CN110004083A - One plant of Burkholderia cepacia and its application - Google Patents
One plant of Burkholderia cepacia and its application Download PDFInfo
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Abstract
The invention discloses one plant of Burkholderia cepacia and its applications, belong to microorganism field.Burkholderia cepacia (Burkholderia cepacia) ZWS15 is preserved in China typical culture collection center on November 6th, 2017, and deposit number is CCTCC NO:M2017661, and preservation address is Wuhan, China Wuhan University.The bacterium can be using cholesterol as sole carbon source, and can grow at 50 DEG C, is resistant to 10% organic solvent, and cholesterol oxidase enzyme activity is up to 200U/L in the crude enzyme liquid obtained using its fermentation.Cholesterol is catalyzed in the binary system existing for organic solvent using Burkholderia cepacia ZWS15 fermentation crude enzyme liquid, finally being collected into the amount of gallbladder Gona-4-en-3-one 3- ketone is about the 5~11% of substrate additive amount, gallbladder Gona-4-ene-3 3, the amount of 6- diketone are about the 10~15% of substrate additive amount.
Description
Technical field
The present invention relates to one plant of Burkholderia cepacia and its applications, belong to microorganism field.
Background technique
Sterol derivative has various physiological functions, can be used for the industries such as medicine, food and feed.The 5- alkene-of sterol
3- ketone derivatives, 4- alkene -3- ketone derivatives, 4- alkene -3,6- derovatives all have the function of that good lipid-metabolism improves, can
As slimming drugs, treat hepatopathy, atherosclerosis and it is anti-in terms of there is significant curative effect.
The ketenes derivative of sterol mainly passes through chemical synthesis or natural product extraction obtains.Such as cholesteric -4- alkene -3,6-
Diketone is using cholesterol as raw material, by Jones reagent oxidation or the synthesis of PCC/ methylene chloride forensic chemistry or source Yu Haiyang
Natural extracts；Beans Gona-4-ene-3,6- diketone can be by obtaining in plant extracts；Gallbladder Gona-4-en-3-one 3- ketone can utilize micro-
The produced cholesterol oxidase of biology is substrate acquisition with cholesterol or derivatives thereof.
Cholesterol oxidase, also known as 3 beta-hydroxy cholesterol oxidases are a kind of difunctional flavo-enzymes, are that degradation gallbladder is solid
The catalyst of alcohol or other natural sterols.It is the analysis tool enzyme of clinical diagnosis, be used to detect the gallbladder in humoral sample
Sterol content, assessment artery sclerosis, thrombosis, cardiovascular disease and other lipid disorders；It can be used as a kind of biological insecticides, energy
Inhibit the growth of lepidopterous larvae；Can be used as lipid-metabolism-improving agent addition reduces the cholesterol level of food in food；It can
Biocatalyst as industrial steroidal drug important intermediate.
The microbial strains reported at present for producing cholesterol oxidase mainly have with subordinate: Nocardia, false unit cell
Pseudomonas, brevibacterium, Rhod, streptomyces, Arthrobacter, Corynebacterium and bacillus etc..Because of sterol substance
It is not soluble in water, especially during the biocatalysis of steroid drugs, need the chaotropic agents such as high temperature and high proportion organic solvent
It participates in, high requirement is proposed to the temperature tolerance of enzyme and microorganism, organic solvent tolerance.More production is studied at present
The bacterial strain of the enzyme is mainly gram-positive bacteria, and generated cholesterol oxidation enzyme heat stability and organic solvent tolerance are not
It is good.
In the reaction that biocatalysis cholesterol generates steroid drugs, reaction product is mainly single cholesteric -4- alkene -3-
Ketone.And the 4- alkene -3,6- derovatives of sterol than the 4- alkene -3- ketone derivatives of sterol there is more preferably lipid-metabolism improvement to make
With and anti-obesic action.
The reported Gram-negative bacteria for producing cholesterol oxidase, such as pseudomonad, burkholderia, color bacillus,
With good organic solvent tolerance, micro organic solvent is added during strain fermentation as sterol chaotropic agent, hair
Ferment is catalyzed 6-8 days and produces a variety of sterol ketenes derivative products, 4- alkene -3,6- derovatives including more preferably sterol.
But above method is to add micro sterol chaotropic agent (organic solvent) in the fermentation medium, can cause to poison to microorganism, shadow
Secretion and the vigor for ringing cholesterol oxidase, a large amount of generations and later separation for being unfavorable for subsequent sterol ketenes derivative are extracted.
Summary of the invention
The first purpose of the invention is to provide one plant of Burkholderia cepacia (Burkholderia cepacia), institutes
It states Burkholderia cepacia and is preserved in China typical culture collection center on November 6th, 2017, deposit number is
CCTCC NO:M2017661, preservation address are Wuhan, China Wuhan University.
The Burkholderia cepacia CCTCC NO:M2017661, has the following properties:
It (1) is Gram-negative bacteria, rod-shaped, size is (0.8~1.1) μm * (1.5~3.0) μm, and bacterium colony circle is smooth
Shape, moisten it is opaque, faint yellow；
(2) the sterols substance such as energy cholesterol degradation, sitosterol, cholesterine；
(3) there is temperature tolerance, can be grown at 50 DEG C；
(3) there is organic solvent resistance, can be grown in the organic solvent of 5%-10%；
(4) nucleotide sequence of 16S rDNA shows the phase belonged to Burkholderia cepacia as shown in SEQ ID NO.1
Like degree up to 99%.
A second object of the present invention is to provide a kind of method for producing cholesterol oxidase, the method is using described
Burkholderia cepacia carry out fermenting and producing.
In one embodiment of the invention, the fermentation is specifically: the Burkholderia cepacia is inoculated in
In culture medium containing cholesterol, cultivated.
In one embodiment of the invention, the concentration of the cholesterol is 0.5-5gL-1。
In one embodiment of the invention, also lead to TritonX-100 containing Qula in the culture medium.
In one embodiment of the invention, (gL is also contained in the culture medium-1): peptone 5-10, yeast powder
2-5, K2HPO40.5-2, KCl 0.1-0.5, NaNO30.5-2, MgSO4100mM, glucose 0.1-1.
Third object of the present invention is to provide a kind of alkene -3 4- of 4- alkene -3- ketone derivatives and sterol for preparing sterol,
The method of 6- derovatives, the method are to be generated using sterol as substrate using Burkholderia cepacia fermentation
Cholesterol oxidase catalytic production.
In one embodiment of the invention, the sterol is cholesterol, cupreol, stigmasterol, brassicasterol, beans
Sterol, ergosterol, lanosterol or cholestanol and its derivative.
In one embodiment of the invention, the preparation can carry out in aqueous phase system or two-phase system.
In one embodiment of the invention, the organic phase in the two-phase system be methanol, ethyl alcohol, acetone, toluene,
Benzene, dimethylbenzene or n-hexane.
The present invention also provides the Burkholderia cepacias to prepare drug, prepare pharmaceutical products, food and feeding
The application in material field.
In one embodiment of the invention, the application is specifically, the Burkholderia cepacia produced
Sterol derivative is used to prepare the drug of slimming drugs, preparation treatment hepatopathy, atherosclerosis and anti-obesity.
In one embodiment of the invention, the application is specifically, the Burkholderia cepacia produced
Cholesterol oxidase adds the cholesterol level that food is reduced in food as lipid-metabolism-improving agent.
Beneficial effects of the present invention:
(1) the Burkholderia cepacia ZWS15 that screens of the present invention, can be using cholesterol as sole carbon source, and can be
50 DEG C of growths, are resistant to 10% organic solvent (methanol, ethyl alcohol, acetone, toluene, dimethylbenzene or n-hexane).
(2) gallbladder of high temperature resistant organic solvent-resistant can be obtained using Burkholderia cepacia ZWS15 fermentation of the invention
Sterol oxidizing ferment, and cholesterol oxidase enzyme activity is up to 200U/L in the crude enzyme liquid that ferments, and finally purifies obtained cholesterol oxidation
The specific enzyme activity of the pure enzyme of enzyme is 15U/mg.
(3) it is catalyzed in the binary system existing for organic solvent using Burkholderia cepacia ZWS15 fermentation crude enzyme liquid
Cholesterol prepares gallbladder Gona-4-en-3-one 3- ketone and gallbladder Gona-4-ene-3 3,6- diketone, and the amount for being finally collected into gallbladder Gona-4-en-3-one 3- ketone is about
The 5~11% of substrate additive amount, gallbladder Gona-4-ene-3 3, the amount of 6- diketone are about the 10~15% of substrate additive amount.
Biomaterial preservation
Burkholderia cepacia (Burkholderia cepacia) ZWS15 provided by the present invention, taxology name
For Burkholderia cepacia ZWS15 Burkholderia cepacia ZWS15, China is preserved on November 6th, 2017
Type Tissue Collection, deposit number are CCTCC NO:M2017661, and preservation address is Wuhan, China Wuhan University.
Detailed description of the invention
Fig. 1: the SDS-PAGE analysis that extracellular cholesterol oxidase isolates and purifies；Lane 4, the cholesterol oxygen purified
Change enzyme.
Fig. 2: crude enzyme liquid catalysate TLC analysis chart；Lane 1, gallbladder Gona-4-en-3-one 3- ketone standard specimen；Lane 2: it is not added with anti-
Answer the crude enzyme liquid of substrate；Lane 3, aqueous phase system catalysate；Lane 4, toluene/crude enzyme liquid two-phase system catalysate.
Fig. 3: catalysate sterling TLC analysis chart；Lane 1, gallbladder Gona-4-en-3-one 3- ketone standard specimen；Lane 2, aqueous phase system is urged
Change the gallbladder Gona-4-en-3-one 3- ketone of the purified acquisition of product crude product；Lane 3, toluene/crude enzyme liquid two-phase system catalysate crude product warp
Purify the gallbladder Gona-4-en-3-one 3- ketone obtained；Lane 4, the gallbladder Gona-4-ene-3 3 of the purified acquisition of aqueous phase system catalysate crude product,
6- diketone；Lane 5, toluene/crude enzyme liquid two-phase system catalysate crude product purified acquisition gallbladder Gona-4-ene-3 3,6- diketone.
Fig. 4: gallbladder Gona-4-en-3-one 3- ketone structural formula and Mass Spectrometric Identification.
Fig. 5: gallbladder Gona-4-en-3-one 3- ketone in catalysate13C-NMR identification.
Fig. 6: gallbladder Gona-4-en-3-one 3- ketone in catalysate1H-NMR identification.
Fig. 7: cholesteric -4- alkene -3,6- diketone structure formula and Mass Spectrometric Identification.
Fig. 8: cholesteric -4- alkene -3,6- diketone in catalysate13C-NMR identification.
Fig. 9: cholesteric -4- alkene -3,6- diketone in catalysate1H-NMR identification.
Specific embodiment
The measurement of cholesterol oxidase enzyme activity: the coupling reaction of cholesterol oxidase and horseradish peroxidase, gallbladder are utilized
The H that sterol oxydasis steroid substrate generates2O2, phenol and 4- amino-peace can be made under the action of horseradish peroxidase
For red benzoquinone imine class compound more raw than forest products, there is maximum absorption band at A500.Concrete operations: 3mL is added in colorimetric cylinder
Detect liquid A (4- amino-antipyrine, 1mmol/L；Phenol, 6mmol/L；Sodium azide, 0.2g/L；Peroxidase, 5000U/
L；Kaliumphosphate buffer, 25mmol/L, pH 7.5)+150 μ L steroid substrate (isopropanol is solvent) of+50 μ L enzyme solution, three kinds of groups
After dividing oscillation to mix, 37 DEG C of water-bath 15min use boiling water boiling 3min immediately, inactivate enzyme, let cool cooling in water；9000rpm, from
Heart 2min；Survey light absorption value at supernatant A500 is taken, parallel laboratory test is surveyed three times, is averaged.The measurement of enzyme activity standard curve is referring to text
Offer (old also cholesterol oxidase affinity purification and stability to β lactamase (D) Wuxi Southern Yangtze University .2013).
The definition of cholesterol oxidase enzyme activity unit: at 37 DEG C, it is catalyzed 1 μm of ol steroid substrate per minute and is catalyzed into production
Enzyme amount needed for object is defined as 1 enzyme activity unit (U).
The calculation formula of conversion ratio: product molar amount/consumption substrate mole * 100%=conversion ratio of generation
The screening of embodiment 1:Burkholderia cepacia ZWS15 and 16SrDNA identification
(1) sample acquisition and processing
Petroleum-contaminated soil (soil sample depth is 5-20cm) is acquired from Karamay Oil Fields in Xinjiang, in enriched medium
In, it is placed in 180rpm enrichment culture 48h on shaking table, temperature is 30 DEG C.
Enriched medium (g/L): glucose 20, yeast extract 10, KH2PO40.05, KNO31, MgSO40.5, FeSO4
0.01, NaCl 0.5, cholesterol 1, Triton X-100 0.5% (v/v).
(2) primary dcreening operation
After pedotheque enrichment culture, enrichment bacterium solution is taken, at 50 DEG C, is cultivated 3 days on primary dcreening operation culture medium flat plate.
Primary dcreening operation culture medium (g/L): K2HPO42, KCl 0.5, NaNO32, TritonX-100 3.4mL, MgSO4
100mM, and the cholesterol of mass volume ratio 0.1% is added as carbon source.
(3) secondary screening
The bacterium colony grown in picking primary dcreening operation culture medium flat plate continues to cultivate 48-72h in secondary screening culture medium at 50 DEG C.
Secondary screening culture medium: added respectively on the basis of primary dcreening operation culture medium 10% (v/v) organic solvent (methanol, ethyl alcohol,
Acetone, toluene, dimethylbenzene, n-hexane).
It finally screens to one plant of bacterium, extracts the strain gene group, design upstream and downstream primer (being shown in Table 1), standard PCR amplification
16S rDNA is sequenced, and the nucleotide sequence of 16S rDNA is as shown in SEQ ID NO.1.With obtained 16S rDNA sequence
Column are compared with the sequence in NCBI, the results show that the nucleotide sequence and Burkholderia of the 16S rDNA of the bacterial strain
(GenBank No.NZ_CP012981.1) similitude of cepacia ATCC 25416 is 99%, is ultimately determined to onion Bai Kehuo
You are moral bacterium Burkholderia cepacia, is Burkholderia cepacia ZWS15 by the Strain Designation.
Burkholderia cepacia (Burkholderia cepacia) ZWS15, taxology are named as Burkholderia
Cepacia ZWS15 Burkholderia cepacia ZWS15 is preserved in China typical culture collection on November 6th, 2017
Center, deposit number are CCTCC NO:M2017661, and preservation address is Wuhan, China Wuhan University.
1 primer sequence table of table
The physio-biochemical characteristics of embodiment 2:Burkholderia cepacia ZWS15
Burkholderia cepacia ZWS15 is inoculated in respectively with cholesterol, sitosterol, stigmasterol, etembonate
The sterols substance such as alcohol, cholestanol, pregnenolone is that 48h is cultivated at 50 DEG C in the culture medium of sole carbon source, is found
Burkholderia cepacia ZWS15 can grow bacterium colony, show that Burkholderia cepacia ZWS15 can benefit
With these carbon sources.
Culture medium prescription (g/L): K2HPO42, KCl 0.5, NaNO32, TritonX-100 3.4mL, MgSO4
100mM, and the sterol substance that mass volume ratio is 0.1% is added as carbon source.
The physio-biochemical characteristics of Burkholderia cepacia ZWS15 are as shown in table 2.
The physio-biochemical characteristics of 2 bacterial strain of table
Note: ND: uncertain；+: indicate that microorganism can degrade the substance；: indicate that microorganism cannot degrade the substance.
Embodiment 3: cholesterol oxidase is prepared using Burkholderia cepacia ZWS15
(1) Burkholderia cepacia (Burkholderia cepacia) ZWS15 inclined-plane culture: is inoculated in LB solid
Culture medium, under the conditions of 37 DEG C, static gas wave refrigerator 20-28 hours；
LB solid medium: yeast powder 5g, peptone 10g, NaCl 10g, agar powder 20g are added in every 1L distilled water.
(2) seed culture: the bacterial strain that step (1) is cultivated, aseptically picking single colonie is inoculated in 50mL LB liquid
In body culture medium, the shaken cultivation 9-13h in 37 DEG C, the shaking table that revolving speed is 200rpm.
LB liquid medium: yeast powder 5g, peptone 10g, NaCl 10g are added in every 1L distilled water.
(3) fermented and cultured: the seed liquor after taking step (2) to activate is seeded to fermented and cultured by the inoculum concentration of 3%-10%
In base, the shaken cultivation 20-28h in 30 DEG C, the shaking table that revolving speed is 200rpm.
Fermentation medium: peptone 10g, yeast powder 5g, K are added in every 1L distilled water2HPO42g, KCl 0.5g, NaNO3
2g, TritonX-100 3.4mL, MgSO4100mM, glucose 0.1g, cholesterol 2g, pH 7.0.
Wherein: cholesterol addition manner is to mix with a certain amount of culture medium, and ultrasonication is handled after adding TritonX-100
10min。
(4) collection of cholesterol oxidase: for the fermentation liquid that step (3) are obtained at 4 DEG C, 8000rpm is centrifuged 5-10min
Cell is removed, taking supernatant liquor is crude enzyme liquid.Measuring cholesterol oxidase enzyme activity in crude enzyme liquid is 200U/L.
(5) cholesterol oxidase isolates and purifies: by mass percent being 50% by the crude enzyme liquid obtained in step (4)
Ammonium sulfate precipitated；Sediment is collected, is dissolved with the phosphate buffer of pH 7.0, removes remnants' using the method for dialysis
Ammonium sulfate；Fermentation liquid is concentrated using the method for ultrafiltration, net cholesterol oxidizing ferment is obtained (such as by DEAE ion-exchange chromatography
Fig. 1 Lane 4).The specific enzyme activity for measuring net cholesterol oxidizing ferment is 15U/mg.
The gene cloning of embodiment 4:Burkholderia cepacia ZWS15 cholesterol oxidase
Bacterial genomes kit extracts the genome of Burkholderia cepacia ZWS15 as template, and design is drawn
Object (being shown in Table 3) carries out PCR amplification.PCR response parameter are as follows: 95 DEG C of initial denaturations 4min, 95 DEG C of denaturation 1min, 60 DEG C of annealing 1min,
72 DEG C of extension 1.5min, 30 circulations, 72 DEG C re-extend 10min.The gene of obtained cholesterol oxidase is sequenced, is obtained
To its nucleotide sequence as shown in SEQ ID NO.2.
3 primer sequence table of table
Embodiment 5: the organic solvent analysis of cholesterol oxidase and thermal stability analysis
(1) measurement of the thermostabilization parameter melting temperature (Tm) of cholesterol oxidase
Melting temperature is measured by differential scanning calorimeter (DSC).When measurement, pure gallbladder obtained in Example 3
Sterol oxidizing ferment enzyme solution, protein concentration 1.0mg/mL, buffer solution system are Tris-HCl buffer (pH 9.0,20mM).Temperature
Degree rate of change is 10 DEG C/min, and temperature change section is 20~90 DEG C.The melting temperature Tm of cholesterol oxidase as the result is shown
Value is 71.03, shows the good thermal stability of the enzyme.
(2) cholesterol oxidase organic solvent tolerance is analyzed
The pure enzyme solution 2U/mL of cholesterol oxidase obtained in Example 3, with 30% (v/v) variety classes (such as acetic acid second
Ester, petroleum ether, butanol, chloroform, benzene, toluene, paraxylene, 1,3,5- trimethylbenzene, hexamethylene, n-hexane, normal octane) it is hydrophobic
Property organic solvent mixing, using add 30% (v/v) water pure enzyme solution as control, be placed in 37 DEG C, revolving speed for 200rpm shaking table
Middle shake culture 24 hours uses cholesterol as substrate, and remaining enzyme activity after measurement mixing, the results are shown in Table 4.Show gallbladder
The opposite residual enzyme activity of sterol oxidizing ferment in organic solvent, which is higher than in aqueous solution, remains enzyme activity, which has good organic molten
Agent tolerance.
Influence of 4 organic solvent of table to cholesteryl ester enzyme stability
Note: Log Pow (Octanol/water Partition Coefficients): the logarithm of distribution coefficient of the substance between octanol/water.Characterization has
One parameter of solvent, the value is bigger, illustrates that the hydrophobicity of organic solvent is stronger.
Embodiment 6: Burkholderia cepacia ZWS15 fermentation crude enzyme liquid prepares sterol derivative in aqueous phase system
Aqueous phase system: 50mL crude enzyme liquid, 2mg/mL cholesterol, 10% (v/v) water.
(1) catalysis reaction
2mg/mL cholesterol, 10% (v/ is added in the cholesterol oxidase crude enzyme liquid 50mL being prepared in Example 3
V) water, 37 DEG C, 200rpm oscillating reactions for 24 hours.By the generation feelings of catalytic mixing product utilization thin layer chromatography (TLC) detection product
Condition, the results showed that containing there are two types of products in catalysate: gallbladder Gona-4-en-3-one 3- ketone and gallbladder Gona-4-ene-3 3,6- diketone (figure
2lane3).Finally being collected into the amount of gallbladder Gona-4-en-3-one 3- ketone is about the 5~8% of substrate additive amount, gallbladder Gona-4-ene-3 3,6- diketone
Amount be about the 8~10% of substrate additive amount.
(2) purifying of catalysate
It is catalyzed reaction solution chloroform extraction, it is dry in vacuum rotary evaporator, obtain catalysate melange.Benefit
It is isolated and purified with preparative TLC, it is dry in vacuum rotary evaporator, obtain each pure product.
(3) identification of purified product
For further accurate Qualitative Identification product, the catalysate sterling of acquisition is subjected to TLC analysis, the results showed that, water
Sterling gallbladder Gona-4-en-3-one 3- ketone (Fig. 3 lane 2) and cholesteric -4- alkene -3,6- two are respectively obtained after phase system catalysate is purified
Ketone (Fig. 3 lane 4)；The gallbladder Gona-4-en-3-one 3- ketone purity wherein obtained is about 90%~95%, gallbladder Gona-4-ene-3 3, and 6- diketone is pure
Degree about 85%~92%.
By gallbladder Gona-4-en-3-one 3- ketone sterling through LC-MS (Fig. 4),13C-NMR (Fig. 5) and1H-NMR identifies (Fig. 6), as a result table
The bright catalysate is gallbladder Gona-4-en-3-one 3- ketone really；By cholesteric -4- alkene -3,6- diketone through LC-MS (Fig. 7),13C-NMR (Fig. 8)
With1H-NMR identifies (Fig. 9), the results showed that the catalysate is gallbladder Gona-4-ene-3 3,6- diketone really.
Embodiment 7: Burkholderia cepacia ZWS15 fermentation crude enzyme liquid prepares sterol derivative in two-phase system
It is identical as preparation process in embodiment 6, except that reaction carries out in a two-phase system.
Two-phase system: 50mL crude enzyme liquid, 2mg/mL cholesterol, 10% (v/v) toluene.
(1) catalysis reaction
2mg/mL cholesterol, 10% (v/ is added in the cholesterol oxidase crude enzyme liquid 50mL being prepared in Example 3
V) toluene, 37 DEG C, 200rpm oscillating reactions for 24 hours.By the generation of catalytic mixing product utilization thin layer chromatography (TLC) detection product
Situation, the results showed that containing there are two types of products in catalysate: gallbladder Gona-4-en-3-one 3- ketone and gallbladder Gona-4-ene-3 3,6- diketone (figure
2lane4).Finally being collected into the amount of gallbladder Gona-4-en-3-one 3- ketone is about the 5~11% of substrate additive amount, gallbladder Gona-4-ene-3 3,6- bis-
The amount of ketone is about the 10~15% of substrate additive amount.
(2) purifying of catalysate
It is catalyzed reaction solution chloroform extraction, it is dry in vacuum rotary evaporator, obtain catalysate melange.Benefit
It is isolated and purified with preparative TLC, it is dry in vacuum rotary evaporator, obtain each pure product.
(3) identification of purified product
For further accurate Qualitative Identification product, the catalysate sterling of acquisition is subjected to TLC analysis, the results showed that, two
Sterling gallbladder Gona-4-en-3-one 3- ketone (Fig. 3 lane 3) and cholesteric -4- alkene -3,6- two are respectively obtained after phase system catalysate is purified
Ketone (Fig. 3 lane5)；The gallbladder Gona-4-en-3-one 3- ketone purity wherein obtained is about 95%~99.99%, gallbladder Gona-4-ene-3 3,6- diketone
Purity is about 95%~99.99%.
Compared with aqueous phase system, Burkholderia cepacia ZWS15 fermentation crude enzyme liquid is catalyzed gallbladder in a two-phase system
Sterol prepare gallbladder Gona-4-en-3-one 3- ketone and cholesteric -4- alkene -3,6- diketone it is more efficient, the substrate transformation rate is higher, product purity
More preferably (it is shown in Table 5).
The preparation of product in the different systems of table 5
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
SEQUENCE LISTING
<110>Southern Yangtze University
<120>one plants of Burkholderia cepacias and its applications
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 1391
<212> DNA
<213>artificial sequence
<400> 1
caagtcgaac ggcagcacgg agtgcttgca cctggtaggc gagtggcgaa cgggtgagta 60
atacatcgga acatgtcctg tagtggggga tagcccggcg aaagccggat taataccgca 120
tacgatctac ggatgaaagc gggggacctt cgggcctcgc gctatagggt tggccgatgg 180
ctgattagct agttggtggg gtaaaggcct accaaggcga cgatcagtag ctggtctgag 240
aggacgacca gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg 300
gggaattttg gacaatgggc gaaagcctga tccagcaatg ccgcgtgtgt gaagaaggcc 360
ttcgggttgt aaagcacttt tgtccggaaa gaaatccttg gctctaatac agtcggggga 420
tgacggtacc ggaagaataa gcaccggcta actacgtgcc agcagccgcg gtaatacgta 480
gggtgcgagc gttaatcgga attactgggc gtaaagcgtg cgcaggcggt ttgctaagac 540
cgatgtgaaa tccccgggct caacctggga actgcattgg tgactggcag gctagagtat 600
ggcagagggg ggtagaattc cacgtgtagc agtgaaatgc gtagagatgt ggaggaatac 660
cgatggcgaa ggcagccccc tgggccaata ctgacgctca tgcacgaaag cgtggggagc 720
aaacaggatt agataccctg gtagtccacg ccctaaacga tgtcaactag ttgttgggga 780
ttcatttcct tagtaacgta gctaacgcgt gaagttgacc gcctggggag tacggtcgca 840
agattaaaac tcaaaggaat tgacggggac ccgcacaagc ggtggatgat gtggattaat 900
tcgatgcaac gcgaaaaacc ttacctaccc ttgacatggt cggaatcccg ctgagaggtg 960
ggagtgctcg aaagagaacc ggcgcacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt 1020
gagatgttgg gttaagtccc gcaacgagcg caacccttgt ccttagttgc tacgcaagag 1080
cactctaagg agactgccgg tgacaaaccg gaggaaggtg gggatgacgt caagtcctca 1140
tggcccttat gggtagggct tcacacgtca tacaatggtc ggaacagagg gttgccaacc 1200
cgcgaggggg agctaatccc agaaaaccga tcgtagtccg gattgcactc tgcaactcga 1260
gtgcatgaag ctggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc 1320
gggtcttgta cacaccgccc gtcacaccat gggagtgggt tttaccagaa gtggctagtc 1380
taaccgcaag g 1391
<210> 2
<211> 1749
<212> DNA
<213>artificial sequence
<400> 2
atgagtcaag acttccgaga cgaaccagcg tcgcgccgcg ctttcctcgc cgacatggcg 60
aagctcgcgg cggccggcat cgtcaccggc tggacaccgc tctaccaggt tgcggcgcac 120
gcgcggacac ccggcgacac accgcccggc ttcccggccg acatcccgct ttacaagcag 180
gcgttcctga actggagcgg cgagatcgcc gtgcaggacg tatggaccgc cgcgccgcgc 240
tccgccgacg acgtcgtcgc aaccgtcaac tgggcgcgcg caaacggcta ccggatacgc 300
ccgcgcggct acacgcacaa ctggtcgccg ctgacgctgg acccgggcgc cggcgccgcg 360
aacctcgtgc tgctcgatac gacgaagtcg ctgacggccg tctccgtcga cacgtcggcg 420
cgtccggcgc gcgtcaccgc ccaaacgggc gtctcgctgg agtcgctgct cgcaacgctc 480
gagcagtacg gcctcggcgt gattgccgcg ccggcaccgg gcgacatcac gctcggcggc 540
gcgctcgcga tcgatgcaca cggcaccgcc gtgccggcgg ccggtgaaac cttgcagccg 600
ggccatacct acggctcgct gagcaatctc gtggtcgcgc tcacggcggt cgtgttcgat 660
ccggcccggc agcaatacgt gctgcgccgg ttcgagcgca ccgatcccga gatcggcgcg 720
tttctcgcgc acatcgggcg ggcgctcgtc gtcgaggtca cgctgacggc aggcccgaac 780
cagcggctac gctgccagag ctacgtcgat attccggcat ccgaactgtt cgccgcggcc 840
ggcgcgacag gccgcacgat cgcgtcgttt ctcgatcgcg cgggccgggt ggaagccatc 900
tggtttccgt ttacgaccaa gccgtggctc aaggtctgga cgccgacgcc cagcaagccg 960
ttcctgtcgc gcgccgtcac gcagccgtac aactatccgt tctccgattc gatctcgcag 1020
tccatctcgg atctcgtcaa gcggatcgtg atcggcggcg aaggcgcatt gacaccgctg 1080
ttcggccaga cgcagctggc catcacgacg gccggtctcg cactcacgct cagcggggac 1140
atctggggct ggtcgcgcac cgtgctgcag tacattcggc caacgacgct gcgcgtcacc 1200
gcgaacggct acgcggtact ggcgcggcgc gccgacgtgc agcgcgtgat cagcgaattc 1260
gtgcagttct atcagaaccg cgtcgacacg tacaaggcgc gcggcgagta tccgatgaac 1320
ggtcccgtcg agatccgcgt caccggtctc gacaagccgg ccgatgccgg cccgggcgcg 1380
gccgtacccg ccttgtccgc gctcaagccg cgccccgacc ggccggaatg ggataccgcc 1440
gtatggttcg acatcctgac gttgccgggc acgccggccg ccgatcgctt ctatcgcgag 1500
atcgagcaat ggatgctcgc gaactacacc ggctcgtatg cgacggtgcg cccggaatgg 1560
tcgaagggct gggcctatac cgacacggcc gcctggcagg acgacacgat gctcaccacc 1620
acgattccga acctgcatcg tgagggccag ccgccgtcga gcagctggga tacggcgcgc 1680
gcgacgctcg agcgctacga tccgcaccgg atcttccggt cgccgctgct ggatcggttg 1740
atgccgtaa 1749
<210> 3
<211> 20
<212> DNA
<213>artificial sequence
<400> 3
agagtttgat cctggctcag 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence
<400> 4
aaggaggtga tccagccgca 20
<210> 5
<211> 34
<212> DNA
<213>artificial synthesized
<400> 5
aattaccgga attcatgagt caagacttcc gaga 34
<210> 6
<211> 33
<212> DNA
<213>artificial synthesized
<400> 6
aattaccgaa gctttcacca tcaaccgatc cag 33
Claims (10)
1. one plant of Burkholderia cepacia (Burkholderia cepacia), which is characterized in that primary gram of Hall of the onion
Moral bacterium is preserved in China typical culture collection center on November 6th, 2017, and deposit number is CCTCC NO:
M2017661, preservation address are Wuhan, China Wuhan University.
2. a kind of method for producing cholesterol oxidase, which is characterized in that the method is using onion described in claim 1
Burkholderia carries out fermenting and producing.
3. according to the method described in claim 2, it is characterized in that, the fermentation is specifically: by the onion Burkholder
Bacterium is inoculated in the culture medium containing cholesterol, is cultivated.
4. according to the method described in claim 3, it is characterized in that, the concentration of the cholesterol is 0.5-5gL-1。
5. a kind of method of 4- alkene -3, the 6- derovatives for the 4- alkene -3- ketone derivatives and sterol for preparing sterol, feature exist
In the method is to carry out fermenting and producing using Burkholderia cepacia described in claim 1 using sterol as substrate.
6. according to the method described in claim 5, it is characterized in that, the sterol is cholesterol, cupreol, stigmasterol, dish
Seed sterol, stigmasterol, ergosterol, lanosterol or cholestanol and its derivative.
7. according to the method described in claim 5, it is characterized in that, the preparation carries out in aqueous phase system or two-phase system.
8. the method according to the description of claim 7 is characterized in that the organic phase in the two-phase system is methanol, ethyl alcohol, third
Ketone, toluene, dimethylbenzene or n-hexane.
9. a kind of microbial bacterial agent, which is characterized in that the microbial bacterial agent contains primary gram of Hall of onion described in claim 1
Moral bacterium.
10. Burkholderia cepacia described in claim 1 or microbial bacterial agent as claimed in claim 9 prepare drug,
Prepare the application of pharmaceutical products, food and field of fodder.
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| CN111471737A (en) * | 2020-04-26 | 2020-07-31 | 江南大学 | Method for preparing sterol derivative by virtue of Burkholderia transformation and application |
| CN112725233A (en) * | 2021-01-08 | 2021-04-30 | 南京科技职业学院 | Strain for producing 2, 5-furandimethanol and application thereof |
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| CN112725233A (en) * | 2021-01-08 | 2021-04-30 | 南京科技职业学院 | Strain for producing 2, 5-furandimethanol and application thereof |
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