CN105969703B - A kind of bacillus licheniformis and its application - Google Patents
A kind of bacillus licheniformis and its application Download PDFInfo
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Abstract
The present invention relates to a kind of bacillus licheniformis and its applications, belong to field of biotechnology.Specifically, the present invention provides the bacillus licheniformis that one kind is capable of fermenting and producing fragrance 3-hydroxy-2-butanone, and utilize the method for this microbial fermentation tobacco industry waste production fragrance 3-hydroxy-2-butanone.The bacillus licheniformis (Bacillcus licheniformis) T8, be from cigar mill's surrounding soil by selective medium it is isolated, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number CGMCC No.12732 on July 1st, 2016.The bacterial strain can be using tobacco fermentation waste as Material synthesis fragrance 3-hydroxy-2-butanone, which can be directly used for fragrance-enhancing tobacco or purified rear as food additives.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of bacillus licheniformis and its application, in particular to one plant
Screen the bacillus licheniformis obtained and the method using strain fermentation tobacco waste production fragrance 3-hydroxy-2-butanone.
Background technique
3-hydroxy-2-butanone (3- hydroxy-2-butanone also known as 3-Hydroxybutanone or methyl vinyl methanol) has particular milk as a kind of
The flavorant of incense oil gas is widely used in food, tobacco, drinks and cosmetic industry.Tobacco industry waste is mainly cigarette
Stalk and scrap, nicotine content with higher, random stacking processing can cause environmental pollution.It is fermented using microbiological treatment
Biological-based chemicals are produced, the utilization rate of tobacco leaf resource can be improved, there are research and development to be worth, can more reduce Buddhist nun's Gu simultaneously
Fourth reduces pollution of a large amount of waste of tobacco industry generation to ecological environment.Therefore, development and application will have huge
Development potentiality and application prospect.
According to current result of study, 3-hydroxy-2-butanone is a kind of important component of cigarette fragrance, and the fragrance of cigarette is mainly by micro-
Biofermentation generates.But it is carbon source that current 3-hydroxy-2-butanone biofermentation, which mainly utilizes glucose, is not yet found discarded with tobacco
Object is the report that fermenting raw materials synthesize 3-hydroxy-2-butanone.The present invention synthesizes the bacterium of 3-hydroxy-2-butanone to obtain using tobacco waste as fermenting raw materials
Strain is starting point, it is intended to establish a kind of new way of tobacco toxic waste higher value application.
Summary of the invention
The present invention is low for current tobacco industry waste utilization rate, lacks although it can generate 3-hydroxy-2-butanone in tobacco fermentation
The status of weary bacterial strain, screening obtain one plant of strain Bacillus licheniformis that can utilize tobacco waste Synthesis 3-hydroxy-2-butanone
(Baclicus lincheniformis), it can be using tobacco processing waste offal as master using the bacillus licheniformis
It wants to grow in the culture medium of nutrient source, and 3-hydroxy-2-butanone can be generated.
To achieve the above object, The technical solution adopted by the invention is as follows:
A kind of bacillus licheniformis (Bacillcus licheniformis) T8, it has been preserved on July 1st, 2016
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number CGMCC No. 12732.
The present invention also provides bacillus licheniformis (Bacillcus licheniformis) T8 answering in production 3-hydroxy-2-butanone
With.
Using bacillus licheniformis (Bacillcus licheniformis) method in T8 production 3-hydroxy-2-butanone, including such as
Lower step:
Bacillus licheniformis is seeded in sterilized LB culture medium according to 1 ~ 5% inoculum concentration and cultivates by step (1), training
Supporting to OD600 is 0.8 ~ 1.2；The component of the LB culture medium are as follows: 8 ~ 12 g/L of peptone, 4 ~ 6 g/L of yeast powder, sodium chloride
1 ~ 4 g/L, pH7.0 ~ 7.5；
The obtained bacillus licheniformis of step (1) culture is seeded to according to 3 ~ 5% inoculum concentration sterilized by step (2)
Using tobacco waste as in the culture medium of carbon source, in shaking table, 30 ~ 37 DEG C are cultivated 24 ~ 36 hours；
It is described using tobacco waste as the component of the culture medium of carbon source are as follows: 1 ~ 3 g/L of tryptone, 1 ~ 3 g/ of beef extract
L, NaCl 3 ~ 6 g/L, MgSO47H2O, 2 ~ 4 g/L, 3 ~ 10 g/L of tobacco waste, pH value 7.0 ~ 7.4；
The revolving speed of the shaking table is 100 ~ 140r/min；
The tobacco waste is that the partial size of offal crushed material is -80 mesh.
It sterilizes 25 ~ 35 minutes it is further preferred that the sterilising conditions of the LB culture medium are 112 DEG C.
It by the sterilising conditions of the culture medium of carbon source of tobacco waste is 110 ~ 120 DEG C it is further preferred that described
Sterilizing 20 ~ 30 minutes.
It is further preferred that being placed in 250 by the culture medium of carbon source of tobacco waste for 40 ~ 80 ml are sterilized
In the triangular flask of ml, then inoculation step (1) cultivates obtained bacillus licheniformis, cultivates in shaking table later.
It is of the present invention to produce 3-hydroxy-2-butanone by primary carbon source of tobacco processing waste --- bacillus licheniformis
(Bacillcus licheniformis) it is isolated by selective medium from cigar mill's surrounding soil.
It is Grain-positive bacillus the present invention relates to bacillus licheniformis, there is gemma, generates ellipticity gemma raw in close, spore
Capsule slightly expands, and the size for observing thallus under the microscope is 0.8 μm of x(1.5~3.5) μm.Somatic cells are in rod-short.In LB
Solid culture primary surface, thallus form opaque light yellow petite, and bacterium colony has fold, and edge is irregular.Above-mentioned bacterial strains
Through universal primer PCR obtain 16S rDNA sequence and the 16S rDNA sequence of other more bacillus licheniformis have 99% it is similar
Property but it is different.
Wherein, the upstream primer of above-mentioned PCR is fD1:5 '-agagtttgatcctggctcag-3 ' (SEQ ID NO.2)；
Downstream primer rP2:5 '-cggctaccttgttacgactt-3 ' (SEQ ID NO.3).
The present invention can be beef extract and peptone by compound nitrogen source in the culture medium of carbon source of tobacco waste, but not office
It is limited to beef extract and peptone.
Compared with prior art, the present invention has the advantages that:
1) present invention screening, which obtains bacillus licheniformis, to be main nutrient source using tobacco waste.
2) bacillus licheniformis being capable of fermented tobacco waste production 3-hydroxy-2-butanone.The 3-hydroxy-2-butanone can be directly used for tobacco increasing
Food additives are used as after fragrant or purified.
Detailed description of the invention
Fig. 1 present invention, which is fermented, obtains the mass spectrogram of 3-hydroxy-2-butanone.
Bacillus licheniformis (Bacillcus licheniformis) T8, it is micro- that China has been preserved on July 1st, 2016
Biological inoculum preservation administration committee common micro-organisms center, deposit number CGMCC No. 12732, preservation address is Beijing
The institute 3 of Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair
Bright range.In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer is that can be obtained by purchase
Conventional products.
The present invention is unless there are specified otherwise, and otherwise percentage is mass percent.
Embodiment 1 produces the screening of 3-hydroxy-2-butanone microorganism using tobacco processing waste
1. culture medium and condition of culture:
Using tobacco waste as the culture medium prescription of carbon source are as follows: 1 ~ 3 g/L of tryptone, beef extract 1 ~ 3 g/L, NaCl 3
~ 6 g/L, MgSO4·7H2O, 2 ~ 4 g/L, 3 ~ 10 g/L of tobacco waste, pH value 7.0 ~ 7.4.Optimum temperature is 30 ~ 37 DEG C,
Liquid amount in the triangular flask of 250ml is 40 ~ 80ml.
The tobacco waste be offal crushed material partial size be -80 mesh, i.e., 80 meshes, extracting screen underflow.
The incubation time of the culture of above-mentioned strain is 24 ~ 36 hours.
The sterilising conditions of above-mentioned culture medium are 112 DEG C and sterilize 30 minutes.
It is 0.1% creatine as screened indicator that mass percentage is added in above-mentioned culture medium.
The agar that mass percentage is 1% ~ 1.5% is added in above-mentioned culture medium is prepared into solid medium for bacterial strain sieve
Choosing.
2. screening technique:
It takes that tobacco factory, soil sample is dissolved according to 5 ml water of 1g sample around cigarette district, then water sample is placed in activation in room temperature 24 hours
It is added in another test tube for filling 9 ml sterile waters, is uniformly mixed from 1 ml is drawn in this test tube with aseptic straw, and so on
It is made 10-1、10-2、10-3、10-4、10-5、10-6The soil of different dilutions.
The dilution soil bacterium solution of various concentration is taken to be applied on solid medium culture dish described in step 1, culture 36
Picking colour developing single colonie is based on 37 DEG C using tobacco waste as the culture of carbon source using described in step 1 after hour, 120 turns/
Min carries out culture 12-24h, is screened and obtains 36 plants of bacterial strain altogether, by Liquid Culture secondary screening, using described in step 1 when secondary screening
Using tobacco waste be the culture of carbon source be based on 37 DEG C, 120 turns/min carries out culture 12-24h, the hair for taking secondary screening culture to obtain
Zymotic fluid dilutes 5-10 times, the fermentation liquid after being diluted.
By NaOH, 1mL mass concentration that 1mL mass concentration is 10% be 0.5% creatine, 1mL mass concentration be 5% 1-
After mixing, the fermentation liquid after 25 μ L dilute is added, later in 30 DEG C of concussion reactions in naphthols and 7mL deionized water thereto
30 min measure its OD520 value, and experiment draws standard curve with the 3-hydroxy-2-butanone standard sample of various concentration every time, pass through mark
The content of directrix curve measurement 3-hydroxy-2-butanone.Optimal t bacteria 8 is obtained by screening.
The strain idenfication of the screening microorganism of embodiment 2
1. culture medium and material
LB culture medium contains following ingredient: 4 ~ 6 g/L of yeast powder, 8 ~ 12 g/L of peptone, sodium chloride 1 ~ 4 g/L, pH
7.0 ~ 7.5,112 DEG C sterilize 25 ~ 35 minutes.
LA culture medium: 40 ~ 100 μ g/mL ampicillins are added in LB culture medium.
LB solid medium: the agar that mass percent is 1.5% is added in above-mentioned LB culture medium.
TAE:50 times of TEA buffer contains 2M tris-acetate, 0.05M EDTA, pH 8.3.
Normal Agarose Gel: 1 times of TAE electrophoretic buffer, the agarose that addition mass percentage is 0.8 ~ 1.2% are solidifying
Glue.
2.16S rDNA is measured
12-1 thallus is incubated in LB culture medium, after 12 ~ 24 hours, centrifugation extracts genomic DNA, then uses concentration
For the electrophoresis detection for the agarose gel electrophoresis progress DNA that mass fraction is 1%.The sample containing genomic DNA is detected to carry out
Pcr gene amplification.
The upstream primer of PCR is fD1:5 '-agagtttgatcctggctcag-3 ' (SEQ ID NO.2)；
Downstream primer rP2:5 '-cggctaccttgttacgactt-3 ' (SEQ ID NO.3).
The reaction system of PCR are as follows: template DNA adds 0.5 μ l and adjusts to suitable concentration, and primer concentration is 0.4 μM, dNTP
Concentration is 0.2 mM, and about 2.5 U of archaeal dna polymerase adds ddH2O to reaction 50 μ l of total volume.
The reaction condition of PCR are as follows: 94 DEG C are denaturalized 30 seconds, and 55 DEG C of degree are annealed 30 seconds, and 72 DEG C extend 1 minute, and totally 30 are followed
Ring.
PCR product is about 1.5 kb or so, and the agarose gel electrophoresis for being 1% through mass percentage utilizes quality percentage
Content is dyed in 0.01% ethidium bromide, is observed under uv analyzer, has the product of light tone band in 1.5kb or so, even
PMD18-T conversion Escherichia coli are connected to, the DH5 ɑ bacterial strain of conversion is coated on LA culture medium that (Luria-Bertani culture medium adds
Add 50 mg/L ampicillins), after 37 DEG C of static gas wave refrigerators, extracts plasmid and utilizeBamHI andSalAfter I digestion, quality is utilized
Percentage composition is that 1% Ago-Gel carries out electrophoresis verifying, the plasmid containing purpose band is sequenced, sequencing result such as SEQ
Shown in ID NO.1.
The 16S rDNA sequence announced in the 16S rDNA sequence of bacterial strain of the invention and GenBank is subjected to homologous ratio
Right, the 16S rDNA sequence and the 16S rDNA sequence of other more bacillus licheniformis are similar but not exactly the same.The bacterium is
Grain-positive bacillus, bacillus have gemma, generate ellipticity gemma raw in close, and sporangiocyst slightly expands, and observe thallus under the microscope
Size be 0.8 μm of x(1.5~3.5) μm.Somatic cells are in rod-short.In LB solid culture primary surface, thallus forms impermeable
Bright light yellow petite, bacterium colony have fold, and edge is irregular.Above-mentioned bacterial strains obtain 16S rDNA sequence through universal primer PCR
It arranges still different from the similitude that the 16S rDNA sequence of other more bacillus licheniformis has 99%.Preliminary Identification changes strain
Bacillus licheniformis (Bacillcus licheniformis), the bacterial strain preservation to Chinese microorganism strain preservation conservator
Meeting common micro-organisms center, deposit number are as follows: CGMCC No.12732.
Embodiment 3 produces 3-hydroxy-2-butanone using tobacco industry waste
Using tobacco waste as the component of the culture medium of carbon source are as follows: 2 g/L of tryptone, beef extract 2 g/L, NaCl 4
G/L, MgSO47H2O, 6 g/L, 7 g/L of tobacco waste, pH value 7.2；Tobacco waste is that the partial size of offal crushed material is -80
Mesh.
The component of LB culture medium are as follows: peptone 10g/L, 5 g/L of yeast powder, sodium chloride 3g/L, pH7.3；
The sterilising conditions of LB culture medium are 112 DEG C and sterilize 30 minutes.
It is 115 DEG C as the sterilising conditions of the culture medium of carbon source using tobacco waste to sterilize 25 minutes.
LB culture medium and using tobacco waste as the culture medium of carbon source be both needed to sterilizing after could use.
Being drawn with oese takes strain inoculated in the test tube of the LB culture medium containing 2 ml, and 120 turns/min is cultivated 12 hours,
To bacillus licheniformis (Bacillcus licheniformis) T8 activated；
The culture medium using tobacco waste as carbon source is taken, according to the bacillus licheniformis after 3% inoculum concentration inoculation activation
(Bacillcus licheniformis) T8, it is inoculated in the test tube equipped with 2ml LB culture medium, growing into cell density is
When OD600 1.0, it is inoculated in equipped with 50 ml according to 4% inoculum concentration using tobacco waste as 250 ml's of the culture medium of carbon source
In shaking flask, in the shaking table of 120 r/min, 37 DEG C of cultures.Culture was developed the color and is measured using creatine and α naphthols after 36 hours
The yield of 3-hydroxy-2-butanone is 5.6 g/L.
The qualitative detection of 3-hydroxy-2-butanone: taking above-mentioned fermentation liquid to be centrifuged 10min through 12000 r/min, after 0.22 μm of film filtering,
Qualitative detection is carried out using GC-MS.Using Agilent HP-INNOWax capillary chromatographic column (30 m × 0.32 mm, 0.25 μ
M.), temperature program is 50 DEG C of maintenances 2 minutes, and then 10 DEG C/min of temperature programmings to 240 DEG C, 240 DEG C maintain 3 minutes.It obtains
As a result mass spectrogram is as shown in Figure 1, through comparing with java standard library as 3-hydroxy-2-butanone.
Embodiment 4 produces 3-hydroxy-2-butanone using tobacco industry waste
Using tobacco waste as the component of the culture medium of carbon source are as follows: 1 g/L of tryptone, beef extract 1 g/L, NaCl 3g/
L, MgSO47H2O, 2g/L, 3 g/L of tobacco waste, pH value 7.0；Tobacco waste is that the partial size of offal crushed material is -80 mesh；
The component of LB culture medium are as follows: peptone 8g/L, yeast powder 4g/L, sodium chloride 1g/L, pH7.0；
The sterilising conditions of LB culture medium are 112 DEG C and sterilize 25 minutes.
It is 110 DEG C as the sterilising conditions of the culture medium of carbon source using tobacco waste to sterilize 20 minutes.
LB culture medium and using tobacco waste as the culture medium of carbon source be both needed to sterilizing after could use.
Being drawn with oese takes strain inoculated in the test tube of the LB culture medium containing 2 ml, and 100 turns/min is cultivated 12 hours,
To bacillus licheniformis (Bacillcus licheniformis) T8 activated；
The culture medium using tobacco waste as carbon source is taken, according to the bacillus licheniformis after 1% inoculum concentration inoculation activation
(Bacillcus licheniformis) T8, it is inoculated in the test tube equipped with 2ml LB culture medium, growing into cell density is
When OD600 0.8, it is inoculated in equipped with 40 ml according to 3% inoculum concentration using tobacco waste as 250 ml's of the culture medium of carbon source
In shaking flask, in the shaking table of 100 r/min, 30 DEG C of cultures.Culture was developed the color and is measured using creatine and α naphthols after 34 hours
The yield of 3-hydroxy-2-butanone is 5.5 g/L.
Embodiment 5 produces 3-hydroxy-2-butanone using tobacco industry waste
Using tobacco waste as the component of the culture medium of carbon source are as follows: 3 g/L of tryptone, beef extract 3 g/L, NaCl 6
G/L, MgSO47H2O, 4 g/L, 10 g/L of tobacco waste, pH value 7.4；Tobacco waste be offal crushed material partial size be-
80 mesh；
The component of LB culture medium are as follows: 12 g/L of peptone, 6 g/L of yeast powder, 4 g/L of sodium chloride, pH7.0 ~ 7.5；
The sterilising conditions of LB culture medium are 112 DEG C and sterilize 35 minutes.
It is 120 DEG C as the sterilising conditions of the culture medium of carbon source using tobacco waste to sterilize 30 minutes.
LB culture medium and using tobacco waste as the culture medium of carbon source be both needed to sterilizing after could use.
Being drawn with oese takes strain inoculated in the test tube of the LB culture medium containing 2 ml, and 140 turns/min is cultivated 12 hours,
To bacillus licheniformis (Bacillcus licheniformis) T8 activated；
The culture medium using tobacco waste as carbon source is taken, according to the bacillus licheniformis after 5% inoculum concentration inoculation activation
(Bacillcus licheniformis) T8, it is inoculated in the test tube equipped with 2ml LB culture medium, growing into cell density is
When OD600 1.2, it is inoculated in equipped with 80 ml according to 5% inoculum concentration using tobacco waste as 250 ml's of the culture medium of carbon source
In shaking flask, in the shaking table of 140 r/min, 35 DEG C of cultures.Culture was developed the color and is measured using creatine and α naphthols after 30 hours
The yield of 3-hydroxy-2-butanone is 5.56g/L.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Sequence table
SEQ ID NO.1
agctatacat gcagtcgagc ggaccgacgg gagcttgctc ccttaggtca gcggcggacg 60
ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga aaccggggct 120
aataccggat gcttgattga accgcatggt tcaatcataa aaggtggctt ttagctacca 180
cttgcagatg gacccgcggc gcattagcta gttggtgagg taacggctca ccaaggcgac 240
gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact 300
cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc 360
cgcgtgagtg atgaaggttt tcggatcgta aaactctgtt gttagggaag aacaagtacc 420
gttcgaatag ggcggcacct tgacggtacc taaccagaaa gccacggcta actacgtgcc 480
agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc gtaaagcgcg 540
cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga gggtcactgg 600
aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc 660
gtagagatgt ggaggaacac cagtggcgta ggcgactctc tggtctgtaa ctgacgctga 720
ggcgcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacga 780
tgagtgctaa gtgttagagg gtttccgccc tttagtgctg cagcaaacgc attaagcact 840
ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg gcccgcacaa 900
gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc 960
ctctgacaac cctagagata gggcttcccc ttcgggggca gagtgacagg tggtgcatgg 1020
ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttga 1080
tcttagttgc cagcattcag ttgggcactc taaggtgact gccggtgaca aaccggagga 1140
aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa 1200
tgggcagaac aaagggcagc gaagccgcga ggctaagcca atcccacaaa tctgttctca 1260
gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga atcgctagta atcgcggatc 1320
agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag 1380
tttgtaacac ccgaagtcgg tgaggtaacc ttttgagcca gccgccgaac gggac 1435
SEQ ID NO.2
agagtttgat cctggctcag 20
SEQ ID NO.3
cggctacctt gttacgactt 20。
SEQ ID NO.1
agctatacat gcagtcgagc ggaccgacgg gagcttgctc ccttaggtca gcggcggacg 60
ggtgagtaac acgtgggtaa cctgcctgta agactgggat aactccggga aaccggggct 120
aataccggat gcttgattga accgcatggt tcaatcataa aaggtggctt ttagctacca 180
cttgcagatg gacccgcggc gcattagcta gttggtgagg taacggctca ccaaggcgac 240
gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac ggcccagact 300
cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac ggagcaacgc 360
cgcgtgagtg atgaaggttt tcggatcgta aaactctgtt gttagggaag aacaagtacc 420
gttcgaatag ggcggcacct tgacggtacc taaccagaaa gccacggcta actacgtgcc 480
agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc gtaaagcgcg 540
cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga gggtcactgg 600
aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc ggtgaaatgc 660
gtagagatgt ggaggaacac cagtggcgta ggcgactctc tggtctgtaa ctgacgctga 720
ggcgcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg ccgtaaacga 780
tgagtgctaa gtgttagagg gtttccgccc tttagtgctg cagcaaacgc attaagcact 840
ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg gcccgcacaa 900
gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg tcttgacatc 960
ctctgacaac cctagagata gggcttcccc ttcgggggca gagtgacagg tggtgcatgg
1020
ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg caacccttga
1080
tcttagttgc cagcattcag ttgggcactc taaggtgact gccggtgaca aaccggagga
1140
aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa
1200
tgggcagaac aaagggcagc gaagccgcga ggctaagcca atcccacaaa tctgttctca
1260
gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga atcgctagta atcgcggatc
1320
agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accacgagag
1380
tttgtaacac ccgaagtcgg tgaggtaacc ttttgagcca gccgccgaac gggac 1435
SEQ ID NO.2
agagtttgat cctggctcag 20
SEQ ID NO.3
cggctacctt gttacgactt 20
Claims (6)
1. a kind of bacillus licheniformis (Bacillcus licheniformis) T8, China is preserved on July 1st, 2016
Microbiological Culture Collection administration committee common micro-organisms center, deposit number CGMCC No. 12732；The lichens gemma
Bacillus can synthesize 3-hydroxy-2-butanone by fermenting raw materials of tobacco waste.
2. bacillus licheniformis described in claim 1 (Bacillcus licheniformis) T8 production 3-hydroxy-2-butanone in
Using.
3. bacillus licheniformis according to claim 2 (Bacillcus licheniformis) T8 production 3-hydroxy-2-butanone
In application, which comprises the steps of:
Bacillus licheniformis is seeded in sterilized LB culture medium according to 1 ~ 5% inoculum concentration and cultivates by step (1), and culture is extremely
OD600 is 0.8 ~ 1.2；The component of the LB culture medium are as follows: 8 ~ 12 g/L of peptone, 4 ~ 6 g/L of yeast powder, sodium chloride 1 ~ 4
G/L, pH7.0 ~ 7.5；
The obtained bacillus licheniformis of step (1) culture is seeded to according to 3 ~ 5% inoculum concentration sterilized with cigarette by step (2)
Careless waste is in the culture medium of carbon source, and in shaking table, 30 ~ 37 DEG C are cultivated 24 ~ 36 hours；
It is described using tobacco waste as the component of the culture medium of carbon source are as follows: 1 ~ 3 g/L of tryptone, 1 ~ 3 g/L of beef extract,
NaCl 3 ~ 6 g/L, MgSO4•7H2O, 2 ~ 4 g/L, 3 ~ 10 g/L of tobacco waste, pH value 7.0 ~ 7.4；
The revolving speed of the shaking table is 100 ~ 140r/min；
The tobacco waste is offal crushed material, and the partial size of the offal crushed material is -80 mesh.
4. bacillus licheniformis according to claim 3 (Bacillcus licheniformis) T8 production 3-hydroxy-2-butanone
In application, which is characterized in that the sterilising conditions of the LB culture medium be 112 DEG C sterilize 25 ~ 35 minutes.
5. bacillus licheniformis according to claim 3 (Bacillcus licheniformis) T8 production 3-hydroxy-2-butanone
In application, which is characterized in that it is described using tobacco waste as the sterilising conditions of the culture medium of carbon source be 110 ~ 120 DEG C sterilizing
20 ~ 30 minutes.
6. bacillus licheniformis according to claim 3 (Bacillcus licheniformis) T8 production 3-hydroxy-2-butanone
In application, which is characterized in that be placed in 250 ml's by the culture medium of carbon source of tobacco waste for 40 ~ 80 ml are sterilized
In triangular flask, then inoculation step (1) cultivates obtained bacillus licheniformis, cultivates in shaking table later.
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