CN102399726A - Sporosarcina and application thereof - Google Patents
Sporosarcina and application thereof Download PDFInfo
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- CN102399726A CN102399726A CN2011103713100A CN201110371310A CN102399726A CN 102399726 A CN102399726 A CN 102399726A CN 2011103713100 A CN2011103713100 A CN 2011103713100A CN 201110371310 A CN201110371310 A CN 201110371310A CN 102399726 A CN102399726 A CN 102399726A
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- 241000186547 Sporosarcina Species 0.000 title claims abstract description 15
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 241000192023 Sarcina Species 0.000 claims description 41
- 241000726221 Gemma Species 0.000 claims description 39
- 241000894006 Bacteria Species 0.000 claims description 18
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 9
- 108010076876 Keratins Proteins 0.000 claims description 8
- 102000011782 Keratins Human genes 0.000 claims description 8
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- 238000012360 testing method Methods 0.000 abstract description 13
- 108010059345 keratinase Proteins 0.000 abstract description 8
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Abstract
The invention discloses Sporosarcina and an application thereof. The Sporosarcina (Sporosarcina guangzhouensis GIMN 1.015<T>=47) is preserved in China Center for Type Culture Collection on October 27th, 2011, and the preservation number is CCTCCNO: M2011367. The 16SrDNA nucleotide sequence of the strain is shown as SEQIDNO:1. The Sporosarcina guangzhouensis can be used for producing keratinase, and through the tests, the keratinase activity reaches 105U/ml after the Sporosarcina is cultured for 48hours, so the Sporosarcina can be used for producing the keratinase, and the Sporosarcina has an important significance on the study and the utilization of the keratinase.
Description
Technical field
The present invention relates to microbial technology field, be specifically related to a kind of gemma sarcina and application thereof.
Background technology
Keratin sulfate wastes such as a large amount of feathers that produce in the extensive poultry farming of modern agriculture and the course of processing every year in the world can be to up to ten million tons, and China also reaches the hundreds of thousands of ton.On the one hand, these feathers become the abundantest Keratin sulfate source of occurring in nature, but also become problem demanding prompt solution in the environmental improvement simultaneously.And on the other hand, the researchist finds that Keratin sulfate has been rich in amounts of protein and amino acid, and its Study on nutrition is shown: contain crude protein in the feather and surpass 80 %, various total amino acid contents surpass 70 %.It is thus clear that if appropriately handle, Keratin sulfate wastes such as feather can reach the purpose of bio-transformation.
The occurring in nature Keratin sulfate does not have the phenomenon of accumulation; Its reason just is that some peculiar microorganism can secrete class of enzymes, is called M-Zyme (keratinase, EC 3.4.21/24/99.11); It can decompose Keratin sulfate specifically, makes it be degraded to polypeptide and amino acid.Find the earliest can decompose keratic mikrobe be horse onyx that Ward in 1899 reports roll into a ball the capsule bacterium (
Onygena equina), found 30 in succession afterwards surplus the keratic mikrobe of kind of degradable, mainly comprise 3 types on fungi, actinomycetes and bacterium.Wherein, the bacterium that produces proteolytic enzyme is many from genus bacillus, and what wherein research was the most deep is Bacillus licheniformis and subtilis, and indivedual M-Zymes that bacterial strain produces are commercially produced, as Bacillus licheniformis PWD-1 (
Bacillius licheniforrmisPWD-1) purifying relief angle protease activity is very high, and trade name is Versazyme
TM
China lags behind abroad in the cultivation of feather keratin decomposer and the research field of screening; Separate and the high-efficiency strain that filters out and few; And be mostly fungi and actinomycetes, thereby screening high yield and the new flora of tool better stability M-Zyme also be a research focus, not only made full use of protein resource; Simultaneously also significantly reduce the environmental pollution that causes thus, had remarkable economical interests and far-reaching social benefit.
Summary of the invention
The objective of the invention is to provides a kind of gemma sarcina to above-mentioned deficiency of the prior art.
Another object of the present invention provides the application of above-mentioned gemma sarcina.
The present invention realizes above-mentioned purpose through following technical scheme:
A kind of gemma sarcina, the classification called after
Sporosarcina guangzhouensisGIMN 1.015
T=47, be called for short Guangzhou gemma sarcina among the present invention, this bacterium is preserved in Chinese typical culture collection center (CCTCC), address: China on October 27th, 2011. Wuhan. Wuhan University, deposit number is CCTCC NO:M2011367.
The 16S rDNA nucleotide sequence of this bacterium is shown in SEQ ID NO:1.
The bacterium colony of this bacterium after cultivating 24h on the flat board is faint yellow, circle, and smooth surface is translucent, neat in edge; Gramstaining is positive, and thalline is shaft-like, has whole body cilium, motion, and no pod membrane, the gemma end is given birth to, ellipse.
Measure through Physiology and biochemistry, the urease test of Guangzhou of the present invention gemma sarcina is positive, and oxidase test is negative; The ability caseinhydrolysate, tool is not water-disintegrable to gelatin, starch, tween 80 and tyrosine; Nitrate reductase is determined as the positive, and phenylalanine(Phe) is determined as feminine gender; Can not utilize D-glucose, D-seminose and D-wood sugar.15 ~ 45 ℃ of growth temperature ranges, and growth pH value scope is 5.5 ~ 11.5.
This bacterial strain has the function of decomposing feather, can produce M-Zyme, and through measuring, this bacterium was cultivated after 48 hours, and protease activity reaches 105U/ml, therefore can be used for producing in the industrial production of M-Zyme, and the research and the utilization of M-Zyme had important value.
Description of drawings
Fig. 1. the transmission electron microscope photo of Guangzhou gemma sarcina (6000 times).
Fig. 2. Guangzhou gemma sarcina and part correlation bacterial strain are according to the phylogenetic tree of 16S rDNA sequence construct.
Fig. 3. the design sketch of Guangzhou brood cell sarcina degradation of feather by using, wherein test tube 47 is for adding the experimental group of Guangzhou brood cell sarcina, and CK is the blank group.
Embodiment
Further explain the present invention below in conjunction with specific embodiment, but the present invention is not constituted any qualification.Except that specified otherwise, be conventional reagent in this area and method steps in following examples.
The separation of embodiment 1 Guangzhou gemma sarcina
Soil sample is taken from GuangZhou, Guangdong Province city Agricultural University Of South China in the school, sneaks into the chicken feather, treats that feather decomposes the back and collects soil sample, dries in the shade naturally, and 4 ℃ of preservations are subsequent use.
Get the above-mentioned soil sample of 10g and put into the triangular flask (band granulated glass sphere) that the 90mL sterilized water is housed, vibration is 30 minutes under 180 rpm rotating speeds, leave standstill 10 minutes after, get 10 times of upper strata liquid dilutions after, process 10 successively
-4, 10
-5, 10
-6The soil diluent is got 0.1mL and is uniformly coated on the NA nutrient agar flat board, and the upset flat board places 30 ℃ of incubators to cultivate 2 days.The different bacterial strain of each colonial morphology is made 2 parallel purification flat boards.Picking primary dcreening operation list bacterium colony places 30 ℃ of shaking tables in the feather supplemented medium, 120rpm cultivates, and compares with nonvaccinated substratum, observes feather degraded situation.
NA nutrient agar prescription: peptone 10.0g, Carnis Bovis seu Bubali cream powder 3.0g, sodium-chlor 5g, agar 20.0g, water 1000ml; PH 7.4 ± 0.2.
Feather supplemented medium prescription: peptone 10.0g, Carnis Bovis seu Bubali cream powder 3.0g, sodium-chlor 5.0g, water 1000ml, 0.1% natural feather, pH 7.4 ± 0.2.
Through the further cultivation of feather supplemented medium, filter out and decompose the most effective bacterial strain of feather, after morphology and Physiology and biochemistry and DNA evaluation, called after
Sporosarcina guangzhouensisGIMN 1.015
T=47, be called for short Guangzhou gemma sarcina.
Biological character and Physiology and biochemistry and the Molecular Identification of embodiment 2 Guangzhou gemma sarcinas
1. morphological specificity
The Guangzhou form of gemma sarcina under Electronic Speculum is shown in Fig. 1, and the bacterium colony of this gemma sarcina after cultivating 24h on the flat board is faint yellow, circle, and smooth surface is translucent, neat in edge; Gramstaining is positive, and thalline is shaft-like, has whole body cilium, motion, and no pod membrane, the gemma end is given birth to, ellipse.
2. physiological and biochemical property
Measure through Physiology and biochemistry, the urease test of Guangzhou of the present invention gemma sarcina is positive, and oxidase test is negative; The ability caseinhydrolysate, tool is not water-disintegrable to gelatin, starch, tween 80 and tyrosine; Nitrate reductase is determined as the positive, and phenylalanine(Phe) is determined as feminine gender; Can not utilize D-glucose, D-seminose and D-wood sugar.15 ~ 45 ℃ of growth temperature ranges, and growth pH value scope is 5.5 ~ 11.5.
The Guangzhou gemma sarcina that embodiment 1 is filtered out (
S. guangzhouensisGIMN1.015
T=47) and the gemma sarcina bacterial strain the highest (concrete title see table 1) with its homology carry out the Microbiological Characteristics analysis, the result sees table 1.
Table 1. Guangzhou sarcina and the gemma sarcina strain microorganism characteristic comparison the highest with its homology
Annotate :+, the positive;-, feminine gender.
LO, faint yellow; Y, yellow; B, cream-coloured; WH, white.
R is shaft-like; S, spherical.
T, end is given birth to; C, central authorities give birth to.
NA can not fetch data; W, a little less than.
Can find out by table 1, Guangzhou of the present invention gemma sarcina, the righttest growth pH9.0 can grow under the pH5.7 condition; But anaerobic growth; Oxidase test is negative; Water-disintegrable to the casein tool; The nitrate reduction test is positive.The bacterial strain the highest with its homology
S. KoreensisDSM 16921
TThere are differences at everyways such as physiological and biochemical property, G+Cmol% and cell walls fatty acid content.
S. KoreensisDSM 16921
TPhysiological and biochemical property be the righttest growth pH 7.0, can not grow under the pH5.7 condition; Non-anaerobic growth; Oxidase test is positive; Tool is not water-disintegrable to casein; The nitrate reduction test is negative; G+Cmol% is 46.5 mol%.And
S. guangzhouensisGIMN1.015
T=47 physiological and biochemical property is the righttest growth pH9.0, can grow under the pH5.7 condition; But anaerobic growth; Oxidase test is negative; Water-disintegrable to the casein tool; The nitrate reduction test is positive; G+Cmol% is 45.59 mol%.In addition,
S. KoreensisDSM 16921
TMain kind of lipid acid and content be respectively iso-C
15: 0(44.4%), anteiso-C
15: 0(38.1%) and iso-C
14: 0(6.5 %), and
S. guangzhouensisGIMN1.015
T=47 is iso-C
15: 0(32.26%), anteiso-C
15: 0(41.71%), iso-C
14: 0(9.16 %), C
16: 1 ω7
cAlcohol (6.04%) and summed feature 4 (comprising iso-C
17:1I and/or anteiso-C
17:1B; 2.64%).
Other character:
The main type of methyl naphthoquinone is MK-7, cell walls lipid acid kind and content such as table 2.
Table 2 Guangzhou sarcina and with the cell walls lipid acid kind of the high gemma sarcina bacterial strain of its homology and content relatively
Annotate: Summed Feature 4 comprises iso-C17:1 I and/or anteiso-C17:1 B.
NA can not fetch data.
3. sequencing and the analysis of Guangzhou gemma sarcina 16S rDNA
(1) the quick preparation of pcr template DNA
With Guangzhou gemma sarcina streak inoculation on the flat board of NA substratum, 30 ℃ of overnight cultures.Get single bacterium colony and be suspended in the 10 μ l sterile distilled waters, in boiling water, heat 5min, centrifugal, get supernatant as pcr template DNA.
(2) 16S rDNA gene PCR amplification
The PCR primer is synthetic by Shanghai Ying Jun company.
PrimerA：5’-AGAGTTTGATCCTGGCTCAG-3’（SEQ?ID?NO:2）；
PrimerB：5’-AAGGAGGTGATCCACCCCCA-3’?（SEQ?ID?NO:3）；
PCR reaction system (TV 50 μ l): 10 times of damping fluid 5 μ l, dNTP (2mmol/l) 4 μ l, PrimerA (10pmol/l) 1 μ l, PrimerB (10pmol/l) 1 μ l, Taq enzyme (2U/l) 0.4 μ l, dna profiling 1 μ l, sterilization ddH
2O 37.4 μ l.
Pcr amplification condition: 94 ℃ of 5min; 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 72 ℃ of 7min.
(3) sequencing
Behind the PCR product purification, serve Hai Yingjun company with 3730 full-automatic sequenator order-checkings, its sequence is shown in SEQ ID NO:1.Known array in this sequence and the GenBank DB carries out the BLAST comparative analysis, and obtains the 16S rDNA sequence of relevant kind from DB, and constructing system is grown tree, is shown in Fig. 2.Find through comparative analysis, Guangzhou of the present invention gemma sarcina and bacterial strain (
Sporosarcina koreensisDSM 16921
T) sibship is nearest, the 16S rDNA gene order of Guangzhou gemma sarcina with
Sporosarcina koreensis DSM 16921
T99.6% homology is arranged, but show Guangzhou gemma sarcina and gemma sarcina type strain through DNA-DNA hybridization
Sporosarcina koreensisDSM 16921
THas only 40.5 ± 0.2% homology.Comprehensive above-mentioned data can prove that they are not same kinds.
Comprehensive 16S rDNA sequential analysis, Microbiological Characteristics analysis and DNA-DNA results of hybridization (the international system bacteriology ICSB of council regulation in 1987; Dna homology property less than 70% or the pyrolysis chain temperature head of hybrid molecule be less than or equal to 2 ℃ and be the boundary line of bacterium kind, Wayne
Et al., 1987), show that gemma sarcina of the present invention is a kind of new bacterium, called after Guangzhou gemma sarcina (
Sporosarcina guangzhouensisGIMN1.015
T=47), this bacterium was preserved in Chinese typical culture collection center (CCTCC), address: China on October 27th, 2011. Wuhan. and Wuhan University, deposit number is CCTCC M2011367.
The M-Zyme that embodiment 3 Guangzhou gemma sarcinas produce produces to be measured
Single bacterium colony of the Guangzhou gemma sarcina that picking embodiment 1 filters out is seeded to the feather supplemented medium, on 30 ℃ vibration shaking table, cultivates 3 ~ 4 days, observes degraded in substratum, whether to occur.
The mensuration of protease hydrolysis activity method is according to document [Radha; S. Gunasekaran, P. (2007). Cloning and expression of keratinase gene in Bacillus megaterium and optimization of fermentation conditions for the production of keratinase by recombinant strain.
Journal of Applied Microbiology 103, 1301-1310] and method, above-mentioned nutrient solution is through 9,000 * g, and 4 ℃, centrifugal 10min promptly obtains crude enzyme liquid.Get 120 μ l enzyme liquid and add 480 μ l and contain 1% (w/v) azo-casein (Azocasein) in 100mM Tris-HCl damping fluid (pH=7.5), 30 ℃ of incubations of mixed solution 30 minutes, add 600 μ l, 10% trichoroacetic acid(TCA)s (TCA) termination reaction after; Ice bath 30 minutes, 12,000 * g; 10min; 4 ℃ centrifugal, and 800 μ l supernatants add the NaOH of 200 μ l 1.8M, measures the OD value at the 420nm place.Calculate Δ OD.The enzyme activity unit definition: under the experiment condition, the absorption value 0.01 required enzyme amount that raises in 420nm place is a unit of activity (U).
Test result finds, the (see figure 3) that is degraded of the feather in the substratum shows that Guangzhou of the present invention gemma sarcina has the ability of degradation of feather by using, and through measuring, this bacterium was cultivated after 48 hours, can feather be degraded, and protease activity reaches 105 U/ml.Therefore can be used for the research and the utilization of keratin degrading.
SEQUENCE?LISTING
< 110>Agricultural University Of South China
< 120>a kind of gemma sarcina and application thereof
<130>
<160> 3
<170> PatentIn?version?3.3
<210> 1
<211> 1421
<212> DNA
< 213>Guangzhou gemma sarcina 16S rDNA
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aataatcggt?tcttccgcat?ggaagaactc?tgaaagacgg?tttcggctgt?cactgcagga 180
tgggcccgcg?gcgcattagc?tagttggtgg?ggtaacggcc?taccaaggcg?acgatgcgta 240
gccgacctga?gagggtgatc?ggccacactg?ggactgagac?acggcccaga?ctcctacggg 300
aggcagcagt?agggaatctt?ccacaatgga?cgaaagtctg?atggagcaac?gccgcgtgag 360
cgaagaaggt?tttcggatcg?taaagctctg?ttgcgaggga?agaacaagta?cgggagtaac 420
tgcccgtacc?ttgacggtac?ctcgtcagaa?agccacggct?aactacgtgc?cagcagccgc 480
ggtaatacgt?aggtggcaag?cgttgtccgg?aattattggg?cgtaaagcgc?gcgcaggcgg 540
tcctttaagt?ctgatgtgaa?agcccacggc?tcaacccgtg?gagggtcatt?ggaaactgga 600
ggacttgagt?acagaagagg?aaagcggaat?tccacgtgta?gcggtgaaat?gcgtagagat 660
gtggaggaac?accagtggcg?aaggcggctt?tctggtctgt?aactgacgct?gaggcgcgaa 720
agcgtgggga?gcaaacagga?ttagataccc?tggtagtcca?cgccgtaaac?gatgagtgct 780
aagtgttagg?gggtttccgc?cccttagtgc?tgcagctaac?gcattaagca?ctccgcctgg 840
ggagtacggc?cgcaaggctg?aaactcaaag?gaattgacgg?ggacccgcac?aagcggtgga 900
gcatgtggtt?taattcgaag?caacgcgaag?aaccttacca?ggtcttgaca?tcccgctgac 960
cggtgtagag?atacgccttt?cccttcgggg?acagcggtga?caggtggtgc?atggttgtcg 1020
tcagctcgtg?tcgtgagatg?ttgggttaag?tcccgcaacg?agcgcaaccc?ttgatcttag 1080
ttgccagcat?tcagttgggc?actctaaggt?gactgccggt?gacaaaccgg?aggaaggtgg 1140
ggatgacgtc?aaatcatcat?gccccttatg?acctgggcta?cacacgtgct?acaatggacg 1200
gtacaaaggg?ctgcgaaccc?gcgaggggga?gccaatccca?taaaaccgtt?cccagttcgg 1260
attgcaggct?gcaactcgcc?tgcatgaagc?cggaatcgct?agtaatcgtg?gatcagcatg 1320
ccacggtgaa?tacgttcccg?ggtcttgtac?acaccgcccg?tcacaccacg?agagtttgta 1380
acacccgaag?tcggtggggt?aacccttacg?gagccagccg?c 1421
<210> 2
<211> 20
<212> DNA
< 213>artificial sequence PrimerA
<400> 2
agagtttgat?cctggctcag 20
<210> 3
<211> 20
<212> DNA
< 213>artificial sequence PrimerB
<400> 3
aaggaggtga?tccaccccca 20
Claims (4)
1. gemma sarcina, the classification called after
Sporosarcina guangzhouensisGIMN 1.015
T=47, this bacterium is preserved in Chinese typical culture collection center on October 27th, 2011, and deposit number is CCTCC NO:M2011367.
2. according to claim 1 a gemma sarcina, the 16S rDNA nucleotide sequence that it is characterized in that this bacterium is shown in SEQ ID NO:1.
3. claim 1 or the 2 said gemma sarcinas application in decomposing Keratin sulfate.
4. claim 1 or the 2 said gemma sarcinas application in producing M-Zyme.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703341A (en) * | 2012-04-23 | 2012-10-03 | 清华大学 | Urease-producing microorganisms and method for solidifying heavy metals in foundation using same |
| CN103173376A (en) * | 2012-11-16 | 2013-06-26 | 清华大学 | Method for preparing high-strength microbial mortar by using urease-producing microbes |
| CN103289920A (en) * | 2012-04-23 | 2013-09-11 | 清华大学 | Urease-producing microbes and curing method for heavy metals in foundation |
| CN103695355A (en) * | 2013-12-23 | 2014-04-02 | 华南农业大学 | Pseudomonas otitidis H3 strain and application thereof |
| CN107828838A (en) * | 2017-12-06 | 2018-03-23 | 东莞理工学院 | South Korea gemma sarcine CGMCC No.5915 are applied and lipopeptid class Surfactin preparation method and composition |
| CN115975862A (en) * | 2022-10-26 | 2023-04-18 | 安徽农业大学 | Sporosarcina korea JZ-2 and application thereof |
-
2011
- 2011-11-21 CN CN 201110371310 patent/CN102399726B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
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| LIN X.，ET AL.: "Comparison of Two Feather-Degrading Bacillus Licheniformis Strains", 《ASIAN-AUST. J. ANIM.SCI.》 * |
| ZHU,H.-H，ET AL.: "Accession No.JF31343.1", 《NCBI GENBANK》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703341A (en) * | 2012-04-23 | 2012-10-03 | 清华大学 | Urease-producing microorganisms and method for solidifying heavy metals in foundation using same |
| CN103289920A (en) * | 2012-04-23 | 2013-09-11 | 清华大学 | Urease-producing microbes and curing method for heavy metals in foundation |
| CN102703341B (en) * | 2012-04-23 | 2014-04-02 | 清华大学 | Urease-producing microorganisms and method for solidifying heavy metals in foundation using same |
| CN103289920B (en) * | 2012-04-23 | 2014-11-26 | 清华大学 | Urease-producing microbes and curing method for heavy metals in foundation |
| CN103173376A (en) * | 2012-11-16 | 2013-06-26 | 清华大学 | Method for preparing high-strength microbial mortar by using urease-producing microbes |
| CN103173376B (en) * | 2012-11-16 | 2014-10-29 | 清华大学 | Method for preparing high-strength microbial mortar by using urease-producing microbes |
| CN103695355A (en) * | 2013-12-23 | 2014-04-02 | 华南农业大学 | Pseudomonas otitidis H3 strain and application thereof |
| CN107828838A (en) * | 2017-12-06 | 2018-03-23 | 东莞理工学院 | South Korea gemma sarcine CGMCC No.5915 are applied and lipopeptid class Surfactin preparation method and composition |
| CN107828838B (en) * | 2017-12-06 | 2020-11-03 | 东莞理工学院 | Korean spore sarcina CGMCC No.5915 application, and preparation method and composition of lipopeptide surfactant |
| CN115975862A (en) * | 2022-10-26 | 2023-04-18 | 安徽农业大学 | Sporosarcina korea JZ-2 and application thereof |
| CN115975862B (en) * | 2022-10-26 | 2023-10-24 | 安徽农业大学 | Korean spore sarcina JZ-2 and application thereof |
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|---|---|
| CN102399726B (en) | 2013-05-01 |
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