CN110172417A - One bacillus and its application - Google Patents
One bacillus and its application Download PDFInfo
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- CN110172417A CN110172417A CN201910374063.6A CN201910374063A CN110172417A CN 110172417 A CN110172417 A CN 110172417A CN 201910374063 A CN201910374063 A CN 201910374063A CN 110172417 A CN110172417 A CN 110172417A
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- C—CHEMISTRY; METALLURGY
- 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
- C05F11/08—Organic fertilisers containing added bacterial cultures, mycelia or the like
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- 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)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
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Abstract
The invention belongs to technical field of microbial fermentation, disclose a bacillus and its application.The bacillus is bacillus (Bacillus sp.) CQUST1, and Guangdong Province's Culture Collection was preserved on September 20th, 2018, and deposit number is GDMCC No:60448.Bacillus of the invention finds that fermentation liquid has significant proteinase activity by transparent circle method.The invention also discloses the relationships and the purposes in agricultural wastes accumulation of the bacillus protease production and fermentation temperature.For stalk mainly by the difficult decomposition such as lignin, cellulose, hemicellulose at being grouped as, naturally decomposed speed is slower.Straw-returning technology can effectively improve soil organic matter content, improve soil physical properties, enhance soil microbial activities, it increases soil fertility, increase soil moisture content, improve crop yield, reduces and polluted caused by agricultural production, the resource utilization for realizing stalk, to promote the healthy and rapid development of circular agriculture.
Description
Technical field
The present invention relates to technical field of microbial fermentation, and in particular to a bacillus CQUST1 and its produces protease
Using.
Background technique
Protease is the class of enzymes of aminosal peptide bond, the enzyme both can by protein molecule inner cut-out, formed compared with
The peptone of small-molecular-weight, can also by C-terminal in protein molecule or the peptide bond hydrolysis of N-terminal, and it is free go out amino acid, to realize hydrolysis
Protein.The basic physiological activity of the organisms such as food digestion, blood pressure control, blood clotting be unable to do without the participation of protease,
Therefore, protease is widely used each neck in human lives such as leather industry, Gelatin Industry, food industry and detergent
Domain (industrial microorganism, 2008,38,4:49-61).Clinically, protease occupies main status in enzyme therapy, is usually used in controlling
Treat indigestion, bronchitis etc., it may also be used for anti-inflammatory detumescence removes necrotic tissue, such as post-operation inflammatory swelling and bladder
Inflammation etc. (life science information, 1991,8,2:8), it is seen that protease has extensive demand prospect.
Currently, protease is broadly divided into two class of endopeptidase and exopeptidase, it is widely present in pluck, plant stem-leaf, fruit
In real, microorganism.The microorganism for generating protease is many kinds of in nature, including bacterium, fungi, actinomyces etc., distribution
Also very extensive.Influence protease factor of production is various, and same microorganism generates more protease under different condition of culture
Type is also different.Industrially use bacillus licheniformis C1213 and 2709, bacillus pumilus 209 and 289 and basophilic
Property the production alkali protease such as bacillus B45, produce neutral and acid protease using aspergillus, head mold, aspergillus oryzae etc., and chestnut
Phytophthora, Mucor are used to produce the enzyme (Qinghai science and technology, 2018,2:73-76) of curds type.In Bacteria Culture, usually with liquid depth
Layer cultivation production in, alkali protease, and use plate method production mold protease (Science Bulletin, 1974,19,1:
34-37)。
Bacillus belongs to gram-positive bacteria, can form gemma, and form has rod-shaped and spherical, is widely present in soil
Earth, water, air and animal intestinal tract etc. can resist extraneous injurious factor, be the industrial main bacteria seed for producing alkali protease.
The culture propagation is very competent, can be proliferated within four hours 100,000 times, and ability is considerably beyond other common strains；There is tolerance simultaneously
The high feature of power, not only resistance to strong acid, resistance to highly basic, some bacterium also -60 DEG C of low-temperature-resistant or+280 DEG C of high temperature resistant.Main applying to
In fertilizer addition, pesticide addition, compost and liquid fertilizer production and the productions activity such as lignocellulose degradation (China Brewing, 2009,
205,4:102-103).In terms of bioengineering, due to getting worse for antibiotic pollution problem, beneficial bacillus is answered
With research, it may be possible to solve the problems, such as an effective scheme of antibiotic.
Summary of the invention
The purpose of the present invention is to provide a bacillus CQUST1 and its applications；Bacillus (the Bacillus
Sp.) tunning of CQUST1 has significant proteinase activity.
Bacillus (Bacillus sp.) CQUST1 of the present invention is isolated from agriculture in the national industry garden of Chongqing Bishan
In industry product Natural compost, pure medium is LB culture medium, the well-grown on LB culture medium；Bacillus CQUST1 table
Face is coarse opaque, and bacterium colony is rod-shaped in yellowish.
The purpose of the present invention is achieved through the following technical solutions:
In a first aspect, the present invention provides a bacillus (Bacillus sp.) CQUST1, it is preserved in the micro- life in Guangdong Province
Object Culture Collection Center, culture presevation number are GDMCC No:60448, and depositary institution address is Xianlie Middle Road, Guangzhou City 100
5 building, the building of compound the 59th, the deposit date is on September 20th, 2018.
Preferably, the pure medium of the bacillus is LB culture medium；The composition of LB culture medium described in every 1L are as follows: pancreas
Peptone 10g, yeast extract 5g, sodium chloride 10g, agar 15g, distilled water are settled to 1L, 7.2,121 DEG C of moist heat sterilizations of pH
30min。
Preferably, the tunning of the bacillus has proteinase activity.
Second aspect, the invention further relates to the purposes of the bacillus.Egg is being prepared including bacillus CQUST1
Purposes in white enzyme.With the purposes in fermentation agricultural wastes compost.
Further, the tunning (protease) of the bacillus can also be used for agricultural wastes compost treatment.
The tunning of the bacillus is that method as follows is prepared: will be from Chongqing Bishan state family property
Isolated strain is inoculated into LB culture medium in industry garden, is incubated overnight 12h in 37 DEG C of insulating boxs；LB culture medium described in every 1L
Composition are as follows: tryptone 10g, yeast extract 5g, sodium chloride 10g, agar 15g, distilled water are settled to 1L, pH 7.2,121
DEG C moist heat sterilization 30min.
The determination of bacillus CQUST1 tunning: selecting suitable single colonie, is inoculated into skimmed milk power plate culture
In base, 12h is incubated overnight in 37 DEG C of -50 DEG C of insulating boxs.Tunning confirmation culture medium used is the culture of skimmed milk power plate
Base, the composition of skimmed milk power plating medium described in every 1L are as follows: tryptone 10g, yeast extract 5g, sodium chloride 10g, agar
15g, skim milk 0.5-1 μ l, distilled water are settled to 1L, 7.2,121 DEG C of moist heat sterilization 30min of pH.
The device have the advantages that are as follows: the present invention provides a bacillus, according to skimmed milk power plate culture
Base transparent circle shows that its fermentation liquid has proteinase activity, can aminosal.The invention also discloses the productions of this bacillus
The relationship of proteinase activity and fermentation temperature, method of proof in control bacillus skimmed milk power plating medium by fermenting
Temperature, it was demonstrated that the relationship of tunning and fermentation temperature with protease production.
For stalk mainly by the difficult decomposition such as lignin, cellulose, hemicellulose at being grouped as, naturally decomposed speed is slower.
Bacillus CQUST1 can be by high polymerization degree cellulose and hemicellulose degradation at the glucan and xylan of small molecule, and small point
Sub- substance is dissolved in water and improves straw-returning efficiency.So as to effectively improve soil organic matter content using straw-returning technology,
Improve soil physical properties, enhance soil microbial activities, increase soil fertility, increase soil moisture content, improves crop yield,
It reduces and is polluted caused by agricultural production, the resource utilization of stalk is realized, to promote the healthy and rapid development of circular agriculture.
Detailed description of the invention
The colonial morphology of bacterial strain CQUST1 is shown in Fig. 1；
The transparent circle situation that bacterial strain CQUST1 occurs in skimmed milk power plating medium is shown in Fig. 2；
Each group tobacco dry weight and weight in wet base arithmetic mean of instantaneous value is shown in Fig. 3.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment 1: the separation of bacillus CQUST1
The sampling of high temperature nuclear reactor fatbits laboratory will be taken back out of Chongqing Bishan national industry garden with sterile chamber, with selective LB
Culture medium is incubated overnight 12h in 37 DEG C of insulating boxs, isolates 113 kinds of strains using stalk as carbon source, bacterium high temperature resistant, and
Secretion activity high cellulase and hemicellulase at high temperature, therefore by the strain isolation, that is, obtain CQUST1.
Embodiment 2: the identification of bacterial strain CQUST1
For bacterial strain isolated in embodiment 1, comprehensive the bacterium morphological feature, physiological and biochemical property, 16S rRNA sequence
Column etc. identify that this plant of bacterium is bacillus.Specific qualification result is as follows:
1. thalli morphology
Bacterial strain CQUST1 well-grown on LB culture medium, coarse opaque, surface folding, bacterium colony be it is rod-shaped, have micro- Huang
Color spore (see Fig. 1).
2. physiological and biochemical property
Bacterial strain CQUST1 can be grown on the LB culture medium of higher temperature, belong to high temperature resistant bacterium, bacterium colony is in 37 DEG C, 40
DEG C, 45 DEG C, 48 DEG C, grow preferably in 50 DEG C of skimmed milk power culture medium.
3.16S rRNA is identified
According to NCBI blast comparative analysis, bacterial strain CQUST1 and bacillus licheniformis
Strain (100.00%) shows highest sequence homology.
The 16S rRNA complete nucleotide sequence overall length of bacillus CQUST1 is 1427bp, and sequence is as follows:
ATAATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGT
GGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTT
CAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTC
ACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACG
GGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCG
GATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAA
GCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGG
GCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTT
GAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAG
GCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCA
CGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCC
TGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAAT
TCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGC
AGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC
TTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGAC
GTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGA
GGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTA
GTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTG
TAACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCGAAGT
Embodiment 3: the relationship of bacillus protease production and fermentation temperature
(1) fermentation of bacillus CQUST1
Isolated strain out of Chongqing Bishan national industry garden is inoculated into LB culture medium, mistake in 37 DEG C of insulating boxs
Night cultivates 12h；
The culture medium is LB culture medium, composition: tryptone 10g, yeast extract 5g, sodium chloride 10g, agar
15g, distilled water are settled to 1L, 7.2,121 DEG C of moist heat sterilization 30min of pH.
(2) determination of bacillus CQUST1 tunning
The suitable single colonie of selection, is inoculated into skimmed milk power plating medium, is incubated overnight in 37 DEG C of -50 DEG C of insulating boxs
12h；
The culture medium is skimmed milk power plating medium, composition: tryptone 10g, yeast extract 5g, sodium chloride
10g, agar 15g, skim milk 0.5-1 μ l, distilled water are settled to 1L, 7.2,121 DEG C of moist heat sterilization 30min of pH.
(3) determination of bacillus CQUST1 protease production and fermentation temperature relationship
After skimmed milk power culture medium is cultivated for 24 hours under different temperatures insulating box respectively, it can be seen on isolation medium
It observes and generates macroscopic hydrolysis circle around bacterial strain.Amount to 5 culture mediums, there are 3 single colonies in each culture, it is seen that 15
A bacterial strain and its permeable Xie Quan of generation, measure its colony diameter and its hydrolytic circle respectively, as a result such as table 1.
1 bacillus CQUST1 bacteria produced proteinase of table falls diameter and hydrolytic circle
The bacterial strain separated is grown on plate containing skimmed milk power, is analyzed the data obtained, hydrolytic circle and
The ratio (R/r) of colony diameter is maximum to be.It can be seen that bacillus CQUST1 protease production at 45 DEG C is most strong, 37
At DEG C -45 DEG C, protease production is in rising trend with the raising of fermentation temperature；It and is more than to produce after 45 DEG C in fermentation temperature
Proteinase activity is declined, and analysis is shown in Table 2.
The ratio of table 2 hydrolytic circle and colony diameter
Embodiment 4: the purposes of bacillus CQUST1
Bacillus CQUST1 is not in the case where adding any inorganic salts, in 8% maize straw powder culture medium
Growth, and promote Corn Stalk Decomposition, can effectively degrade agricultural production waste.
Embodiment 5:
Bacillus CQUST1 can be with the microcrystalline cellulose of 0.5%-3%, sodium carboxymethylcellulose, corn stover, green pepper
Residual branch of residual branch, tomato etc. is sole carbon source growth, therefore the agricultural production waste that can be used for degrading.Plant tobacco in mode
For material, 0.1g or so tobacco seed is weighed, is seeded in and soaks soil surface, components of soil is not A group: vermiculite: corn stalk
Stalk compost: perlite=6:3:1, V/V；B group: vermiculite: Nutrition Soil: perlite=6:3:1, V/V；C group: vermiculite: sandy soil: pearl
Rock=6:3:1, V/V, every group totally 12 tobacco, surface are covered to move back for preservative film 5 days and be removed, transplanted after Tobacco Seed Germination 7 days
Into plastics flowerpot, continued growth started to compare tobacco dry weight and weight in wet base after 6 weeks, and in triplicate, Fig. 3 show each group for the experiment
Dry weight and weight in wet base arithmetic mean of instantaneous value, it was demonstrated that the biological tobacco amount highest of addition CQUST1 compost treatment group.
Sequence table
<110>Chongqing University of Science and Technology
<120>one bacillus and its application
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1427
<212> DNA
<213> Bacillus sp.
<220>
<221> misc_feature
<222> (1)..(1427)
<223> 16S rRNA
<400> 1
ataatgcagt cgagcggaca gatgggagct tgctccctga tgttagcggc ggacgggtga 60
gtaacacgtg ggtaacctgc ctgtaagact gggataactc cgggaaaccg gggctaatac 120
cggatggttg tttgaaccgc atggttcaaa cataaaaggt ggcttcggct accacttaca 180
gatggacccg cggcgcatta gctagttggt gaggtaacgg ctcaccaagg caacgatgcg 240
tagccgacct gagagggtga tcggccacac tgggactgag acacggccca gactcctacg 300
ggaggcagca gtagggaatc ttccgcaatg gacgaaagtc tgacggagca acgccgcgtg 360
agtgatgaag gttttcggat cgtaaagctc tgttgttagg gaagaacaag taccgttcga 420
atagggcggt accttgacgg tacctaacca gaaagccacg gctaactacg tgccagcagc 480
cgcggtaata cgtaggtggc aagcgttgtc cggaattatt gggcgtaaag ggctcgcagg 540
cggtttctta agtctgatgt gaaagccccc ggctcaaccg gggagggtca ttggaaactg 600
gggaacttga gtgcagaaga ggagagtgga attccacgtg tagcggtgaa atgcgtagag 660
atgtggagga acaccagtgg cgaaggcgac tctctggtct gtaactgacg ctgaggagcg 720
aaagcgtggg gagcgaacag gattagatac cctggtagtc cacgccgtaa acgatgagtg 780
ctaagtgtta gggggtttcc gccccttagt gctgcagcta acgcattaag cactccgcct 840
ggggagtacg gtcgcaagac tgaaactcaa aggaattgac gggggcccgc acaagcggtg 900
gagcatgtgg tttaattcga agcaacgcga agaaccttac caggtcttga catcctctga 960
caatcctaga gataggacgt ccccttcggg ggcagagtga caggtggtgc atggttgtcg 1020
tcagctcgtg tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttgatcttag 1080
ttgccagcat tcagttgggc actctaaggt gactgccggt gacaaaccgg aggaaggtgg 1140
ggatgacgtc aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggaca 1200
gaacaaaggg cagcgaaacc gcgaggttaa gccaatccca caaatctgtt ctcagttcgg 1260
atcgcagtct gcaactcgac tgcgtgaagc tggaatcgct agtaatcgcg gatcagcatg 1320
ccgcggtgaa tacgttcccg ggccttgtac acaccgcccg tcacaccacg agagtttgta 1380
acacccgaag tcggtgaggt aaccttttag gagccagccg ccgaagt 1427
Claims (6)
1. a bacillus (Bacillus sp.) CQUST1, is preserved in Guangdong Province's Culture Collection, deposit number
For GDMCC No:60448.
2. bacillus (Bacillus sp.) CQUST1 as described in claim 1, it is characterised in that: bud is cultivated in the purifying
The culture medium of spore bacillus (Bacillus sp.) CQUST1 is LB culture medium；The composition of LB culture medium described in every 1L are as follows: pancreas egg
White peptone 10g, yeast extract 5g, sodium chloride 10g, agar 15g, distilled water are settled to 1L, 7.2,121 DEG C of moist heat sterilizations of pH
30min。
3. bacillus (Bacillus sp.) CQUST1 described in claim 1 is preparing the purposes in protease.
4. use of bacillus (Bacillus sp.) CQUST1 described in claim 1 in fermentation agricultural wastes compost
On the way.
5. purposes as described in claim 3 or 4, it is characterised in that: the fermented and cultured process of the bacillus CQUST1 are as follows:
Strain is inoculated into LB culture medium, is incubated overnight 12h in 37 DEG C of insulating boxs.
6. purposes as claimed in claim 5, it is characterised in that: the composition of LB culture medium described in every 1L are as follows: tryptone 10g,
Yeast extract 5g, sodium chloride 10g, agar 15g, distilled water are settled to 1L, pH7.2,121 DEG C of moist heat sterilization 30min.
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| CN110172417B (en) | 2023-04-21 |
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