CN109613162A - A method of induction marine actinomycete cryptiogene expression generates novel substance - Google Patents
A method of induction marine actinomycete cryptiogene expression generates novel substance Download PDFInfo
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Abstract
The invention belongs to gene inductive technology fields, disclose a kind of method that induction marine actinomycete cryptiogene expression generates novel substance；It is diluted with taking its intestinal tissue to be placed in sterile water after sterile wash 3 times with no Herba Artemisiae Scopariae seawater flushing Anthocidaris crassispina, is drawn 200 μ L, be coated on the Gao Shi culture medium flat plate containing 50 μ g/mL potassium bichromates and 20 μ g/mL kanamycins, obtain actinomycetes strain；It is fermented on a small scale to bacterial strain, the ethyl acetate extract of 2 plants of bacterial strain fermentation liquors is filtered out using plate filter paper enzyme, and carry out screening active ingredients；Bacterial strain after detection induction generates new secondary metabolite.The present invention uses physics and chemical method for the first time to induce marine actinomycete cryptiogene expression to generate novel substance, and the method for combining with activity rating and establishing induction and identification marine actinomycete production novel substance is tested using spectroscopy.
Description
Technical field
The invention belongs to gene inductive technology fields more particularly to a kind of induction marine actinomycete cryptiogene expression to generate
The method of novel substance.
Background technique
Currently, the prior art commonly used in the trade be such that microorganism generate active material in more than 50% from
Actinomyces, it is scientific in past 50 years since ability of the actinomyces in terms of generating novel active metabolite is very prominent
Family have been devoted to separate novel actinomyces from terrestrial microbial source.However, being separated from terrestrial actinomyces in recent years
The quantity of the new compound arrived in lasting reduction, therefore people need to find from undeveloped actinomyces habitat it is novel
Actinomyces, the source as exploitation novel active secondary metabolite.Due to the particularity of marine environment, marine actinomycete is in life
It is had differences in physiological-biochemical characteristic and genetic background characteristic with terrestrial actinomyces, marine actinomycete is enabled to generate structure novel
Active metabolite.Marine actinomycete is distributed in different marine environment and habitat, and having that numerous studies prove can be
Novel actinomyces are found from anywhere in ocean, including from deep seafloor to coral reef, from bottom sediment to ocean without ridge
Vertebrate and plant, the trace that marine actinomycete can be all found from the surface of ocean to seabed abyss.
In recent years, obligate marine actinomycete Salinispora category bacterial strain, which is reported, can generate the secondary of various structures multiplicity
Metabolin, the category actinomyces are proved to be the source of new chemical structure material abundance, including potential protease inhibitors
SalinosporamideA, new terpenoid, amino acid derivativges and polyene macrolides.
Marine actinomycete has important application value in aquaculture, and existing research marine actinomycete is applied to aquatic products
Cultivation pathogenic bacterium causes the prevention of disease.The prior art one is reported is drawn using marine actinomycete treatment and prevention kinds of pathogenic vibrio
The shrimp disease risen, is able to suppress the formation of vibrios mycoderm, and antibacterial substance is produced in cultivating pool water system.The prior art two is reported
Road marine streptomyces are applied to promote the growths of Penaeus monodon as probiotics.Three researcher of the prior art is from ocean unwrapping wire
Antibacterial material is extracted in bacterium, and it is fed into litopenaeus vannamei together with food, and the prawn of tool white point syndrome virus is generated
Antiviral effect.
Mariculture industry development in recent years is swift and violent, and many aquiculture diseases are serious and uncontrollable, and from marine organisms
And its research and development is rapid with finding newtype drug molecule in symbiotic microorganism, marine microorganism is applied to aquatic products by existing research
Cultivation pathogenic bacterium causes the prevention of disease, it may be possible to which its effect for generating bioactive molecule, this is the control of seawater aquiculture disease
Provide good research direction.Meanwhile microorganism has the advantages that growth and breeding is fast, is easy to cultivate, and can pass through breeding, culture
The technologies such as condition control improve the content of its effective component, and can realize industrialization large-scale production using Fermentation Engineering,
To fundamentally solve the problems, such as shortage of raw materials.
In conclusion problem of the existing technology is:
(1) yield of active ingredients that wild actinomycetes strain fermentation generates is often lower, even if sometimes can be isolated
Noval chemical compound, but because measure the work without being enough to complete its activity identification etc. very little.
(2) many secondary metabolic pathways under conventional culture conditions in actinomyces are silencings, so more difficult find it
The ability for producing new active substance, to limit the discovery of original new drug lead compound on source.
Solve the difficulty and meaning of above-mentioned technical problem:
Newtype drug molecule is found from marine organisms and its symbiotic microorganism, it can be to caused by aquaculture pathogenic bacteria
Disease is prevented and treated.
Microorganism has the advantages that growth and breeding is fast, is easy to cultivate, and can be mentioned by technologies such as breeding, condition of culture controls
The content of its high effective component, and industrialization large-scale production can be realized using Fermentation Engineering, to fundamentally solve former
The problem of material shortage.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of generations of induction marine actinomycete cryptiogene expression
The method of novel substance.
The invention is realized in this way a method of induction marine actinomycete cryptiogene expression generates novel substance are as follows:
Step 1: it is first embathed Anthocidaris crassispina 3 times with no Herba Artemisiae Scopariae seawater, 10 minutes every time, is subsequently placed in 75% alcohol and sterilizes
10 minutes, after washed 3 times with sterile saline, aseptic filter paper is carefully solved with sterile scissors and scalpel again after blotting surface moisture
Cut open, take out enteron aisle and in sterile saline and smash to pieces, with sterile saline gradient dilution, draw 200 μ L, be coated on containing
On the Gao Shi culture medium flat plate of 50 μ g/mL potassium bichromates and 20 μ g/mL kanamycins, it is subsequently placed in 28 DEG C of incubators and cultivates 15
It, during which inspects periodically, and when there is doubtful actinomyces bacterium colony to grow, careful picking single colonie streak inoculation is cultivated to fresh ISP2
It is cultivated on base plate, obtains pure culture bacterial strain through 2~3 scribing line purifying picking single colonie.
Step 2: by the strain inoculated isolated and purified in the liquid Gao Shi culture medium and malt extract of 200mL
(ME) culture medium (1L contains the component of following quality: 10~15g of malt extract, 10~15g of glucose, 1~2g of peptone,
PH value is 7.0~7.2) in, with 160~210rmin at 26~28 DEG C-110~14d of shaken cultivation, obtains fermentation liquid；It crosses
Filter fermentation liquid obtains bacterium solution and thallus, and bacterium solution is extracted 2~3 times with isometric ethyl acetate, thallus chloroform methanol (volume ratio
It is that the extract being concentrated under reduced pressure to give at 1:1) extraction 2~3 times, 40~60 DEG C is screened for anti-pathogenic activity；
Step 3: the ethyl acetate extract of 2 plants of bacterial strain fermentation liquors is filtered out to lithosporic fish disease using plate filter paper enzyme
Former vibrio alginolyticus strain has growth inhibitory activity, and antibacterial circle diameter is 8~17mm；Using two kinds of culture mediums in step 2,
The optimization that fermentation condition is carried out to the above-mentioned active bacterial strain that screening obtains by 18 DEG C of low temperature stimulations, changes fermentation medium not
Same carbon source (glucose or sucrose), nitrogen source (peptone or yeast extract) or addition rare earth element etc., it is small-scale to carry out 200mL
Fermentation；
Step 4: handling the fermentation liquid ethyl acetate extract after inducing and before induction using ODS C-18 reversed-phase column, into
Row HPLC-MS-UV analysis, in conjunction with its hydrogen spectrum analysis, the bacterial strain after detection induction generates new secondary metabolite.It was found that using changing
(1L contains the component of following quality, malt extract 10g, sucrose 20g, peptone 2g, rare earth element 1mg, pH to good ME culture medium
Value is detected as 3,4- for that 7.2) can generate new active constituent with inducible strain Streptomyces spectabilis HDa1
Dimethoxy -1- naphthalene amino acid (3,4-dimethoxy-1-naphthamide, compound 1) and p-O-3,3- dimethyl-allyl -
Benzamide (p-O- (3,3-dimethylallyl)-benzamide, compound 2).
Further, the plate filter paper enzyme is that extract sample is made into concentration with dimethyl sulfoxide is the molten of 5mg/mL
Liquid adds 50 μ L of sample solution on the sterilizing filter paper disk of diameter 6mm, then is affixed on to have applied and be put down for the culture medium for trying bacteria suspension
On plate, place 20min after place into incubator, 28 DEG C dark culture 7 days, survey its inhibition zone size.
Further, the inducer is rare earth element.
Another object of the present invention is to provide a kind of detection method of novel substance, this method are as follows:
Step 1: be utilized respectively screening and culturing medium and add inducer culture medium to 2 plants of actinomyces in the training optimized
Large scale fermentation is carried out under the conditions of supporting, fermentation liquid and mycelium are extracted 3 times with ethyl acetate, chloroform methanol respectively, then anti-
Bacterium screening active ingredients guidance under, using a variety of chromatographic separation technologies by the active constituent of novel substance from fermentation liquid and thallus crude extract
Separation and purification comes out；
Step 2: integrated use Modern spectroscopy technology determines the chemical structure of monomeric compound；
Step 3: further carrying out activity rating to the reactive compound that above structure determines using plate filter paper enzyme,
Real antibacterial, disease-resistant new active substance may be desirably to obtain with other interference by excluding false positive.
Further, the chromatographic separation technology includes: normal pressure positive, reversed-phase silica gel column chromatography, gel chromatography, half preparation
HPLC。
Further, the Modern spectroscopy technology includes: that one-dimensional, two-dimentional, nuclear magnetic resonance technique, high resolution mass spectrum technology, X are penetrated
Line single crystal diffraction technology and ultraviolet, infrared, optically-active, circular dichroism spectra.
In conclusion advantages of the present invention and good effect are as follows: about the method that induction actinomyces produce novel substance, Qian Renzhu
Induction research is carried out using single chemistry or gene genetic operating method, and the present invention uses low temperature induction and addition for the first time
The method that different nutrient sources combines utilizes to induce marine actinomycete cryptiogene expression to generate novel substance and improves ME culture
Base inducible strain Streptomyces spectabilis HDa1 generates new active constituent, is detected as 3,4- dimethoxy -1-
Naphthalene amino acid (3,4-dimethoxy-1-naphthamide, compound 1) and p-O-3,3- dimethyl-allyl-benzamide (p-
O- (3,3-dimethylallyl)-benzamide, compound 2), to combine foundation using spectroscopy test and activity rating
The method that induction and identification marine actinomycete produce novel substance.
Detailed description of the invention
Fig. 1 is the method flow that induction marine actinomycete cryptiogene expression provided in an embodiment of the present invention generates novel substance
Figure；
Fig. 2 is the detection method flow chart of novel substance provided in an embodiment of the present invention.
Fig. 3 is that newly occur two main components 1 and 2 in crude extract 2 after induction provided in an embodiment of the present invention to illustrate
Figure.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is described in detail with reference to the accompanying drawing.
As shown in Figure 1, induction marine actinomycete cryptiogene expression provided in an embodiment of the present invention generates the side of novel substance
Method are as follows:
S101: Anthocidaris crassispina is first embathed 3 times with no Herba Artemisiae Scopariae seawater, 10 minutes every time, is subsequently placed in 75% alcohol and is sterilized 10
Minute, then washed 3 times with sterile saline, aseptic filter paper is carefully solved with sterile scissors and scalpel again after blotting surface moisture
Cut open, take out enteron aisle and in sterile saline and smash to pieces, with sterile saline gradient dilution, draw 200 μ L, be coated on containing
On the Gao Shi culture medium flat plate of 50 μ g/mL potassium bichromates and 20 μ g/mL kanamycins, it is subsequently placed in 28 DEG C of incubators and cultivates 15
It, during which inspects periodically, and when there is doubtful actinomyces bacterium colony to grow, careful picking single colonie streak inoculation is cultivated to fresh ISP2
It is cultivated on base plate, obtains pure culture bacterial strain through 2~3 scribing line purifying picking single colonie；
S102: being fermented 200mL purified actinomyces on a small scale using liquid Gao Shi culture medium and ME culture medium, filtering hair
Zymotic fluid obtains bacterium solution and thallus, and bacterium solution is extracted 2~3 times with isometric ethyl acetate, thallus with chloroform methanol (volume ratio 1:
1) extract being concentrated under reduced pressure to give at extracting 2~3 times, 40~60 DEG C is screened for anti-pathogenic activity；
S103: the ethyl acetate extract of 2 plants of bacterial strain fermentation liquors is filtered out to grouper cause of disease using plate filter paper enzyme
Vibrio alginolyticus strain has growth inhibitory activity, and antibacterial circle diameter is 8~17mm；Using above two culture medium, to screening
The active bacterial strain arrived carries out the optimization of fermentation condition, by 18 DEG C of low temperature stimulations, changes the different carbon source (grape of fermentation medium
Sugar or sucrose), nitrogen source (peptone or yeast extract) or addition rare earth element etc., carry out 200mL and ferment on a small scale；
S104: the fermentation liquid ethyl acetate extract after inducing and before induction is handled using ODS C-18 reversed-phase column, is carried out
HPLC-MS-UV analysis, in conjunction with its hydrogen spectrum analysis, the bacterial strain after detection induction generates new secondary metabolite.It was found that utilizing improvement
(1L contains the component of following quality, malt extract 10g, sucrose 20g, peptone 2g, rare earth element 1mg, pH value to ME culture medium
For 7.2) new active constituent can be generated with inducible strain Streptomyces spectabilis HDa1, it is detected as 3,4- bis-
Methoxyl group -1- naphthalene amino acid (3,4-dimethoxy-1-naphthamide, compound 1) and p-O-3,3- dimethyl-allyl-benzene
Formamide (p-O- (3,3-dimethylallyl)-benzamide, compound 2).
Plate filter paper enzyme provided in an embodiment of the present invention is that extract sample is made into concentration with dimethyl sulfoxide to be
The solution of 5mg/mL adds 50 μ L of sample solution on the sterilizing filter paper disk of diameter 6mm, then is affixed on and has been applied for trying bacteria suspension
Culture medium flat plate on, place 20min after place into incubator, 28 DEG C dark culture 7 days, survey its inhibition zone size.
Inducer provided in an embodiment of the present invention is rare earth element.
As shown in Fig. 2, the detection method of novel substance provided in an embodiment of the present invention are as follows:
S201: be utilized respectively screening and culturing medium and add inducer culture medium to 2 plants of actinomyces in the culture optimized
Under the conditions of carry out large scale fermentation, fermentation liquid and mycelium are extracted 3 times with ethyl acetate, chloroform methanol respectively, then in antibacterial
Under screening active ingredients guidance, the active constituent of novel substance is divided from fermentation liquid and thallus crude extract using a variety of chromatographic separation technologies
It is come out from purification；
S202: integrated use Modern spectroscopy technology determines the chemical structure of monomeric compound；
S203: activity rating, row further are carried out to the reactive compound that above structure determines using plate filter paper enzyme
Except false positive may be desirably to obtain real antibacterial, disease-resistant new active substance with other interference.
Chromatographic separation technology provided in an embodiment of the present invention include: normal pressure positive, reversed-phase silica gel column chromatography, gel chromatography,
Half preparation HPLC.
Modern spectroscopy technology provided in an embodiment of the present invention includes: one-dimensional, two-dimentional, nuclear magnetic resonance technique, high resolution mass spectrum
Technology, X-ray single crystal diffraction technology and ultraviolet, infrared, optically-active, circular dichroism spectra.
Application effect of the invention is explained in detail below with reference to experiment.
Below in experiment, mass spectrograph is Agilent 6210TOF LC-MS (high-resolution) mass spectrograph.NMR spectrometer with superconducting magnet
For BrukerAVIII-500.Silica gel for thin layer chromatography GF254 and column chromatography silica gel (200-300 mesh) are Qingdao Haiyang chemical industry
Factory's product.Reverse phase ODS filler and Sephadex LH-20 are U.S.'s Merck Products.Water is dual distilled water, other examinations
Agent is that analysis is pure.In biological activity test experiment, to grouper cause of disease vibrio alginolyticus strain inhibitory activity test model reference
Nature Protocol, 2008,3,163-175 (Agar and broth dilution methods to determine
The minimal inhibitory concentration (MIC) ofantimicrobial substances.Wiegand,
I.；Hilpert, K.；Hancock, R.E.W.).Positive antibacterials chlorobenzene Buddhist nun examines (Florfenicol) purchased from Haikou Shengjing city section
Skill Co., Ltd.
The fermentation of 1 bacterial strain Streptomyces spectabilis HDa1 and screening active ingredients
The sea urchin enteron aisle actinomyces Streptomyces spectabilis HDa1 fungus block that 5 pieces of diameters are 1cm is connect respectively
Kind is in liquid ME culture medium and the liquid ME culture medium of improvement, each 200mL, in 28 DEG C, 210rmin-1Shaken cultivation 13d,
Obtain fermentation liquid.With isometric ethyl acetate extractive fermentation liquid, repetition extraction 3 times, combined ethyl acetate extracting solution and 60
It is concentrated to dryness at DEG C, respectively obtains crude extract 1 (before induction) and 2 (after inductions).It is sieved using plate filter paper enzyme
Publishing existing crude extract 2 has growth inhibitory activity to grouper cause of disease vibrio alginolyticus strain.
The detection and analysis of 2 reactive compounds
(1) the fermentation liquid ethyl acetate extract 1 and 2 after being induced using the processing of ODS C-18 reversed-phase column and before induction, into
(mobile phase is methanol-water to row high-efficient liquid phase analysis, with linear gradient elution, specific steps are as follows: 0~35min, volume fraction are
0%~100% methanol；35~50min, the methanol that volume fraction is 100%；50~51min, volume fraction be 100%~
0% methanol；51~56min, the methanol that volume fraction is 0%；Ultraviolet detection wavelength is 254nm), the bacterial strain after detection induction
New secondary metabolite is generated, as a result, it has been found that newly there are two main components 1 and 2 in the crude extract 2 after induction (see Fig. 3).
(2) the sea urchin enteron aisle actinomyces Streptomyces spectabilis HDa1 fungus block that 5 pieces of diameters are 1cm is connect
Kind is in ME culture medium, 28 DEG C, 210rmin-1Shaken cultivation 3d is inoculated into the ME culture medium of 10L according to 5% inoculum concentration,
28 DEG C, 210rmin-1Shaken cultivation 13d, obtains fermentation liquid.With isometric ethyl acetate (10L) extractive fermentation liquid, repeat
It extracts 3 times, combined ethyl acetate extracting solution is simultaneously concentrated to dryness at 60 DEG C, and crude extract is obtained.To the crude extract
(B) silica gel column chromatography is carried out, with the chloroform-methanol ladder that volume ratio is 100:0,100:1,100:2,100:4,100:8,0:100
Degree elution, obtains B1, B2, B3, B4, B5 and B6 totally 6 sub- fractions.ODS column chromatography and half preparation HPLC column are carried out to fraction B 2
Chromatography obtains compound 1 and compound 2.Pass through high resolution mass spectrum, nuclear magnetic resonance (including one-dimensional, two dimensional NMR experiments) etc.
A variety of Wave Spectrum means have determined that the structure of the above compound is 3,4- dimethoxy -1- naphthalene amino acid (3,4-dimethoxy-1-
Naphthamide, compound 1) and p-O-3,3- dimethyl-allyl-benzamide (p-O- (3,3-dimethylallyl)-
Benzamide, compound 2).Their minimum inhibitory concentration, positive drug chlorobenzene Buddhist nun are tested using known dilution gradient method
The minimum inhibitory concentration examined is 5.0 μ g/mL, and the minimum inhibitory concentration of compound 1 and 2 is respectively 5.0 and 7.81 μ g/mL, with sun
Property medicine activity quite.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (6)
1. a kind of method that induction marine actinomycete cryptiogene expression generates novel substance, which is characterized in that the induction ocean
The method of actinomyces cryptiogene expression generation novel substance are as follows:
Step 1: with no Herba Artemisiae Scopariae seawater flushing Anthocidaris crassispina, after 75% alcohol disinfecting, with it is sterile washing 3 times after take its enteron aisle group
It knits and is placed in sterile water, dilute, draw 200 μ L, be coated on the height containing 50 μ g/mL potassium bichromates and 20 μ g/mL kanamycins
On family name's culture medium flat plate, 28 DEG C are cultivated 15 days, in picking single colonie streak inoculation to ISP2 culture medium flat plate, through 3 single colonies
Scribing line purifying obtains actinomycetes strain；
Step 2: being fermented 1L purified actinomyces on a small scale using liquid Gao Shi culture medium and malt extract culture medium, mistake
Filter fermentation liquid obtains bacterium solution and thallus, is extracted with ethyl acetate after bacterium solution is concentrated, and thallus is extracted with chloroform methanol, after concentration
The extract arrived is screened for anti-pathogenic activity；
Step 3: filtering out the ethyl acetate extract of 2 plants of bacterial strain fermentation liquors using plate filter paper enzyme, utilizes the above culture medium
And fermented on a small scale above-mentioned 2 plants of active bacterial strains by optimum culture condition and addition inducer, it is filtered to remove thallus, with acetic acid second
Ester extractive fermentation liquid obtains crude extract, and carries out screening active ingredients；
Step 4: the fermentation crude extract after inducing and before induction is handled using ODS C-18 reversed-phase column, carries out HPLC-MS-UV points
Analysis, in conjunction with its hydrogen spectrum analysis, the bacterial strain after detection induction generates new secondary metabolite.
2. the method that induction marine actinomycete cryptiogene expression generates novel substance as described in claim 1, which is characterized in that
The plate filter paper enzyme is that extract sample is made into the solution that concentration is 5mg/mL with dimethyl sulfoxide, adds sample solution 50
μ L is on the sterilizing filter paper disk of diameter 6mm, then is affixed on and has been applied on the culture medium flat plate for trying bacteria suspension, and 20min is placed
After place into incubator, 28 DEG C dark culture 7 days, survey its inhibition zone size.
3. the method that induction marine actinomycete cryptiogene expression generates novel substance as described in claim 1, which is characterized in that
The inducer is rare earth element.
4. the method that induction marine actinomycete cryptiogene expression generates novel substance as described in claim 1, which is characterized in that
The detection method of the novel substance are as follows:
Step 1: be utilized respectively screening and culturing medium and add inducer culture medium to 2 plants of actinomyces in the culture item optimized
Large scale fermentation is carried out under part, fermentation liquid and mycelium are extracted 3 times with ethyl acetate, chloroform methanol respectively, then living in antibacterial
Property screening guidance under, the active constituent of novel substance is separated from fermentation liquid and thallus crude extract using a variety of chromatographic separation technologies
Purification comes out；
Step 2: integrated use Modern spectroscopy technology determines the chemical structure of monomeric compound；
Step 3: activity rating further is carried out to the reactive compound that above structure determines using plate filter paper enzyme, is excluded
False positive may be desirably to obtain real antibacterial, disease-resistant new active substance with other interference.
5. the method that induction marine actinomycete cryptiogene expression generates novel substance as claimed in claim 4, which is characterized in that
The chromatographic separation technology includes: normal pressure positive, reversed-phase silica gel column chromatography, gel chromatography, half preparation HPLC.
6. the method that induction marine actinomycete cryptiogene expression generates novel substance as claimed in claim 4, which is characterized in that
The Modern spectroscopy technology include: one-dimensional, two dimensional NMR technology, high resolution mass spectrum technology, X-ray single crystal diffraction technology with
And ultraviolet, infrared, optically-active, circular dichroism spectra.
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