CN105087390A - High-yield itaconic acid strains and high-throughput screening method thereof based on deep-hole plate - Google Patents

High-yield itaconic acid strains and high-throughput screening method thereof based on deep-hole plate Download PDF

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CN105087390A
CN105087390A CN201510475563.0A CN201510475563A CN105087390A CN 105087390 A CN105087390 A CN 105087390A CN 201510475563 A CN201510475563 A CN 201510475563A CN 105087390 A CN105087390 A CN 105087390A
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succinic acid
methylene
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杨静
蒋剑春
卫民
张宁
赵剑
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Institute of Chemical Industry of Forest Products of CAF
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Institute of Chemical Industry of Forest Products of CAF
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Abstract

The invention discloses high-yield itaconic acid strains and a high-throughput screening method thereof based on a deep-hole plate. The method includes: the protoplast mutagenesis of the strains, selective flat plate primary screening, high-throughput screening based on the deep-hole cell culture plate, secondary shake flask screening and genetic stability observation. The method has the advantages that traditional screening methods are improved, work intensity is lowered effectively, and strain screening efficiency is increased.

Description

A kind of high-throughput screening method of the methylene-succinic acid superior strain based on deep-well plates and bacterial strain
Technical field
The present invention relates to microorganism strains filtration technical field, especially a kind of method of the high flux screening methylene-succinic acid superior strain based on Protoplast Mutation, Deep-hole cell culture plate.
Background technology
Methylene-succinic acid formal name used at school is methene succsinic acid, methylene-succinic acid, is the fifth-largest organic acid in the world.Because molecular memory closes double bond and two active carboxyls insatiable hunger, make methylene-succinic acid to carry out addition, polyreaction, form polymerization macromolecule, wherein the particularly important is the esterification of methylene-succinic acid.The esterification of standard can produce methylene-succinic acid diester, and productive rate is very high.Itaconic ester class is the important source material of synthetic resins, plastics, softening agent; In addition, methylene-succinic acid and amine react the N-alkyl pyrrolidone generated is the important compound of a class.Methylene-succinic acid and ester class thereof produce the industrial raw material such as chemical fibre, synthetic resins, plastics, rubber, medicine, tensio-active agent, foodstuffs without toxicity wrapping material, weedicide, scale remover, tackiness agent such as acrylic fibers, is widely used in chemical industry.
The production method of methylene-succinic acid has chemical synthesis, citric acid decomposition method and microbe fermentation method.Wherein microbe fermentation method is because raw material sources are extensive, cost is low, working condition is gentle, is the main method of producing methylene-succinic acid at present both at home and abroad.In the production process of methylene-succinic acid, how to obtain superior strain quickly and efficiently most important.At present, itaconic acid producing strain mainly contains the terreus (Asp.terreus) belonging to terreus group and the methylene-succinic acid aspergillus (Asp.itaconicus) belonging to Aspergillus amstelodami group.Applying maximum in actual production is terreus, but the general output of Asp.terreus wild strain is all very low, therefore uses the method for selection by mutation day by day to increase to the research obtaining superior strain.And bacterial screening workload is huge and efficiency is very low after mutagenesis, be restrict the important factor that industrial microorganism produces fermentation at present.High Throughput Screening Assay, due to possess trace, efficient, sensitive, accurate, the feature such as can to repeat, become the technical way of microbe to screen.Yet there are no the relevant report of the high-throughput screening method being applicable to screening methylene-succinic acid high productive mutant.
This research is intended to the high-throughput screening method setting up a kind of efficient rapid screening methylene-succinic acid superior strain.By selection by mutation, in conjunction with high throughput method screening methylene-succinic acid superior strain, both can increase work efficiency, reduce costs, and screening speed can be increased substantially again.
Summary of the invention
The object of this invention is to provide a kind of high-throughput screening method and bacterial strain of the methylene-succinic acid superior strain based on deep-well plates.
Technical scheme of the present invention is: a kind of methylene-succinic acid superior strain, for terreus (Aspergillusterreus), depositary institution is: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number is CGMCCNo.11039, and preservation date is on July 2nd, 2015.
The high-throughput screening method based on deep-well plates of methylene-succinic acid superior strain according to claim 1, comprises the Protoplast Mutation of bacterial strain; The dull and stereotyped primary dcreening operation of selectivity; Based on the high flux screening of Deep-hole cell culture plate; Shaking flask is sieved again and genetic stability is investigated, concrete steps are: the first step, the protoplasma mutagenesis of bacterial strain: after strain culturing, filtration and infiltration pressure stablizer rinses the mycelium obtained, collection mycelium moves in aseptic triangular flask and adds enzyme liquid enzymolysis, filter and remove mycelia fragment, collected by centrifugation protoplastis, is resuspended in homeo-osmosis agent after homeo-osmosis agent cleaning; Then mutagenesis is carried out to the protoplastis obtained and obtain protoplasma body fluid; Described Protoplast Mutation employing method is any one in ultraviolet mutagenesis, DES mutagenesis or ultrasonic wave mutagenesis;
Second step, the dull and stereotyped primary dcreening operation of selectivity: utilize homeo-osmosis agent to prepare regeneration culture medium, upper panel liquid is put into 40 ~ 45 DEG C of water-baths in advance and is incubated, protoplasma body fluid is added the mixing of upper panel liquid, rapid dumps is on the lower floor's regenerated plate solidified, be inverted, incubator temperature 35 ~ 37 DEG C, cultivate 3 ~ 5d; Dull and stereotyped screening factor is: in tetrabromo-mcresolsulfonphthalein, high sugar, meta-bolites methylene-succinic acid or metabolic precursor thereof succsinic acid any one;
3rd step, high flux screening based on Deep-hole cell culture plate: picking regenerated plate list bacterium colony is chosen in the deep-well plates that 1.2 ~ 2.0mL fermention medium is housed, under 150 ~ 250r/min, shaking culture 2 ~ 4d, centrifugal, get supernatant liquor and survey methylene-succinic acid content, be connected in the deep-well plates that solid slant culture base is housed correspondingly simultaneously, in 25 ~ 28 DEG C of constant temperature culture 3 ~ 5d, use disc seal film phonograph seal, 4 DEG C of Refrigerator stores; Select bacterial strain according to measurement result to carry out shaking flask and sieve again;
4th step, shaking flask is sieved again and genetic stability is investigated: will carry out bacterial strain that shaking flask sieves again colony inoculation corresponding from slant medium to containing in the shaking flask of seed culture medium by picking out, shaking table is cultivated and obtained kind of a liquid, carries out shaking flask and sieves again; The condition that shaking flask is sieved again is: inoculum size 5% ~ 10%, 35 ~ 37 DEG C, and 3 ~ 5d cultivated by 150 ~ 250r/min shaking table; Continuous passage cultivated for 5 generations, investigated genetic stability, and screening obtains the most stable bacterial strain.
Enzyme liquid described in the first step is: cellulase 4 ~ 8mg/ml, helicase 2 ~ 4mg/ml, the mixed enzyme of N,O-Diacetylmuramidase 0.5 ~ 1.5mg/ml.
Described homeo-osmosis agent is the NaCl aqueous solution of 0.4 ~ 0.65mol/L.
Fermentative medium formula described in 3rd step is: glucose 60 ~ 100g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L, regulate pH3.0-3.5; Solid slant culture based formulas is PDA substratum.
Described ultraviolet mutagenesis method is: draw monospore suspension 5 ~ 8mL, join in the culture dish of the diameter 9cm be put on magnetic stirring apparatus, with the ultra violet lamp 50 ~ 200s of 15 ~ 20W under the distance of 15 ~ 20cm.Irradiate while stirring, make spore absorb ultraviolet light wave uniformly.
Described DES mutagenesis is: with the phosphoric acid buffer of 0.1 ~ 0.2mol/L, pH value 5 ~ 8, configure the DES solution that volume fraction is 0.5 ~ 2.5% respectively, respectively get above DES solution and bacteria suspension equivalent and join mixing in sterile test tube, shaken at room temperature process 5 ~ 10min, adds the NaS of 0.5 ~ 1mL, 1 ~ 2%wt after mutagenic treatment terminates 2o 3termination reaction.
Described ultrasonic wave mutagenesis is: pipette 2 ~ 4mL bacteria suspension with liquid-transfering gun, is placed in the sterile centrifugation tube of bacterium of having gone out, and uses supersonic cell disintegrating machine, adopts the power of 150 ~ 200W, acts on 5 ~ 60min respectively.
In second step middle plateform screening factor, described height sugar regeneration culture medium formula is: glucose 160 ~ 250g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L; Tetrabromo-mcresolsulfonphthalein regeneration culture medium formula is: glucose 40 ~ 60g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L, the tetrabromo-mcresolsulfonphthalein solution 30-50ml/L of 0.2%; Meta-bolites methylene-succinic acid regeneration culture medium formula is: glucose 40 ~ 60g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L, methylene-succinic acid 10 ~ 50g/L; Metabolic precursor thereof succsinic acid regeneration culture medium formula is: glucose 40 ~ 60g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L, succsinic acid 10 ~ 50g/L.
In 3rd step, selected deep-well plates is 24 holes or 48 hole depth orifice plates; Fermention medium liquid amount is 1.3 ~ 4mL, and solid slant culture base liquid amount is 1.8 ~ 4.5mL.
Beneficial effect:
The present invention is according to the own metabolism characteristic of methylene-succinic acid producing strains, and design screening is dull and stereotyped, utilizes deep-well plates primary dcreening operation, the pattern that shaking flask is sieved again, substantially increases screening operation efficiency.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
In following examples, the content of methylene-succinic acid all adopts high performance liquid chromatography (HPLC) to measure.Chromatographic condition is as follows: chromatographic instrument: Agillent1100 high performance liquid chromatograph; Chromatographic column: Bio-RadAminexHPX-87H; Moving phase: 0.005mol/L sulfuric acid, flow velocity: 0.6mL/min; Column temperature: 55 DEG C; Tester: differential refraction tester; Sample size: 10 μ L.External standard method.
The high-throughput screening method of methylene-succinic acid superior strain of the present invention, concrete steps are:
Prepared by protoplastis
The terreus kind liquid of 8-12h (35-37 DEG C, 150-250r/min) will be cultivated, filter mycelium with four layers of aseptic lens wiping paper, rinse several times with homeo-osmosis agent, thieving paper suck dry moisture, and collect mycelium and move in aseptic triangular flask.Add enzyme liquid (cellulase 0.4 ~ 0.8%, helicase 0.2 ~ 0.4%, the mixed enzyme of N,O-Diacetylmuramidase 0.05 ~ 0.15z% (w/v)) by 8-10mL/g wet mycelium, slowly jolt to disperse mycelium ball, at 33-35 DEG C of enzymolysis 2-4h.Filter through 4 ~ 6 layers of sterile microscope lens wiping paper, remove mycelia segment, the centrifugal 8 ~ 10min of 300 ~ 700g, collect protoplastis, wash 2 ~ 3 times with homeo-osmosis agent, be resuspended in homeo-osmosis agent.
Protoplast Mutation
Protoplast Mutation method therefor is ultraviolet mutagenesis, DES mutagenesis or ultrasonic wave mutagenesis, adopts double-layer agar technique to carry out protoplast regeneration after mutagenesis.
As ultraviolet mutagenesis method is: draw monospore suspension 5 ~ 8mL, join in the culture dish of the diameter 9cm be put on magnetic stirring apparatus, with the ultra violet lamp 50 ~ 200s of 15 ~ 20W under the distance of 15 ~ 20cm.Irradiate while stirring, make spore absorb ultraviolet light wave uniformly.
As DES mutafacient system is: configure with the phosphoric acid buffer (pH value 5 ~ 8) of 0.1 ~ 0.2mol/L the DES solution that volume fraction is 0.5 ~ 2.5% respectively.Respectively get above DES solution and bacteria suspension equivalent and join mixing in sterile test tube, shaken at room temperature process 5 ~ 10min, add after mutagenic treatment terminates 0.5 ~ 1mL, 1 ~ 2% NaS 2o 3termination reaction.
As ultrasonic wave mutagenesis is: pipette 2 ~ 4mL bacteria suspension with liquid-transfering gun, be placed in the sterile centrifugation tube of bacterium of having gone out, use supersonic cell disintegrating machine, adopt the power of 150 ~ 200W, act on 5 ~ 60min respectively.
The dull and stereotyped primary dcreening operation of protoplastis selectivity
Utilize 0.4 ~ 0.65mol/LNaCl as homeo-osmosis agent preparation regeneration culture medium, upper panel liquid is put into 40 ~ 45 DEG C of water-baths in advance and is incubated, and protoplasma body fluid is added the mixing of upper panel liquid, rapid dumps is on the lower floor's regenerated plate solidified.Dull and stereotyped screening factor is: tetrabromo-mcresolsulfonphthalein, or high sugar, or meta-bolites methylene-succinic acid, or metabolic precursor thereof succsinic acid.
Regeneration culture medium formula as sugared in height is: glucose 160 ~ 250g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L.
As tetrabromo-mcresolsulfonphthalein regeneration culture medium formula is: glucose 40 ~ 60g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L, the tetrabromo-mcresolsulfonphthalein solution 30-50ml/L of 0.2%.
As meta-bolites methylene-succinic acid regeneration culture medium formula is: glucose 40 ~ 60g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L, methylene-succinic acid 10 ~ 50g/L.
As metabolic precursor thereof succsinic acid regeneration culture medium formula is: glucose 40 ~ 60g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L, succsinic acid 10 ~ 50g/L.
Picking regenerated plate list bacterium colony is chosen in the deep-well plates that 1.2 ~ 2.0mL fermention medium is housed, under 150 ~ 250r/min, and shaking culture 2 ~ 4d, centrifugal, get supernatant liquor and survey methylene-succinic acid content.Be connected in the deep-well plates that solid slant culture base is housed correspondingly simultaneously, in 25 ~ 28 DEG C of constant temperature culture 3 ~ 5d, use disc seal film phonograph seal, 4 DEG C of Refrigerator stores.
The formula of fermention medium is: glucose 60 ~ 100g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L, adjust ph to 3.0 ~ 3.5.Slant culture based formulas is PDA substratum.
Shaking flask is sieved again
According to measurement result by the corresponding bacterial strain in 48 hole slant mediums, preparation kind of liquid, carries out shaking flask and sieves again.Condition is: inoculum size 5% ~ 10%, 35 ~ 37 DEG C, and 3 ~ 5d cultivated by 150 ~ 250r/min shaking table.Continuous passage cultivated for 5 generations, investigated genetic stability, and screening obtains the most stable bacterial strain.
Prepared by embodiment 1 protoplastis
By bacterial strain aspergillus terreus (numbering 2433, purchased from Chinese industrial Microbiological Culture Collection administrative center (CICC)) cultivate 12h (37 DEG C, 200r/min), mycelium is filtered with four layers of aseptic lens wiping paper, several times are rinsed with homeo-osmosis agent, thieving paper suck dry moisture, collects mycelium and moves in aseptic triangular flask.Add enzyme liquid (cellulase 0.6%, helicase 0.3%, the mixed enzyme of N,O-Diacetylmuramidase 0.1% (w/v)) by 10mL/g wet mycelium, slowly jolt to disperse mycelium ball, at 35 DEG C of enzymolysis 3.5h.Filter through 6 layers of sterile microscope lens wiping paper, remove mycelia segment, the centrifugal 10min of 500g, collect protoplastis, wash 2 ~ 3 times with homeo-osmosis agent, be resuspended in homeo-osmosis agent.
Protoplast Mutation
Draw monospore suspension 5mL, join in the culture dish of the diameter 9cm be put on magnetic stirring apparatus, with the ultra violet lamp 150s of 18W under the distance of 15cm.Irradiate while stirring, make spore absorb ultraviolet light wave uniformly.
The dull and stereotyped regeneration of screening and primary dcreening operation
Spore suspension is suitably diluted rear painting tetrabromo-mcresolsulfonphthalein regenerated plate.Choosing the large bacterium colony of transparent circle chooses to containing in 48 hole depth orifice plates of 1.4mL fermention medium, under 200r/min, and shaking culture 3d, centrifugal, get supernatant liquor and survey methylene-succinic acid content.Be connected in the deep-well plates that 1.8mL slant medium is housed correspondingly simultaneously, in 28 DEG C of constant temperature culture 3d, use disc seal film phonograph seal, 4 DEG C of Refrigerator stores.
Tetrabromo-mcresolsulfonphthalein regeneration culture medium formula is: glucose 45g/L, (NH 4) 2sO 45g/L, MgSO 4.7H 2o1g/L, KH 2pO 41g/L, FeSO 47H 2o0.04g/L, ZnSO 47H 2o0.05g/L, corn steep liquor 1g/L, the tetrabromo-mcresolsulfonphthalein solution 40ml/L of 0.2%.
Fermentative medium formula is: glucose 80g/L, (NH 4) 2sO 45g/L, MgSO 4.7H 2o1g/L, KH 2pO 41g/L, FeSO 47H 2o0.04g/L, ZnSO 47H 2o0.05g/L, corn steep liquor 3g/L, regulate pH to 3.5.
Through deep-well plates primary dcreening operation, obtain 20 strain methylene-succinic acid output comparatively control strain (18.07g/L) improve the bacterial strain of more than 40%.The results are shown in Table 1.Wherein there is the increasing amount of 5 strains at more than 130% (AtYSZ-03, AtYSZ-21, AtYSZ-33, AtYSZ-38, AtYSZ-73), shaking flask is carried out to it and sieves again, investigate genetic stability.
Shaking flask is sieved and genetic stability again
Bacterium colony corresponding in picking slant medium, be seeded in the 100mL shaking flask containing 30mL seed culture medium, 37 DEG C, 12h cultivated by the shaking table of 200r/min, the inoculum size of 5% is seeded in the 250mL shaking flask containing 100mL fermention medium subsequently, 37 DEG C, and 5d cultivated by the shaking table of 200r/min, continuous passage cultivated for 5 generations, investigated genetic stability.Wherein AtYSZ-38 and AtYSZ-73 maintains high yield level substantially, has good genetic stability, and after continuous passage 5 generation, itaconic acid fermentation ability improves 154.5% and 132.7% respectively.Final Integrated Selection obtain performance best carry out preservation for AtYSZ-38 bacterial classification, preserving number is CGMCC:11039.
The morphological observation of bacterial strain:
Bacterium colony quality felted; Intimate surface, has radial rill; Edge sawtooth shape; Bacterium colony salmon is yellowish pink; Reverse side has xanthein.The most substrata of conidiophore; Conidium is radial, pencil; Top capsule is subsphaeroidal, and surface 1/2 can be educated; Conidial fructification is double-deck; Conidium is subsphaeroidal, and wall is smooth.

Claims (10)

1. a methylene-succinic acid superior strain, it is characterized in that, for terreus (Aspergillusterreus), depositary institution is: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preserving number is CGMCCNo.11039, and preservation date is on July 2nd, 2015.
2. the high-throughput screening method based on deep-well plates of methylene-succinic acid superior strain according to claim 1, is characterized in that: the Protoplast Mutation comprising bacterial strain; The dull and stereotyped primary dcreening operation of selectivity; Based on the high flux screening of Deep-hole cell culture plate; Shaking flask is sieved again and genetic stability is investigated, concrete steps are: the first step, the protoplasma mutagenesis of bacterial strain: after strain culturing, filtration and infiltration pressure stablizer rinses the mycelium obtained, collection mycelium moves in aseptic triangular flask and adds enzyme liquid enzymolysis, filter and remove mycelia fragment, collected by centrifugation protoplastis, is resuspended in homeo-osmosis agent after homeo-osmosis agent cleaning; Then mutagenesis is carried out to the protoplastis obtained and obtain protoplasma body fluid; Described Protoplast Mutation employing method is any one in ultraviolet mutagenesis, DES mutagenesis or ultrasonic wave mutagenesis;
Second step, the dull and stereotyped primary dcreening operation of selectivity: utilize homeo-osmosis agent to prepare regeneration culture medium, upper panel liquid is put into 40 ~ 45 DEG C of water-baths in advance and is incubated, protoplasma body fluid is added the mixing of upper panel liquid, rapid dumps is on the lower floor's regenerated plate solidified, be inverted, incubator temperature 35 ~ 37 DEG C, cultivate 3 ~ 5d; Dull and stereotyped screening factor is: in tetrabromo-mcresolsulfonphthalein, high sugar, meta-bolites methylene-succinic acid or metabolic precursor thereof succsinic acid any one;
3rd step, high flux screening based on Deep-hole cell culture plate: picking regenerated plate list bacterium colony is chosen in the deep-well plates that 1.2 ~ 2.0mL fermention medium is housed, under 150 ~ 250r/min, shaking culture 2 ~ 4d, centrifugal, get supernatant liquor and survey methylene-succinic acid content, be connected in the deep-well plates that solid slant culture base is housed correspondingly simultaneously, in 25 ~ 28 DEG C of constant temperature culture 3 ~ 5d, use disc seal film phonograph seal, 4 DEG C of Refrigerator stores; Select bacterial strain according to measurement result to carry out shaking flask and sieve again;
4th step, shaking flask is sieved again and genetic stability is investigated: will carry out bacterial strain that shaking flask sieves again colony inoculation corresponding from slant medium to containing in the shaking flask of seed culture medium by picking out, shaking table is cultivated and obtained kind of a liquid, carries out shaking flask and sieves again; The condition that shaking flask is sieved again is: inoculum size 5% ~ 10%, 35 ~ 37 DEG C, and 3 ~ 5d cultivated by 150 ~ 250r/min shaking table; Continuous passage cultivated for 5 generations, investigated genetic stability, and screening obtains the most stable bacterial strain.
3. the high-throughput screening method based on deep-well plates of described methylene-succinic acid superior strain according to claim 2, it is characterized in that, enzyme liquid described in the first step is: cellulase 4 ~ 8mg/ml, helicase 2 ~ 4mg/ml, the mixed enzyme of N,O-Diacetylmuramidase 0.5 ~ 1.5mg/ml.
4. the high-throughput screening method based on deep-well plates of described methylene-succinic acid superior strain according to claim 2, it is characterized in that, described homeo-osmosis agent is the NaCl aqueous solution of 0.4 ~ 0.65mol/L.
5. the high-throughput screening method based on deep-well plates of described methylene-succinic acid superior strain according to claim 2, it is characterized in that, the fermentative medium formula described in the 3rd step is: glucose 60 ~ 100g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L, regulate pH3.0-3.5; Solid slant culture based formulas is PDA substratum.
6. the high-throughput screening method based on deep-well plates of described methylene-succinic acid superior strain according to claim 2, it is characterized in that, described ultraviolet mutagenesis method is: draw monospore suspension 5 ~ 8mL, join in the culture dish of the diameter 9cm be put on magnetic stirring apparatus, with the ultra violet lamp 50 ~ 200s of 15 ~ 20W under the distance of 15 ~ 20cm.Irradiate while stirring, make spore absorb ultraviolet light wave uniformly.
7. the high-throughput screening method based on deep-well plates of described methylene-succinic acid superior strain according to claim 2, it is characterized in that, described DES mutagenesis is: with the phosphoric acid buffer of 0.1 ~ 0.2mol/L, pH value 5 ~ 8, configure the DES solution that volume fraction is 0.5 ~ 2.5% respectively, respectively get above DES solution and bacteria suspension equivalent and join mixing in sterile test tube, shaken at room temperature process 5 ~ 10min, adds the NaS of 0.5 ~ 1mL, 1 ~ 2%wt after mutagenic treatment terminates 2o 3termination reaction.
8. the high-throughput screening method based on deep-well plates of described methylene-succinic acid superior strain according to claim 2, it is characterized in that, described ultrasonic wave mutagenesis is: pipette 2 ~ 4mL bacteria suspension with liquid-transfering gun, be placed in the sterile centrifugation tube of bacterium of having gone out, use supersonic cell disintegrating machine, adopt the power of 150 ~ 200W, act on 5 ~ 60min respectively.
9. the high-throughput screening method based on deep-well plates of described methylene-succinic acid superior strain according to claim 2, is characterized in that, in second step middle plateform screening factor, described height sugar regeneration culture medium formula is: glucose 160 ~ 250g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L; Tetrabromo-mcresolsulfonphthalein regeneration culture medium formula is: glucose 40 ~ 60g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L, the tetrabromo-mcresolsulfonphthalein solution 30-50ml/L of 0.2%; Meta-bolites methylene-succinic acid regeneration culture medium formula is: glucose 40 ~ 60g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L, methylene-succinic acid 10 ~ 50g/L; Metabolic precursor thereof succsinic acid regeneration culture medium formula is: glucose 40 ~ 60g/L, (NH 4) 2sO 41 ~ 5g/L, MgSO 4.7H 2o1 ~ 3g/L, KH 2pO 41 ~ 3g/L, FeSO 47H 2o0.02 ~ 0.05g/L, ZnSO 47H 2o0.01 ~ 0.1g/L, corn steep liquor 1 ~ 3g/L, succsinic acid 10 ~ 50g/L.
10. the high-throughput screening method based on deep-well plates of described methylene-succinic acid superior strain according to claim 2, is characterized in that, in the 3rd step, selected deep-well plates is 24 holes or 48 hole depth orifice plates; Fermention medium liquid amount is 1.3 ~ 4mL, and solid slant culture base liquid amount is 1.8 ~ 4.5mL.
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CN106086091A (en) * 2016-06-17 2016-11-09 青岛琅琊台集团股份有限公司 A kind of itaconic acid concentrates the condensed water method for itaconic acid fermentation
CN106282047A (en) * 2016-04-05 2017-01-04 中国科学院上海高等研究院 There is the screening technique of the Azotica of bio-fertilizer application potential
CN108384815A (en) * 2018-05-22 2018-08-10 中国林业科学研究院林产化学工业研究所 A method of preparing itaconic acid using lignocellulosic material
CN108624503A (en) * 2018-03-20 2018-10-09 华东师范大学 A kind of efficient high-throughput screening method of alpha-glucosidase superior strain

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OKABE M等: "Biotechnological production of itaconic acid and its biosynthesis in Aspergillus terresu", 《APPLIED MICROBIOLOGY AND BIOTECHNOLOGY》 *
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106282047A (en) * 2016-04-05 2017-01-04 中国科学院上海高等研究院 There is the screening technique of the Azotica of bio-fertilizer application potential
CN106086091A (en) * 2016-06-17 2016-11-09 青岛琅琊台集团股份有限公司 A kind of itaconic acid concentrates the condensed water method for itaconic acid fermentation
CN108624503A (en) * 2018-03-20 2018-10-09 华东师范大学 A kind of efficient high-throughput screening method of alpha-glucosidase superior strain
CN108384815A (en) * 2018-05-22 2018-08-10 中国林业科学研究院林产化学工业研究所 A method of preparing itaconic acid using lignocellulosic material
CN108384815B (en) * 2018-05-22 2022-03-04 中国林业科学研究院林产化学工业研究所 Method for preparing itaconic acid by using lignocellulose raw material

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Application publication date: 20151125