CN110373346A - Bacillus subtilis Bacillus sp.A-5 and application thereof - Google Patents
Bacillus subtilis Bacillus sp.A-5 and application thereof Download PDFInfo
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Abstract
The present invention provides a kind of bacillus subtilisBacillusSp.A-5 and application thereof.The bacillus subtilisBacillus sp. The deposit number of A-5 is CCTCC NO:M2019157.Using bacillus subtilisBacillus sp. It is more than 75% that A-5 fermentation, which prepares gamma-polyglutamic acid conversion ratio, and molecular weight is more than 4000 KDa, conversion ratio significantly larger than in the prior art.
Description
Technical field
The present invention relates to a kind of bacillus subtilis Bacillus sp.A-5 and application thereof, belong to bioengineering field.
Background technique
Gamma-polyglutamic acid is that one kind is formed by connecting by several glutamic acid monomers (D type or L-type) by γ-amido bond
A kind of anionic polypeptide polymer, can be synthesized by chemical method or microbial fermentation, molecular weight mostly 100-1000kDa it
Between, average molecular weight is about 1.23 × 104KDa.Gamma-polyglutamic acid does not have typical peptide chain structure, and basic framework is in straight chain
Threadiness.A kind of novel high polymer green bio product of the gamma-polyglutamic acid as no pollution to the environment, have degradability,
The unique physicochemical property such as film forming, fibre forming property, plasticity, caking property, can be applied to that medical manufacture, food processing are fresh-keeping, change
Many fields such as cosmetic, manufacturing industry, agricultural.
The advantages of microbial fermentation production prepares gamma-polyglutamic acid is that production cost is low, and environmental pollution is small, controllability
By force.The zymotechnique of gamma-polyglutamic acid is conducted extensive research both at home and abroad, including liquid deep layer fermenting and solid fermentation
Formula and culture process based on.The production bacterial strain of gamma-polyglutamic acid is divided into glutamic acid dependent form and glutamic acid independent form, by
It is lower in the gamma-polyglutamic acid yield of the latter, therefore producing and studying is mostly glutamic acid dependent form bacterial strain.Glutamic acid dependent form bacterium
Strain needs to add a certain amount of glutamic acid in the fermentation medium as precursor, and glutamic acid additive amount is larger, and conversion ratio is lower,
Utilization rate keeps production cost high, seriously hinders the application development of gamma-polyglutamic acid less than 50%.Importantly, point
The size of son amount and the purposes of gamma-polyglutamic acid are closely related.Such as the gamma-polyglutamic acid of low molecular weight is most suitable for being applied to agriculture
In industry, and it is then ineffective to be used in other field.And it is used as the gamma-polyglutamic acid in terms of sewage treatment and requires molecular weight especially big
(1500KDa or more), otherwise the effect of flocculated impurities can significantly reduce.And it is bacterial strain self-characteristic in previous microbial fermentation, logical
Gamma-polyglutamic acid catabolic enzyme present in tolerance problem and fermentation system generates hydrolysis etc. to gamma-polyglutamic acid, so that
Gamma-polyglutamic acid has that yield, molecular weight are unstable in mass production.Therefore, it explores fermentation and produces high molecular weight γ-
Polyglutamic acid and its stable optimal culture conditions given full expression to are basis and the key of fermenting and producing.
Summary of the invention
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide a kind of bacillus subtilises
Bacillus sp.A-5 and application thereof, for solving the problems, such as that gamma-polyglutamic acid production efficiency is low in the prior art.
In order to achieve the above objects and other related objects, the present invention provides a kind of bacillus subtilis Bacillus sp.A-
The deposit number of 5, the bacillus subtilis Bacillus sp.A-5 are CCTCC NO:M2019157.
The microorganism that the present invention uses is that inventor screens from Homemade natto and obtains.Its secretion is carried out simultaneously
It isolates and purifies, passes through dissolubility test, ultraviolet-visible spectrum scanning (UV) and chemical identification, Fourier's infrared scan spectrum point
Analysis (FTIR), gel permeation chromatography (GPC), high performance liquid chromatography (HPLC) and UPLC-Q-TOF-MS carry out purified product
Structural Identification finally determines that its secretion is gamma-polyglutamic acid.
Bacillus strain of the invention delivers Zhejiang University's extremophile microorganism laboratory in December, 2018 and carries out form
Characterization, qualification result are as follows:
Morphological feature are as follows: 37 DEG C of incubator overnight incubations, bacterium colony are in irregular cycle, and milky, edge is irregular, impermeable
It is bright；Gram's staining is positive；Microscopically observation is in the shape of a rod, can form gemma；Size is (1.20~3.00) μ m (0.45
~1.40) μm, both ends blunt circle.
The Main Physiological Characteristics of A-5 bacterial strain are as follows: alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase
Enzyme, naphthol-AS-BI-phosphohydrolase are the positive；Nitrate can be restored, arginine, urea, β-Portugal are hydrolyzed
The ability of polyglycoside has gelatin liquefaction ability, and assimilates glucose, and maltose and gluconic acid reactant salt are the positive, V-P examination
It tests, catalase test and methyl red test are the positive.
Through Morphological Identification and the development tree analysis of 16s rRNA extension increasing sequence, determines that the bacterial strain is bacillus subtilis, press
According to Intemational Nomenclature rule: generic name+kind of name+strain name is named the bacterial strain, generic name, kind name, strain name be respectively Bacillus,
Sp. and A-5, it is named as Bacillus sp.A-5, deposit number are as follows: CCTCC NO:M2019157.The withered grass bud that the present invention separates
The nucleotide sequence of spore bacillus Bacillus sp.A-5 bacterial strain is as shown in SEQ ID NO.1.
Bacillus subtilis Bacillus sp.A-5 of the invention is on March 15th, 2019 in Chinese Typical Representative culture
Collection (abbreviation CCTCC) registers preservation, and deposit number is CCTCC NO:M2019157.
Another aspect of the present invention provides above-mentioned bacillus subtilis Bacillus sp.A-5 and is used to prepare γ --
The purposes of polyglutamic acid.
Another aspect of the present invention provides a kind of method for preparing gamma-polyglutamic acid, and the method is using upper
Bacillus subtilis Bacillus sp.A-5 fermentation is stated to be made.
Further, the described method comprises the following steps: actication of culture, Spawn incubation, strain fermentation and extraction are collected
Gamma-polyglutamic acid.
Further, the actication of culture can use following methods: by Bacillus sp.A-5 streak inoculation in inclined-plane
On culture medium, at 30~38 DEG C cultivate 12~for 24 hours.Further, the slant medium contains beef extract, peptone, chlorine
Change sodium, pH value 7.0~7.4, such as can be beef extract 3.0g/L, peptone 10g/L, sodium chloride 5.0g/L, pH value 7.2 it is oblique
Face culture medium.
Further, the Spawn incubation can use following methods: single colonie is inoculated in containing appropriate glutamate
In the seed culture medium of (10-80g/L), cultivate at 30-38 DEG C to the logarithm middle and later periods.Further, the seed culture medium
Carbon source is at least one of glucose, sucrose, starch, glycerol or citric acid, and preferably carbon source is glucose；Nitrogen source is yeast
Extract, beef extract, soy peptone, tryptone or NH4At least one of Cl, preferably nitrogen source are yeast extract；Nothing
Machine salt is K2HPO4Or MgSO4At least one of or two kinds.Further, culture medium shaking speed is 150- when the culture
220rpm。
Further, the strain fermentation can use following methods: strain solution be accessed in fermentation medium, inoculation
1-5%, shaking flask liquid amount 50-100mL/500mL, shaking speed 150-220rpm are measured, cultivates 24-48h at 30-38 DEG C.
Such as fermentation medium includes following components: carbon source 20-40g/L, nitrogen source 3-7g/L, sodium glutamate 10-80g/L,
Inorganic salts 0.1-2g/L, remaining is water, pH value 7.0-7.2.
Further, the fermentation medium carbon source be glucose, sucrose, starch, glycerol or citric acid at least
One kind, preferably carbon source are glucose；Nitrogen source is yeast extract, beef extract, soy peptone, tryptone or NH4In Cl
At least one, preferably nitrogen source are yeast extract；Inorganic salts are K2HPO4Or MgSO4At least one of or two kinds.
Such as fermentation medium includes following components: carbon source 20-40g/L, nitrogen source 3-7g/L, sodium glutamate 10-40g/L,
Inorganic salts 0.1-2g/L, remaining is water, pH value 7.0-7.2.
Further, it is described extract collect the method for gamma-polyglutamic acid can be with are as follows: fermentation liquid is acidified to pH 2.0-
4.0, reduce fermentation broth viscosity；10000-12000rpm, 4 DEG C of centrifugation 10-20min remove thallus.
Further, the method also includes that will extract the gamma-polyglutamic acid collected purifying, the method for the purifying is
Gleanings are dissolved using the organic solvent of pre-cooling, collect precipitating, gamma-polyglutamic acid finished product is made in vacuum freeze drying.More into one
Step ground, the organic solvent are at least one of ethyl alcohol, methanol, isopropanol, preferably ethyl alcohol.
As described above, bacillus subtilis Bacillus sp.A-5 and application thereof of the invention, has below beneficial to effect
Fruit:
Preparing gamma-polyglutamic acid conversion ratio using bacillus subtilis Bacillus sp.A-5 fermentation is more than 75%, molecule
Amount is more than 4000KDa, conversion ratio significantly larger than in the prior art.
Bacterial strain preservation information of the present invention is as follows:
Strain name: bacillus subtilis Bacillus sp.A-5；
Deposit number are as follows:: CCTCC NO:M2019157；
Preservation date: on March 15th, 2019；
Depositary institution's title: China typical culture collection center；
Depositary institution's abbreviation: CCTCC；
Depositary institution address: wuchang, wuhan Luo Jia Shan street Wuhan University Life Science College.
Detailed description of the invention
Fig. 1 is shown as the colonial morphology of 1Bacillus of embodiment of the present invention sp.A-5 on LB plate.
Fig. 2 is shown as Bacillus sp.A-5 first of the present invention and opens scanning electron microscope (SEM) photograph.
Fig. 3 is shown as Bacillus sp.A-5 second of the present invention and opens scanning electron microscope (SEM) photograph.
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.
Before further describing the specific embodiments of the present invention, it should be appreciated that protection scope of the present invention is not limited to down
State specific specific embodiment；It is also understood that term used in the embodiment of the present invention is specific specific in order to describe
Embodiment, rather than limiting the scope of protection of the present invention；In description of the invention and claims, unless in text
In addition explicitly point out, singular "one", " one " and " this " include plural form.
When embodiment provides numberical range, it should be appreciated that except non-present invention is otherwise noted, two ends of each numberical range
Any one numerical value can be selected between point and two endpoints.Unless otherwise defined, the present invention used in all technologies and
Scientific term is identical as the normally understood meaning of those skilled in the art of the present technique.Except specific method, equipment used in embodiment,
Outside material, grasp and record of the invention according to those skilled in the art to the prior art can also be used and this
Any method, equipment and the material of the similar or equivalent prior art of method described in inventive embodiments, equipment, material come real
The existing present invention.
Unless otherwise stated, disclosed in this invention experimental method, detection method, preparation method be all made of this technology neck
Molecular biology, biochemistry, chromatin Structure and the analysis of domain routine, analytical chemistry, cell culture, recombinant DNA technology and
The routine techniques of related fields.These technologies have perfect explanation in the prior art, and for details, reference can be made to Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring
Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic
updates；The series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe,
CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；
METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.),
Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119,
Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1
A kind of synthesis gamma-polyglutamic acid bacterial strain, screens from Homemade natto and obtains, classification naming is bacillus subtilis
Bacterium Bacillus sp.A-5.The bacterial strain is on March 15th, 2019 at China typical culture collection center (abbreviation CCTCC)
Preservation is registered, deposit number is CCTCC NO:M2019157.
Bacillus strain of the invention delivers Zhejiang University's extremophile microorganism laboratory in December, 2018 and carries out form
Characterization, qualification result are as follows:
Morphological feature are as follows: 37 DEG C of incubator overnight incubations, bacterium colony are in irregular cycle, and milky, edge is irregular, impermeable
It is bright；Gram's staining is positive；Microscopically observation is in the shape of a rod, can form gemma；Size is (1.20~3.00) μ m (0.45
~1.40) μm, both ends blunt circle.As shown in Figure 1.
The Main Physiological Characteristics of A-5 bacterial strain are as follows: alkaline phosphatase, esterase (C4), esterase lipase (C8), acid phosphatase
Enzyme, naphthol-AS-BI-phosphohydrolase are the positive；Nitrate can be restored, arginine, urea, β-Portugal are hydrolyzed
The ability of polyglycoside has gelatin liquefaction ability, and assimilates glucose, and maltose and gluconic acid reactant salt are the positive, V-P examination
It tests, catalase test and methyl red test are the positive.
Scanning electron microscope result such as Fig. 2 and 3.
The analysis of 16S rDNA sequence: 16S rDNA major part sequence about 1.5kb is measured, as shown in SEQ ID NO.1.By institute
It obtains and carries out BLAST in sequence and Genbank database, construct with 16S phylogenetic evolution tree.The result shows that the bacterial strain and withered grass
Bacillus (Bacillus sp.) homology is 99%.Combining form, cytochemistry ingredient and 16S rDNA sequence research knot
Its classification and orientation can be bacillus subtilis, be named as Bacillus sp.A-5 by fruit.
Bacillus sp.A-5 16S rDNA, SEQ ID NO.1:
cctaaaggttacctcaccgacttccgggtgttacaaactctcgtggtgtgacgggcggtgtgtacaag
gcccgggaacgtattcaccgcggcatgctgatccgcgattactagcgattccagcttcacgcagtcgagttgcaga
ctgcgatccgaactgagaacagatttgtgggattggcttaacctcgcggtttcgctgccctttgttctgtccattg
tagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccaccttcctccggtttgtcaccgg
cagtcaccttagagtgcccaactgaatgctggcaactaagatcaagggttgcgctcgttgcgggacttaacccaac
atctcacgacacgagctgacgacaaccatgcaccacctgtcactctgcccccgaaggggacgtcctatctctagga
ttgtcagaggatgtcaagacctggtaaggttcttcgcgttgcttcgaattaaaccacatgctccaccgcttgtgcg
ggcccccgtcaattcctttgagtttcagtcttgcgaccgtactccccaggcggagtgcttaatgcgttagctgcag
cactaaggggcggaaaccccctaacacttagcactcatcgtttacggcgtggactaccagggtatctaatcctgtt
cgctccccacgctttcgctcctcagcgtcagttacagaccagagagtcgccttcgccactggtgttcctccacatc
tctacgcatttcaccgctacacgtggaattccactctcctcttctgcactcaagttccccagtttccaatgaccct
ccccggttgagccgggggctttcacatcagacttaagaaaccgcctgcgagccctttacgcccaataattccggac
aacgcttgccacctacgtattaccgcggctgctggcacgtagttagccgtggctttctggttaggtaccgtcaagg
taccgccctattcgaacggtacttgttcttccctaacaacagagctttacgatccgaaaaccttcatcactcacgc
ggcgttgctccgtcagactttcgtccattgcggaagattccctactgctgcctcccgtaggagtctgggccgtgtc
tcagtcccagtgtggccgatcaccctctcaggtcggctacgcatcgtcgccttggtgagccgttacctcaccaact
agctaatgcgccgcgggtccatctgtaagtggtagccgaagccaccttttatgtttgaaccatgcggttcaaacaa
ccatccggtattagccccggtttcccggagttatcccagtcttacaggcaggttacccacgtgttactcacccgtc
cgccgctaacatcagggagcaagctcccatctgtccgctcga
Bacillus of the invention belongs to Firmicutes Firmicutes, bacillus guiding principle Bacilli, bacillus head
Bacillales, Bacillaceae Bacillaceae, bacillus Bacillus.
Embodiment 2
(1) gamma-polyglutamic acid solution can be with cationic surfactant cetyl trimethyl bromine under suitable concentration
The sodium hydroxide solution for changing ammonium (CTAB) reacts to form suspension.
Its principle is that in the solution, the oxygen atom pairing of carboxyl, forms not in the nitrogen-atoms and gamma-polyglutamic acid of CTAB
It is dissolved in the gamma-polyglutamic acid-CTAB complex compound of water.In this experiment, the suspension that gamma-polyglutamic acid and CTAB are formed is in 250nm
The absorbance size and gamma-polyglutamic acid concentration at place are proportional, and have good linear relationship.Configuration a certain concentration ladder first
The gamma-polyglutamic acid titer of degree is stored at room temperature 3-4min, measures it after mixing with isometric CTAB sodium hydroxide solution
Absorbance size at 250nm, and drawing standard curve is y=0.0166x-0.134 (R2=0.9996).
Absorbance of the 1 various concentration gamma-polyglutamic acid of table at 250nm
The gamma-polyglutamic acid finished product of preparation is redissolved in isometric deionized water, dilute after suitable multiple as to
Survey liquid.
The NaOH solution of configuration 8%, and as solvent, is added CTAB, be configured to after CTAB dissolution 6% CTAB it is molten
Liquid.2mL gamma-polyglutamic acid prepare liquid is taken, accurate that 2mL CTAB solution is added, the timing from when being added is sufficiently vibrated, standing
Then 3-4min measures absorbance at this time at wavelength 250nm.By to bacillus subtilis Bacillus in embodiment 1
The analysis for the sediment that sp.A-5 fermentation obtains finds that absorbance of the sediment at 250nm and standard items are completely the same.Pass through
Standard curve obtains the concentration of fermentation gamma-polyglutamic acid, concentration > 30g/L.
(2) the gamma-polyglutamic acid finished product of preparation is redissolved in isometric deionized water, using gel permeation chromatography
(GPC) gamma-polyglutamic acid molecular weight is measured.Mobile phase 50mM NaCl and acetonitrile (4:1), flow velocity 0.7mL/min.The poly- paddy of γ-
Loading after 0.45 μm of membrane filtration of propylhomoserin solution is that standard items make standard curve with polyethylene glycol (PEG), and measurement γ-is poly-
Glutamate molecule amount, molecular weight > 4000KDa.
Embodiment 3
The method for preparing gamma-polyglutamic acid using the gamma-polyglutamic acid synthesis bacterium plant height effect in embodiment 1, steps are as follows:
(1) slant activation: Bacillus sp.A-5 streak inoculation is trained at 37 DEG C on slant medium with oese
It supports overnight.Slant medium component are as follows: beef extract 3.0g/L, peptone 10g/L, sodium chloride 5.0g/L, pH value 7.2.
(2) seed culture: single colonie is chosen from slant medium and is inoculated in the seed culture medium containing suitable glutamic acid
In, 200rpm, overnight incubation is to the logarithm middle and later periods at 37 DEG C.Seed culture medium component are as follows: glucose 10g/L, yeast extract
2g/L, sodium glutamate 10g/L, 7.2,115 DEG C of sterilizing 15min of pH value.
(3) fermented and cultured: the seed liquor obtained in step (2) is linked into fermentation medium, inoculum concentration 5%, shaking flask
Liquid amount 100mL/ Flask volume 500mL, shaking speed 200rpm cultivate 36-48h at 37 DEG C.Fermentation medium component are as follows: Portugal
Grape sugar 40g/L, yeast extract 7g/L, sodium glutamate 40g/L, K2HPO42g/L, MgSO40.25g/L, 7.2,115 DEG C of pH value
Sterilize 15min.
(4) extraction and purification of gamma-polyglutamic acid:
1. fermentation liquid is acidified to pH 2.0-4.0, fermentation broth viscosity is reduced；
2. centrifugal rotational speed 10000-12000rpm, 4 DEG C of centrifugation 10-20min take supernatant；
3. fermentation liquid is precipitated using 3 times of volume pre-cooling dehydrated alcohols, product precipitating is collected；By sediment vacuum refrigeration
It is dry, by analyzing (CTAB method) using sediment in the same manner as in Example 2, absorbance and mark of the sediment at 250nm
Quasi- product are completely the same, it was demonstrated that gamma-polyglutamic acid finished product is made.
Wherein ferment the gamma-polyglutamic acid yield 31.51g/L of preparation, and conversion ratio is more than 75%, and molecular weight is about
4600KDa。
Embodiment 4
The method for preparing gamma-polyglutamic acid using the gamma-polyglutamic acid synthesis bacterium plant height effect in embodiment 1, steps are as follows:
(1) slant activation: Bacillus sp.A-5 streak inoculation is trained at 37 DEG C on slant medium with oese
It supports overnight.Slant medium component are as follows: beef extract 3.0g/L, peptone 10g/L, sodium chloride 5.0g/L, pH value 7.2.
(2) seed culture: single colonie is chosen from slant medium and is inoculated in the seed culture medium containing suitable glutamic acid
In, 200rpm, overnight incubation is to the logarithm middle and later periods at 37 DEG C.Seed culture medium component are as follows: glucose 10g/L, yeast extract
2g/L, sodium glutamate 10g/L, 7.2,115 DEG C of sterilizing 15min of pH value.
(3) fermented and cultured: the seed liquor obtained in step (2) is linked into fermentation medium, inoculum concentration 5%, shaking flask
Liquid amount 100mL/ Flask volume 500mL, shaking speed 200rpm cultivate 36-48h at 37 DEG C.Fermentation medium component are as follows: Portugal
Grape sugar 40g/L, soy peptone 5g/L, sodium glutamate 40g/L, K2HPO42g/L, MgSO40.25g/L, 7.2,115 DEG C of pH value
Sterilize 15min.
(4) extraction and purification of gamma-polyglutamic acid:
1. fermentation liquid is acidified to pH 2.0-4.0, fermentation broth viscosity is reduced；
2. centrifugal rotational speed 10000-12000rpm, 4 DEG C of centrifugation 10-20min take supernatant；
3. fermentation liquid is precipitated using 3 times of pre- cold methanols of volume, product precipitating is collected；Sediment vacuum refrigeration is done
It is dry, by analyzing (CTAB method) using sediment in the same manner as in Example 2, absorbance and standard of the sediment at 250nm
Product are completely the same, it was demonstrated that, it is believed that gamma-polyglutamic acid finished product is made.
Wherein ferment the gamma-polyglutamic acid yield 29.38g/L of preparation, conversion ratio 73.45%, and molecular weight 4600KDa is left
It is right.
Above embodiment is can not to be interpreted as in order to illustrate embodiment disclosed by the invention to limit of the invention
System.In addition, in various modifications and invention listed herein method, composition variation, do not departing from the scope of the present invention
Be obvious for those skilled in the art under the premise of spirit.Although having combined of the invention a variety of specific
Preferred embodiment has carried out specific description to the present invention, it is to be understood that, the present invention should not be limited only to these specific embodiments.
In fact, various obviously modify as described above for those skilled in the art to obtain invention all should include
Within the scope of the invention.
Sequence table
<110>Academy of Agricultural Sciences, Shanghai City
<120>bacillus subtilis Bacillus sp. A-5 and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1402
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
cctaaaggtt acctcaccga cttccgggtg ttacaaactc tcgtggtgtg acgggcggtg 60
tgtacaaggc ccgggaacgt attcaccgcg gcatgctgat ccgcgattac tagcgattcc 120
agcttcacgc agtcgagttg cagactgcga tccgaactga gaacagattt gtgggattgg 180
cttaacctcg cggtttcgct gccctttgtt ctgtccattg tagcacgtgt gtagcccagg 240
tcataagggg catgatgatt tgacgtcatc cccaccttcc tccggtttgt caccggcagt 300
caccttagag tgcccaactg aatgctggca actaagatca agggttgcgc tcgttgcggg 360
acttaaccca acatctcacg acacgagctg acgacaacca tgcaccacct gtcactctgc 420
ccccgaaggg gacgtcctat ctctaggatt gtcagaggat gtcaagacct ggtaaggttc 480
ttcgcgttgc ttcgaattaa accacatgct ccaccgcttg tgcgggcccc cgtcaattcc 540
tttgagtttc agtcttgcga ccgtactccc caggcggagt gcttaatgcg ttagctgcag 600
cactaagggg cggaaacccc ctaacactta gcactcatcg tttacggcgt ggactaccag 660
ggtatctaat cctgttcgct ccccacgctt tcgctcctca gcgtcagtta cagaccagag 720
agtcgccttc gccactggtg ttcctccaca tctctacgca tttcaccgct acacgtggaa 780
ttccactctc ctcttctgca ctcaagttcc ccagtttcca atgaccctcc ccggttgagc 840
cgggggcttt cacatcagac ttaagaaacc gcctgcgagc cctttacgcc caataattcc 900
ggacaacgct tgccacctac gtattaccgc ggctgctggc acgtagttag ccgtggcttt 960
ctggttaggt accgtcaagg taccgcccta ttcgaacggt acttgttctt ccctaacaac 1020
agagctttac gatccgaaaa ccttcatcac tcacgcggcg ttgctccgtc agactttcgt 1080
ccattgcgga agattcccta ctgctgcctc ccgtaggagt ctgggccgtg tctcagtccc 1140
agtgtggccg atcaccctct caggtcggct acgcatcgtc gccttggtga gccgttacct 1200
caccaactag ctaatgcgcc gcgggtccat ctgtaagtgg tagccgaagc caccttttat 1260
gtttgaacca tgcggttcaa acaaccatcc ggtattagcc ccggtttccc ggagttatcc 1320
cagtcttaca ggcaggttac ccacgtgtta ctcacccgtc cgccgctaac atcagggagc 1380
aagctcccat ctgtccgctc ga 1402
Claims (10)
1. a kind of bacillus subtilis Bacillus sp.A-5, the deposit number of the bacillus subtilis Bacillus sp.A-5
For CCTCC NO:M2019157.
2. bacillus subtilis Bacillus sp.A-5 according to claim 1, it is characterised in that: the withered grass gemma
The nucleotide sequence of bacillus Bacillus sp.A-5 is as shown in SEQ ID NO.1.
3. bacillus subtilis Bacillus sp.A-5 according to claim 1, it is characterised in that: the withered grass gemma
Bacillus Bacillus sp.A-5 is milky, and edge is irregular, opaque, in the shape of a rod, can form gemma；Size be (1.20~
3.00) μ m (0.45~1.40) μm, both ends blunt circle.
4. the bacillus subtilis Bacillus sp.A-5 as described in claims 1 to 3 any one is used to prepare the poly- paddy of γ-
The purposes of propylhomoserin.
5. a kind of prepare γ -- the method for polyglutamic acid, the method are using withered as described in claims 1 to 3 any one
Careless bacillus sp.A-5 fermentation is made.
6. the method according to claim 5 for preparing gamma-polyglutamic acid, it is characterised in that: the method includes following steps
It is rapid:
Gamma-polyglutamic acid is collected in actication of culture, Spawn incubation, strain fermentation and extraction.
7. the method according to claim 6 for preparing gamma-polyglutamic acid, it is characterised in that: the actication of culture use with
Lower method: cultivate 12 by bacillus subtilis Bacillus sp.A-5 streak inoculation on slant medium, at 30~38 DEG C~
24h。
8. the method according to claim 6 for preparing gamma-polyglutamic acid, it is characterised in that: the Spawn incubation use with
Lower method: single colonie is inoculated in the seed culture medium containing 10-80g/L glutamate, and culture is into logarithm at 30-38 DEG C
Later period.
9. the method according to claim 6 for preparing gamma-polyglutamic acid, it is characterised in that: the strain fermentation use with
Lower method: strain solution being accessed in fermentation medium, inoculum concentration 1-5%, shaking speed 150-220rpm, is trained at 30-38 DEG C
Support 24-48h.
10. the method according to claim 6 for preparing gamma-polyglutamic acid, it is characterised in that: the method also includes mentioning
The gamma-polyglutamic acid of collection is taken to purify, the method for the purifying is the organic solvent dissolution gleanings using pre-cooling, and it is heavy to collect
It forms sediment, gamma-polyglutamic acid finished product is made in vacuum freeze drying, and the organic solvent is ethyl alcohol, methanol, at least one in isopropanol
Kind.
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