bacteria and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110564631A (en) * | 2019-08-09 | 2019-12-13 | 辽宁省农业科学院 | yeast for producing cellulase at low temperature and screening method thereof |
| CN110564631B (en) * | 2019-08-09 | 2022-11-15 | 辽宁省农业科学院 | Yeast for producing cellulase at low temperature and screening method thereof |
| CN111879741A (en) * | 2020-07-15 | 2020-11-03 | 安徽师范大学 | Method for detecting activity of alpha-glucosidase |
| CN111879741B (en) * | 2020-07-15 | 2023-03-28 | 安徽师范大学 | Method for detecting activity of alpha-glucosidase |
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