CN106434417A - High-temperature-resistant cellulase producing bacterium and application thereof - Google Patents
High-temperature-resistant cellulase producing bacterium and application thereof Download PDFInfo
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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
- 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
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F11/00—Other organic fertilisers
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- C05—FERTILISERS; MANUFACTURE THEREOF
- C05F—ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C05F17/00—Preparation of fertilisers characterised by biological or biochemical treatment steps, e.g. composting or fermentation
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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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- 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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- 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 relates to a high-temperature-resistant cellulase producing bacterium and application thereof, and belongs to the technical field of microbiological screening and application. The strain is separated from a mixture of cow dung and straw in an earthworm farm, identified as bacillus subtilis, named as AW1601 and preserved in the China General Microbiological Culture Collection Center on June 24, 2016, and the number is CGMCC No.12650. The strain has the high cellulase producing capacity at the high culture temperature of 50 DEG C to 55 DEG C, the fermentation cycle is short in liquid and solid environments, the degrading degree of straw substrates is high, the problems that a cellulase preparation is high in cost, a high temperature composting strain is low in activity, and the fiber composition degrading rate of the substrates is low when an edible fungus culture medium or coarse fodder is prepared are well solved, and the strain can serve as a novel cellulase producing bacterium and be widely used for recycling agricultural biomass solid waste.
Description
Technical field
The invention belongs to microbe to screen and applied technical field and in particular to one plant of high temperature resistant cellulase-producing bacterium and its
Application, is mainly used in cellulase-producing and the resource of straw-stalk type cultural solid garbage.
Background technology
Cellulose is to be distributed the carbohydrate the widest, reserves are maximum in nature.Plant cell wall mainly by cellulose,
Hemicellulose and lignin composition, wherein the minimum composition unit of cellulose and hemicellulose be glucose, xylose, galactose and
Arabinose.Only by these pentoses and hexose by extracting in Plant fiber and separating, cellulose resource just can be had
The utilization of effect.However, as a kind of Renewable resource, cellulose is not fully developed and efficient utilization, such as agricultural solid
The utilization rates such as the straw in body garbage, Caulis et Folium Oryzae and bagasse are not high.
The straw enormous amount that China produces with rural activity every year, wherein corn straw is the most normal in northern area
See.After waxiness, the cellulose of corn straw and hemicellulose total content can reach more than the 50% of its dry weight, through effective
Process can be used in producing fertilizer, feedstuff, life fuel and edible fungi base material etc..
There is funguses and the antibacterial that can produce cellulase in a large number, this kind of microbial resources are separated, sieve in nature
After choosing and domestication, typically there is cellulose degradation efficiency high, gentle to person poultry harmless, action condition, give up in cultural solid
Waste resource aspect has broad application prospects.Therefore, constantly screen the bacterial strain of High Cellulase Production and develop corresponding
Technology application recycles significant for China's agricultural resource.
Content of the invention
It is an object of the invention to provide one plant of high temperature resistant cellulase-producing bacterium and its application, with cellulose for high temperature during substrate
Culture is obtained in that higher enzymatic productivity, improves straw-stalk type cultural castoff compost or produces the recycling such as culture medium of edible fungus
Efficiency.
High temperature resistant cellulase producing strain in the present invention is bacillus subtilises（Bacillus subtilis）, name
For AW1601, on June 24th, 2016 is preserved in BeiChen West Road, Chaoyang District, BeiJing City institute of microbiology of Chinese Academy of Sciences China Microbiological
Culture presevation administration committee common micro-organisms center, preserving number is CGMCC No.12650.
Strains A W1601 is isolatable from vermiculture field cattle manure and straw mixture, and Gram’s staining is the positive, and bacterium colony is in breast
White, circle are no raised, and single thalline becomes shaft-like, and after LB culture medium culturing 24 h, thalline is long 1.5-2.5 μm.Using primer 2 7F
Carry out the PCR amplification of 16S rRNA gene with 1448R, obtain rDNA sequence and submit to GenBank to obtain strain sequence number
JQ973708, BLAST sequence analysis have 99% homology with least 30 kinds bacillus subtilises, identify that AW1601 is withered
Careless bacillus cereuss（Bacillus subtilis）.
During using LB culture medium, Strain survival condition is：Temperature 20-75 DEG C, pH 6.5-8.0.
Corn straw is used as carbon source（The g containing straw 10.0 in every 1000 mL water, NaCl 5.0 g, KH2PO41.0
G, MgSO40.2 g, peptone 1.0 g, yeast powder 1.0 g）, the optimal condition of enzyme production of strains A W1601 is temperature 50 C, pH
7.0.After cultivating 3 d, CMC enzyme activity reaches 138.0 IU/mL, and FPA enzyme activity reaches 178.4 IU/mL.
It is used corn straw as substrate, concentration of substrate 5%-10%, optimum temperature during liquid fermentation is 50 DEG C, through 7
D degradation of substrates 28.4%, in substrate, cellulose and hemicellulose level reduce 61.5% and 59.9% respectively；Solid fermentation optimal
Temperature is 55 DEG C, and through 7 d degradation of substrates 19.5%, in substrate, cellulose and hemicellulose level decrease 37.2% He respectively
41.1%.
Strains A W1601 according to the present invention belongs to new discovery strain, is located away from vermiculture field cattle manure and corn straw mixing
Thing, Lumbricuss, by crushing, processing with digestion thus improving microorganism growth substrate to mixture, change microorganism in mixture
Group's composition and activity, therefore strains A W1601 are to corn straw using having very strong specificity, efficient degradation substrate
In fibre composition.Cellulose-degrading bacteria typically took from ruminant feces in the past, rot dead leaves, branch or remote mountains humus
Abundant soil, for straw specificity compare weaker.
Strains A W1601 has high-cellulose enzyme enzymatic productivity under higher cultivation temperature, compares room temperature bacterium, and it is applied to
Biomass solid waste compost, culture medium of edible fungus material have more advantage in producing, and 3 d reach producing enzyme peak value, and CMC enzyme activity reaches
138.0 IU/mL, filter paper enzyme activity reaches 178.4 IU/mL, is suitably applied in the production of cellulase, shortens and produces week
Phase, reduces cost.
Brief description
Fig. 1 is strains A W1601 Congo red flat board coloration result.
Fig. 2 is the form of strains A W1601 under ultramicroscope, and wherein A amplification is 3000 times, and B is 9000 times.
Fig. 3 is the impact to strains A W1601 CMC enzyme activity for the temperature and time.
Fig. 4 is the impact to strains A W1601 FPA enzyme activity for the temperature and time.
Fig. 5 is the impact to strains A W1601 degrading maize straws effect for the temperature.
Specific embodiment
Embodiment 1：The separation of bacterial strain, screening and identification
Strains A W1601 is located away from Tianjin vermiculture field cattle manure and straw mixture.Soil sampling 5 g, aseptic water filtration is gone
Except residue, collect filtrate, take supernatant according to 1/10,1/100,1/1000,1/10000 times of gradient dilution, diluent is applied
On LB solid medium, 37 DEG C of constant temperature culture, after bacterium colony, line separates repeatedly, moves to the training of LB test tube after obtaining pure bacterial strain
Foster 4 DEG C of preservations of base.
By bacterial strain difference dibbling after purification in sodium carboxymethyl cellulose plating medium（CMC-Na 15 g, NaCl 5
G, KH2PO41 g, MgSO40.2 g, peptone 1.0 g, yeast powder 1.0 g, agar 18 g, deionized water 1000 mL,
pH7.2）On, each cultivates 3 samples of basic point, 37 DEG C of quiescent culture 4 d, dyes 20 min with 1 mg/mL Congo red dye liquor, discards
1 mol/L NaCl solution is added, after washing 20 min, measurement bacterium colony and transparent loop diameter, are represented with D and H respectively after dye liquor
（Fig. 1）.Each bacterial strain cellulase-producing ability is tentatively judged according to H/D, the transparent loop diameter of AW1601 is maximum after measured, is 27.4
Mm, H/D are 6.0, and therefore AW1601 is carried out with strain identification and further producing enzyme research.
Strains A W1601 Gram’s staining shows the positive, and bacterium colony is creamy white, circle is no raised, 3000 times of ultramicroscope and
Photo such as Fig. 2A and 2B after 9000 times of amplifications, during using LB culture medium, Strain survival condition is temperature 20-75 DEG C, pH 6.5-
8.0, it is initially identified as bacillus subtilises（Bacillus subtilis）.16S rRNA is carried out using primer 2 7F and 1448R
The PCR amplification of gene, the rDNA sequence obtaining submits to GenBank to obtain strain sequence JQ973708, carries out same according to BLAST
Source property compares the homology with least 30 kinds bacillus subtilises with 99%, finally identifies that AW1601 is bacillus subtilises
（Bacillus subtilis）.
Embodiment 2：The condition of enzyme production of strains A W1601
Make corn straw culture medium：Straw 10.0 g, NaCl 5.0 g, KH2PO41.0 g, MgSO40.2 g, albumen
Peptone 1.0 g, yeast powder 1.0 g, 1000 mL deionized waters, 121 DEG C of sterilizing 20 min.Due to measuring strains A W1601 not
With the growth curve under pH, show that pH is that when 7, growth is the most vigorous, therefore corn straw culture medium pH modulates to 7.
With 3 mL sterilized water, thalline is made bacteria suspension, takes 0.5 mL to be inoculated in respectively in culture medium, in 30 DEG C, 35
DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 120 r/min shaking table shaken cultivation at 55 DEG C.Every 24 h take bacterium solution, are centrifuged 15 under 5000 r/min
Min, collects supernatant and measures enzyme activity.Catalytic substrate per minute for cellulase is hydrolyzed generation 1 μ g glucose and is defined as an enzyme
Unit of activity, is represented with IU/mL.
The determination step of CMC enzyme activity：Take CMC buffer 2.0 mL in color comparison tube, add 0.5 mL to dilute 10 times
Enzyme liquid, 30 min in 50 DEG C of thermostat water baths, add 3.0 mL DNS reagent, boiling water bath 10 min, be settled to after cooling
25.0 mL, survey OD value at 540 nm.The determination step of FPA enzyme activity：No. 1 filter paper of 50 mg Xinhua is put into color comparison tube, plus
Enter 0.5 mL and dilute 10 times of enzyme liquid, add 2.0 mL HAc-NaAc buffer, 60 min in 50 DEG C of thermostat water baths, plus
Enter 3.0 mL DNS solution, boiling water bath 10 min, after cooling, be settled to 25.0 mL, survey OD value at 540 nm.After recording OD value
Consult glucose standard curve, obtain enzymolysis gained glucose content, be converted into corresponding enzyme activity force value.
Fig. 3 and Fig. 4 cultivates the enzyme activity force curve of 5 d at different temperatures for AW1601, as seen from the figure, cultivates 3 at 50 DEG C
After d, CMC enzyme activity reaches 138.0 IU/mL, and FPA enzyme activity reaches 178.4 IU/mL.Therefore optimal condition of enzyme production is temperature 50
DEG C, pH 7.0.
Embodiment 3：Liquid fermentation
Strain AW1601 is connected to LB culture medium by slant medium, at 37 DEG C, 120 r/min cultivate 24 h and make seed liquor.
5 g straws are loaded in the shaking flask of 100 mL distilled water, strain inoculum concentration be 3%, fermentation temperature be set as 35 DEG C,
40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C and 60 DEG C, shaking table culture 7 d under 120 r/min.
Difference weight method calculates degradation of substrates rate, and formula is：
Wherein, X is degradation rate；M1 is experimental group substrate weight (g)；M2 is blank group substrate weight (g)；M is liquid fermentation straw
The initial addition of stalk.
Van Soest method is to mensure cellulose and hemicellulose level, before processing corn stalk fiber element and half after measured
Content of cellulose is respectively 34.2% and 25.0%.
As Fig. 5, through 7 d fermentation substrates, at 50 DEG C, degradation rate is maximum, reaches 28.4%, cellulose and half fiber in substrate
Cellulose content is respectively 18.4% and 14.0%, decreases 61.5% and 59.9% respectively.
Embodiment 4：Solid fermentation
Strain AW1601 is connected to LB culture medium by slant medium, at 37 DEG C, 120 r/min cultivate 24 h and make seed liquor.
By crushed stalk to 0.5-1.0 cm, 100 g are taken straw to be dried as fermentation substrate, adjustment moisture content to 55%, bacterium
Planting inoculum concentration is 5%, and fermentation temperature is set as 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C, 55 DEG C and 60 DEG C, standing for fermentation 7 d.
Difference weight method calculates degradation of substrates rate, and formula is：
Wherein, X is degradation rate；M1 is experimental group substrate weight (g)；M2 is blank group substrate weight (g)；M is liquid fermentation straw
The initial addition of stalk.
Van Soest method is to mensure cellulose and hemicellulose level, before processing corn stalk fiber element and half after measured
Content of cellulose is respectively 34.2% and 25.0%.
As Fig. 4, through 7 d fermentation substrates, at 55 DEG C, degradation rate is maximum, reaches 19.5%, cellulose and half fiber in substrate
Cellulose content is respectively 26.7% and 18.3%, decreases 37.2% and 41.1% respectively.
Nucleotide or amino sequence
Bacillus subtilises（Bacillus subtilis）The DNA sequence of AW1601：
1 GCATGGGGGT GCTATACATG CAAGTCGAGC GGACAGATGG GAGCTTGCTC CCTGATGTTA
61 GCGGCGGACG GGTGAGTAAC ACGTGGGTAA CCTGCCTGTA AGACTGGGAT AACTCCGGGA
121 AACCGGGGCT AATACCGGAT GGTTGTTTGA ACCGCATGGT TCAAACATAA AAGGTGGCTT
181 CGGCTACCAC TTACAGATGG ACCCGCGGCG CATTAGCTAG TTGGTGAGGT AACGGCTCAC
241 CAAGGCAACG ATGCGTAGCC GACCTGAGAG GGTGATCGGC CACACTGGGA CTGAGACACG
301 GCCCAGACTC CTACGGGAGG CAGCAGTAGG GAATCTTCCG CAATGGACGA AAGTCTGACG
361 GAGCAACGCC GCGTGAGTGA TGAAGGTTTT CGGATCGTAA AGCTCTGTTG TTAGGGAAGA
421 ACAAGTACCG TTCGAATAGG GCGGTACCTT GACGGTACCT AACCAGAAAG CCACGGCTAA
481 CTACGTGCCA GCAGCCGCGG TAATACGTAG GTGGCAAGCG TTGTCCGGAA TTATTGGGCG
541 TAAAGGGCTC GCAGGCGGTT TCTTAAGTCT GATGTGAAAG CCCCCGGCTC AACCGGGGAG
601 GGTCATTGGA AACTGGGGAA CTTGAGTGCA GAAGAGGAGA GTGGAATTCC ACGTGTAGCG
661 GTGAAATGCG TAGAGATGTG GAGGAACACC AGTGGCGAAG GCGACTCTCT GGTCTGTAAC
721 TGACGCTGAG GAGCGAAAGC GTGGGGAGCG AACAGGATTA GATACCCTGG TAGTCCACGC
781 CGTAAACGAT GAGTGCTAAG TGTTAGGGGG TTTCCGCCCC TTAGTGCTGC AGCTAACGCA
841 TTAAGCACTC CGCCTGGGGA GTACGGTCGC AAGACTGAAA CTCAAAGGAA TTGACGGGGG
901 CCCGCACAAG CGGTGGAGCA TGTGGTTTAA TTCGAAGCAA CGCGAAGAAC CTTACCAGGT
961 CTTGACATCC TCTGACAATC CTAGAGATAG GACGTCCCCT TCGGGGGCAG AGTGACAGGT
1021 GGTGCATGGT TGTCGTCAGC TCGTGTCGTG AGATGTTGGG TTAAGTCCCG CAACGAGCGC
1081 AACCCTTGAT CTTAGTTGCC AGCATTCAGT TGGGCACTCT AAGGTGACTG CCGGTGACAA
1141 ACCGGAGGAA GGTGGGGATG ACGTCAAATC ATCATGCCCC TTATGACCTG GGCTACACAC
1201 GTGCTACAAT GGACAGAACA AAGGGCAGCG AAACCGCGAG GTTAAGCCAA TCCCACAAAT
1261 CTGTTCTCAG TTCGGATCGC AGTCTGCAAC TCGACTGCGT GAAGCTGGAA TCGCTAGTAA
1321 TCGCGGATCA GCATGCCGCG GTGAATACGT TCCCGGGCCT TGTACACACC GCCCGTCACA
1381 CCACGAGAGT TTGTAACACC CGAAGTCGGT GAGGTAACCT TTAGGAGCCA GCCGCCGAAG
1441 GTGAACAT
Claims (5)
1. one plant of high temperature resistant cellulase-producing bacterium and its application, is characterized in that：This is high temperature resistant, and cellulase producing strain is entitled
AW1601, is identified as bacillus subtilises（Bacillus subtilis）, it is preserved in Chinese microorganism strain preservation management committee
Member's meeting common micro-organisms center, deposit number is CGMCC No.12650.
2. one plant of high temperature resistant cellulase-producing bacterium according to claim 1, is producing cellulase and straw-stalk type cultural life
Apply in the resource of material solid garbage.
3. one plant of high temperature resistant cellulase-producing bacterium according to claim 1 and its application, is characterized in that：This strain enzyme-producing is trained
Foster base is the g containing straw 10.0 in every 1000 mL water, NaCl 5.0 g, KH2PO4 1.0 g, MgSO4 0.2 g, peptone 1.0
G, yeast powder 1.0 g, pH 7.0, producing enzyme temperature 50 C, revolution 120 rpm, incubation time 3-4 days.
4. one plant of high temperature resistant cellulase-producing bacterium according to claim 1 and its application, is characterized in that：This bacterial strain liquid is sent out
Ferment concentration of substrate is 5%-10%, inoculum concentration 3%, temperature 50 C, 7 days time.
5. one plant of high temperature resistant cellulase-producing bacterium according to claim 1 and its application, is characterized in that：This bacterial strain solid-state is sent out
Ferment substrate moisture content 55%, inoculum concentration 5%, 55 DEG C of temperature, 7 days time.
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| CN108148777A (en) * | 2018-01-10 | 2018-06-12 | 上海成土养木环保科技有限公司 | A kind of bacillus subtilis, complex micro organism fungicide, organic substrate and its preparation and application |
| CN109136153A (en) * | 2018-09-29 | 2019-01-04 | 中国科学院成都生物研究所 | One plant of salt tolerant bacillus for having a Plant growth promotion |
| CN109402014A (en) * | 2018-11-20 | 2019-03-01 | 江南大学 | A kind of bacillus of cellulase-producing and its application |
| CN109439574A (en) * | 2018-11-08 | 2019-03-08 | 唐山师范学院 | Cellulase producing bacteria and its application |
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| CN109824453A (en) * | 2018-10-26 | 2019-05-31 | 博白县三桦石柳生态农业开发有限责任公司 | Cassava residue organic fertilizer and its fermentation preparation containing bacillus subtilis |
| CN109824455A (en) * | 2018-10-26 | 2019-05-31 | 博白县三桦石柳生态农业开发有限责任公司 | Filter mud of sugarcane organic fertilizer containing bacillus subtilis and preparation method thereof |
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| CN114395506A (en) * | 2022-01-12 | 2022-04-26 | 福建省农业科学院农业生物资源研究所 | High-temperature-resistant cellulase-producing bacillus subtilis and culture method and application thereof |
| CN114560726A (en) * | 2022-04-04 | 2022-05-31 | 广东省农业科学院茶叶研究所 | Method for promoting efficient utilization of soil organic waste by virtue of earthworm and cellulose decomposition bacteria |
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| CN115637243A (en) * | 2022-10-31 | 2023-01-24 | 广西壮族自治区兽医研究所 | Bacterial strain for high-yield cellulase, screening method and application thereof |
| CN114480223B (en) * | 2022-03-21 | 2023-08-29 | 河北科技大学 | Geobacillus thermodenitrificans HX-4 and method for producing cellulase by using same and application of Geobacillus thermodenitrificans HX-4 |
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