CN104726381B - One plant of bacterial strain for producing L lysines and its method for producing L lysines - Google Patents
One plant of bacterial strain for producing L lysines and its method for producing L lysines Download PDFInfo
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Abstract
The present invention relates to the bacterial strain of one plant of production L lysine, its Classification And Nomenclature is ETEC ZY0217（Escherichia coliZY0217）, it is preserved in China typical culture collection center（CCTCC）, deposit number：CCTCC NO：M 2015079.The present invention also provides the mutagenesis screening method of the bacterial strain and the method using bacterial strain production L lysines.ETEC（Escherichia coli）L lysine contents are 70 g/L or so in 7 after fermentation liquid of ZY0217 bacterial strains continuous passage, no significant changes, and genetic stability is good.
Description
Technical field
The present invention relates to biological technical field, and in particular to the bacterial strain and its production 1B of one plant of production 1B
Method.
Background technology
1B (L-Lysine, Lys) is that organism sustains life one of the essential amino acid of activity, is in the world only
Inferior to the second largest amino acid kind of monosodium glutamate, and the important as precursors of the new material such as bio-based nylon.The production of 1B
Method includes extraction process, chemical synthesis/enzyme process and microbe fermentation method.Microbe fermentation method is current industrial production lysine
Most important method, fermentation raw material is easy to get extensively and cheap, such as starch (tapioca, cornstarch), molasses (cane suger
Honey, beet molasses etc.).
Microbe fermentation method is by metabolic control fermentation, artificially controls or changes the metabolic pathway of microorganism to realize
The production of 1B.Ion beam bioengineering technology has been widely used in biology as a kind of brand-new mutation breeding technologies
In terms of mutation breeding.When Low energy nitrogen ions act on microorganism, the structure and permeability that can make microorganism wall/film change
Become, and cause gene damage, and then make microbial gene sequences and its metabolism network significant changes, ultimately result in microorganism generation
Mutation.This induced-mutation technique has been respectively applied to the production strain breeding thereof such as glutamic acid, Pfansteihl so that glutamic acid, Pfansteihl difference
Improve 35%, 46%.
The content of the invention
The first object of the present invention there is provided it is a kind of can simply, efficiently, safety in production 1B bacterial strain.
Second purpose of the invention is to provide a kind of acquisition methods of above-mentioned bacterial strains.
3rd purpose of the invention is to provide a kind of method that 1B is produced using above-mentioned bacterial strains.
In order to realize the first object of the present invention, the technical solution adopted by the present invention is as follows：
A kind of bacterial strain for producing 1B, its Classification And Nomenclature are ETEC ZY0217 (Escherichia
Coli ZY0217), it is preserved in China typical culture collection center CCTCC, deposit number：CCTCC NO：M 2015079.
The morphology and physiological and biochemical property of bacterial strain of the present invention are as follows：
Colony colour：Canescence.
Aerobic mode：Aerobic growth.
Bacterium colony size：0.2~0.9 × 1~5 μm.
Suitable growth temperature：34-40℃.
Suitable growth pH：6.5-7.2.
Thalli morphology：Circular colonies, there is metallic luster.
Gram's staining：It is negative.
Above-mentioned ETEC ZY0217 acquisition methods, be using E.coli A301 as starting strain, by it is ultraviolet-
Lithium chloride complex mutation, low energy ion beam implantation mutagenesis, just, secondary screening and obtain.
Specifically, the acquisition methods of the ETEC ZY0217 comprise the following steps：
1) prepared by bacteria suspension
E.coli A301 and addition lithium chloride sterilized water are mixed and made into bacteria suspension, bacterial concentration 106Individual/mL；
2) UV- LiCl complex mutation
The bacteria suspension of step 1) is transferred in the sterile empty flat board containing magnetic rotor, under stir speed (S.S.) 50-100rpm,
Carry out ultraviolet irradiation mutagenesis, after take bacteria suspension be coated on addition lithium chloride LB plating mediums in, lucifuge is fallen at 34-40 DEG C
Put culture 1-2d；
3) low energy ion beam implantation mutagenesis
Bacterial strain after being cultivated in step 2) is washed, and bacteria suspension is made and is coated on sterile empty plate, after drying, is carried out
N~+ implantation, elute, be applied on LB plating mediums afterwards, lucifuge is inverted culture 1-2d at 34-40 DEG C；
4) primary dcreening operation
The single bacterium colony that the culture of picking step 3) obtains, is seeded on slant medium and activates, and culture is inverted at 34-40 DEG C
After 1-2d, stir culture 7-10h in seed culture medium is moved to；
Some cultured bacterium colonies are inoculated into 1L fermentation tanks respectively by certain inoculum concentration, while are passed through air, are stirred
Ferment 24-62h, detects 1B content in zymotic fluid, just sifts out the bacterium colony that 1B content is more than 65g/L；
In fermentation process, zymotic fluid pH is controlled using ammoniacal liquor, 2mol/L hydrochloric acid；
5) secondary screening
The single bacterium colony that step 4) primary dcreening operation obtains is seeded on slant medium and activated, culture 1- is inverted at 34-40 DEG C
After 2d, stir culture 7-10h in seed culture medium is moved to；
Some cultured bacterium colonies are inoculated into 5L fermentation tanks respectively by certain inoculum concentration, while are passed through air, are stirred
Ferment 48-120h；1B content in zymotic fluid is detected, filters out the high bacterium colony of 1B yield；
Repeat step 1) to step 5), finally filter out the target that 1B content in zymotic fluid is 70g/L and the above
Strain Escherichia coli ZY0217.
In above-mentioned Escherichia coli ZY0217 acquisition methods, the addition of lithium chloride is sterile water quality in the step 1)
2-4%, preferably 2.5%；
In the step 2) addition of lithium chloride be LB plating medium quality 2-4%, preferably 2.5%；The purple
External exposure condition：30W uviol lamps；Irradiation distance 25cm；Irradiation time 10-90s, preferably 30s；
Low energy N+ ions condition in the step 3)：2-4Kv/cm × 2-5min (is 2-4Kv/cm in electric-field intensity
Under conditions of, mutagenesis 2-5min), be preferably：4Kv/cm×3min；Implantation dosage is 15 × 1014-20×1014ions/cm2；It is excellent
Select 15 × 1014ions/cm2。
The step 4) and 5) in, mixing speed is 150-250rpm in seed culture；Inoculum concentration is 1-5% (v/v)；Hair
Mixing speed is 200-900rpm in ferment.
In the present invention, the LB culture mediums, following mass percent component is included：Tryptone 1%, yeast extract
0.5%th, sodium chloride 1%, remaining is water, pH 6.8-7.2.
The seed culture medium includes following mass percent component：It is carbon source 0.1%-0.2%, nitrogen source 3%-8%, inorganic
Salt 0.5%-1.3%, remaining is water, pH 6.8-7.2；Wherein, the carbon source is glucose and/or sucrose；The nitrogen source is egg
One or more of mixing in white peptone, yeast extract or ammonium sulfate；The inorganic salts are sylvite, magnesium salts, molysite, manganese salt, sodium
One or more of mixing in salt or calcium salt；Preferred seed culture medium, it is made up of following mass percent component：Glucose
0.05%th, sucrose 0.15%, peptone 3%, yeast extract 3%, ammonium sulfate 2%, dipotassium hydrogen phosphate 0.3%, magnesium sulfate
0.1%th, ferrous sulfate 0.05%, manganese sulfate 0.03%, sodium dihydrogen phosphate 0.25%, calcium carbonate 0.17%, remaining is water, pH
6.8-7.2。
Fermentation medium in the fermentation tank includes following mass percent component：
Carbon source 1.0%-2%, nitrogen source 0.5%-2%, inorganic salts 0.1%-1%, remaining is water, pH 6.8-7.2；Wherein,
The carbon source is glucose and/or sucrose；The nitrogen source is groundnut meal, soybean cake powder, corn steep liquor, yeast extract or sulfuric acid
One or more of mixing in ammonium；The inorganic salts are the one or more in sylvite, magnesium salts, molysite, manganese salt, sodium salt or calcium salt
Mixing.
It is preferred that fermentation medium, is made up of following mass percent component：Glucose 0.5%, sucrose 0.3%, corn steep liquor
0.4%th, groundnut meal 0.4%, soybean cake powder 0.4%, ammonium sulfate 0.2%, dipotassium hydrogen phosphate 0.08%, magnesium sulfate 0.02%,
Ferrous sulfate 0.04%, manganese sulfate 0.05%, sodium dihydrogen phosphate 0.15%, calcium carbonate 0.13%, remaining is water, pH 6.8-
7.2.Fermentation process is using ammoniacal liquor, 2mol/L hydrochloric acid control zymotic fluid pH.
A kind of method that 1B is produced using Escherichia coli ZY0217, is comprised the following steps：
1) inclined-plane culture
ZY0217 is inoculated in slant medium, 34-40 DEG C of cultivation temperature, incubation time 1-2d；
2) seed culture
The bacterial strain ZY0217 of step 1) inclined-plane culture is inoculated in seed culture medium, cultivation temperature is 34-40 DEG C, stirring
Cultivate 7-10h；
3) fermented and cultured
Bacterial strain ZY0217 after step 2) is cultivated is inoculated into fermentation medium, and cultivation temperature is 34-40 DEG C；Lead to simultaneously
Enter air, stirring fermentation 24-62h.
In the method using bacterial strain ZY0217 production 1Bs, the slant medium is LB culture mediums, is specifically matched somebody with somebody
Side is same as above.
In the step 2), mixing speed 150-250rpm；Seed culture based formulas is same as above.
In the step 3), mixing speed 200-900rpm；Inoculum concentration is 1-5% (v/v)；Fermentative medium formula is same
On.
As a preferred embodiment of Escherichia coli ZY0217 of the present invention production 1Bs, the slant medium
Formula：LB culture mediums, mass percent component are：Tryptone 1%, yeast extract 0.5%, sodium chloride 1%, remaining is
Water, pH 6.8-7.2；
The formula of the seed culture medium：Sucrose 0.2%, peptone 2%, ammonium sulfate 2%, yeast extract 1%, phosphoric acid
Hydrogen dipotassium 0.4%, magnesium sulfate 0.3%, ferrous sulfate 0.02%, manganese sulfate 0.07%, remaining is water, pH 6.8-7.2；
The formula of the fermentation medium：
Glucose 1.5%, corn steep liquor 0.5%, ammonium sulfate 0.5%, dipotassium hydrogen phosphate 0.05%, manganese sulfate 0.02%, sulphur
Sour ferrous iron 0.04%, magnesium sulfate 0.05%, remaining is water, pH 6.8-7.2.
Beneficial effect of the present invention：
For the present invention using Low energy N+ ions and UV- LiCl complex mutation are combined, final screening obtains one
Strain produces the Escherichia coli ZY0217 of 1B, and 1B content is 70g/L left in 7 after fermentation liquid of bacterial strain continuous passage
The right side, no significant changes, genetic stability are good；1B is produced using the bacterial strain, L- in tunning can be improved by a relatively large margin
Lysine content.
Brief description of the drawings
Fig. 1 is Escherichia coli ZY0217 mutagenesis screening flow chart.
Biomaterial of the present invention, its Classification And Nomenclature are ETEC ZY0217 (Escherichia
ColiZY0217), it is preserved in China typical culture collection center on March 3rd, 2015 (abbreviation CCTCC, address are：Chinese
Wuhan Wuhan Universitys), deposit number：CCTCC NO：M 2015079.
Embodiment
Embodiment is set forth below the present invention is further described, but and is not so limited present disclosure.
Embodiment 1：This example demonstrates that using E.coli A301 as starting strain mutagenesis screening Escherichia coli ZY0217's
Method.
In the present embodiment：
1) LB culture mediums, it is made up of following mass percent component：Tryptone 1%, yeast extract 0.5%, chlorination
Sodium 1%, remaining is water, pH 6.8-7.2.
2) seed culture medium, it is made up of following mass percent component：Glucose 0.05%, sucrose 0.15%, peptone
3%th, yeast extract 3%, ammonium sulfate 2%, dipotassium hydrogen phosphate 0.3%, magnesium sulfate 0.1%, ferrous sulfate 0.05%, manganese sulfate
0.03%th, sodium dihydrogen phosphate 0.25%, calcium carbonate 0.17%, remaining is water, pH 6.8-7.2.
3) fermentation medium, it is made up of following mass percent component：Glucose 1%, sucrose 1%, corn steep liquor 1%, flower
Raw cake powder 0.4%, soybean cake powder 0.4%, ammonium sulfate 0.2%, dipotassium hydrogen phosphate 0.08%, magnesium sulfate 0.02%, ferrous sulfate
0.04%th, manganese sulfate 0.05%, sodium dihydrogen phosphate 0.15%, calcium carbonate 0.13%, remaining is water, pH 6.8-7.2.Fermented
Cheng Caiyong ammoniacal liquor, 2mol/L hydrochloric acid control zymotic fluid pH.
With recombination bacillus coli E.coli A301, (Chen Kequan etc., nitrogen source is to restructuring Escherichia coli fermentation production L-arginine
Influence, biological processing, the 6th phase of volume 9 in November, 2011, P11-14) it is starting strain.The starting strain system applicant gathers around
Bacterial strain disclosed in the first open source literature having, by applicant's voluntarily preservation, applicant from present patent application day it is hereby stated that exempt from
Take to the public and send the starting strain.
As shown in figure 1, obtain comprising the following steps that for Escherichia coli ZY0217：
1) prepared by bacteria suspension
The 37 DEG C of incubated 1d fresh inclined-planes of E.coli A301 are taken, add sterilized water 15mL, lower bacterial strain is scraped and is transferred to band
Have in the 250mL triangular flasks of bead, the sterilized water containing 2% lithium chloride is added into triangular flask bacteria suspension is made, adjust bacterium
Concentration is 106Individual/mL；
2) UV- LiCl complex mutation
The bacteria suspension of 15mL steps 1) is taken, is transferred in the sterile empty flat board containing magnetic rotor, in 50rpm mixing speeds
Under, it is placed under 30W uviol lamps progress ultraviolet irradiation mutagenesis, irradiation time 10s, mutagenesis at 25cm and takes bacteria suspension to be coated with after terminating
In the LB plating mediums containing 2.5% lithium chloride, lucifuge is inverted culture 1d at 37 DEG C；
3) low energy ion beam implantation mutagenesis
With sterile water wash step 2) in cultivate after Escherichia coli 2 times, and bacteria suspension is made, takes 0.1mL bacteria suspensions equal
It is even to be coated on sterile empty plate, dried up with sterile wind, in 3Kv/cm × 2min, implantation dosage is 20 × 1014ions/cm2Bar
N~+ implantation is carried out to Escherichia coli under part, after ion implanting, plate is taken out, aseptically with 1mL sterilized waters
Elution, is applied on LB plating mediums, and lucifuge is inverted culture 1d at 37 DEG C；
4) primary dcreening operation
Some single bacterium colonies that step 3) mutagenesis is obtained, which are seeded on slant medium, to be activated, and culture 1d is inverted at 37 DEG C
Afterwards, the ring activated strains of picking one are distinguished into its seed culture medium with oese, mixing speed 150rpm, after cultivating 10h, then press
1% (v/v) inoculum concentration is linked into the fermentation medium in 1L fermentation tanks, while is passed through air, mixing speed 200rpm, hair
Ferment 60h.1B content in zymotic fluid is detected, primary dcreening operation goes out the single bacterium colony that 1B content in zymotic fluid is more than 65g/L.
5) secondary screening
The single bacterium colony of step 4) primary dcreening operation is seeded on slant medium and activated, after culture 1d is inverted at 37 DEG C, with connecing
The ring activated strains of kind ring picking one are into seed culture medium, mixing speed 200rpm, after cultivating 10h, by 5% (v/v) inoculation
Amount is linked into the fermentation medium in 5L fermentation tanks, while is passed through air, mixing speed 900rpm, and ferment 62h；Detection fermentation
1B content in liquid, secondary screening go out the single bacterium colony that 1B content is high in zymotic fluid.
The single bacterium colony that step 5) secondary screening obtains is configured to bacteria suspension, repeat step 1 again) to step 5), when SBA-40E gives birth to
When 1B content is 70g/L and the above in thing sensing instrument measure zymotic fluid, corresponding bacterial strain is aimed strain large intestine bar
Bacterium ZY0217.
Embodiment 2
Escherichia coli ZY0217 is obtained according to the mutagenesis screening method of embodiment 1.
Wherein, lithium chloride addition is the 2% of LB plating medium quality；Time of ultraviolet irradiation is 90s；Low energy nitrogen ions
Injection condition is 4Kv/cm × 5min, and implantation dosage is 15 × 1014ions/cm2 .。
Embodiment 3
Escherichia coli ZY0217 is obtained according to the mutagenesis screening method of embodiment 1.
Wherein, lithium chloride addition is the 2.5% of LB plating medium quality；Time of ultraviolet irradiation is 10s；Low Energy Nitrogen from
Sub- injection condition is 42Kv/cm × 4min, and implantation dosage is 20 × 1014ions/cm2 .。
The morphology and physiological and biochemical property for the Escherichia coli NT 1003 that above-described embodiment 1-3 is obtained are as follows：
Colony colour：Canescence.
Aerobic mode：Aerobic growth.
Bacterium colony size：0.2~0.9 × 1~5 μm
Suitable growth temperature：34-40℃.
Suitable growth pH：6.5-7.2.
Thalli morphology：Circular colonies, there is metallic luster.
Gram's staining：It is negative.
Embodiment 1-3 Escherichia coli ZY0217 genetic stability test
Using dextrose and saccharose in the fermentation medium of carbon source, to detect the mutant strain large intestine bar that embodiment 1-3 is obtained
Bacterium ZY0217 genetic stability, strain passage fermentation test result are as shown in table 1.1B content is by SBA- in zymotic fluid
40E bio-sensings instrument determines.
The Escherichia coli ZY0217 of table 1 genetic stability experiment
It is from experimental result as can be seen that steady by 7 passages, mutant strain Escherichia coli ZY0217 1B yield
It is fixed, there is preferable genetic stability.
Embodiment 4：This example demonstrates that the method for Escherichia coli ZY0217 production 1Bs
Culture medium prescription used in the present embodiment：
Slant medium：LB culture mediums, mass percent component are：Tryptone 1%, yeast extract 0.5%, chlorination
Sodium 1%, remaining is water, pH 6.8-7.2.
Seed culture medium：Glucose 0.1%, sucrose 0.1%, peptone 2%, ammonium sulfate 1%, dipotassium hydrogen phosphate 0.3%,
Magnesium sulfate 0.03%, ferrous sulfate 0.05%, manganese sulfate 0.02%, sodium dihydrogen phosphate 0.05%, calcium carbonate 0.05%, remaining is
Water, pH 6.8-7.2.
Fermentation medium：Glucose 1.5%, sucrose 0.5%, yeast extract 0.5%, ammonium sulfate 1%, potassium dihydrogen phosphate
0.05%th, manganese sulfate 0.02%, magnesium sulfate 0.02%, sodium chloride 0.01%, remaining is water, pH 6.8-7.2.
1) inclined-plane culture
The Escherichia coli ZY0217 that embodiment 1 obtains is inoculated in slant medium, 1d is activated under the conditions of 34 DEG C；
2) seed culture
The bacterial strain ZY0217 of step 2) inclined-plane culture is inoculated in seed culture medium, liquid amount is in 500mL triangular flasks
20mL, cultivation temperature are 37 DEG C, and 10h is cultivated under 250rpm shaking speeds；
3) fermented and cultured
The bacterial strain ZY0217 of step 2) seed culture is inoculated into fermentation medium, inoculum concentration is 5% (v/v), and 5L is sent out
Liquid amount is 2L in fermentation tank, and cultivation temperature is 34 DEG C, while is passed through air, to keep dissolved oxygen environment suitable in fermentation system,
Mixing speed controls zymotic fluid pH, fermentation time 62h in 900rpm, fermentation process using ammoniacal liquor, 2mol/L hydrochloric acid.Through SBA-
It is 73g/L that 40E type bio-sensing instrument, which measures 1B content in zymotic fluid,.
Embodiment 5：This example demonstrates that the method for Escherichia coli ZY0217 production 1Bs
Culture medium prescription used in the present embodiment：
Slant medium：LB culture mediums, mass percent component are：Tryptone 1%, yeast extract 0.5%, chlorination
Sodium 1%, remaining is water, pH 6.8-7.2.
The formula of the seed culture medium：Sucrose 0.2%, peptone 2%, ammonium sulfate 2%, yeast extract 1%, phosphoric acid
Hydrogen dipotassium 0.6%, magnesium sulfate 0.41%, ferrous sulfate 0.02%, manganese sulfate 0.06%, remaining is water, pH 6.8-7.2；
The formula of the fermentation medium：Glucose 1.5%, corn steep liquor 0.5%, ammonium sulfate 0.5%, dipotassium hydrogen phosphate
0.05%th, manganese sulfate 0.02%, ferrous sulfate 0.04%, magnesium sulfate 0.05%, remaining is water, pH 6.8-7.2.
1) inclined-plane culture
The Escherichia coli ZY0217 that embodiment 1 obtains is inoculated in slant medium, 1d is activated under the conditions of 40 DEG C；
2) seed culture
The bacterial strain ZY0217 of step 2) inclined-plane culture is inoculated in seed culture medium, liquid amount is in 500mL triangular flasks
20mL, cultivation temperature are 37 DEG C, and 10h is cultivated under 250rpm shaking speeds；
3) fermented and cultured
The bacterial strain ZY0217 of step 2) seed culture is inoculated into fermentation medium, inoculum concentration is 5% (v/v), and 5L is sent out
Liquid amount is 2L in fermentation tank, and cultivation temperature is 34 DEG C, while is passed through air, to keep dissolved oxygen environment suitable in fermentation system,
Mixing speed controls zymotic fluid pH, fermentation time 50h in 900rpm, fermentation process using ammoniacal liquor, 2mol/L hydrochloric acid.Through SBA-
It is 68g/L that 40E type bio-sensing instrument, which measures 1B content in zymotic fluid,.
Embodiment 6：This example demonstrates that the method for Escherichia coli ZY0217 production 1Bs
Slant medium：LB culture mediums, mass percent component are：Tryptone 1%, yeast extract 0.5%, chlorination
Sodium 1%, remaining is water, pH 6.8-7.2.
Seed culture medium：Glucose 0.1%, peptone 4%, ammonium sulfate 4%, dipotassium hydrogen phosphate 0.3%, magnesium sulfate
0.3%th, ferrous sulfate 0.2%, manganese sulfate 0.2%, sodium dihydrogen phosphate 0.1%, calcium carbonate 0.2%, remaining is water, pH 6.8-
7.2。
Fermentation medium：Glucose 0.5%, sucrose 0.5%, yeast extract 1%, ammonium sulfate 1%, potassium dihydrogen phosphate
0.5%th, manganese sulfate 0.2%, magnesium sulfate 0.2%, sodium chloride 0.1%, remaining is water, pH 6.8-7.2.
1) inclined-plane culture
The Escherichia coli ZY0217 that embodiment 1 obtains is inoculated in slant medium, 2d is activated under the conditions of 34 DEG C；
2) seed culture
The bacterial strain ZY0217 of step 2) inclined-plane culture is inoculated in seed culture medium, liquid amount is in 500mL triangular flasks
20mL, cultivation temperature are 34 DEG C, and 7h is cultivated under 150rpm shaking speeds；
3) fermented and cultured
The bacterial strain ZY0217 of step 2) seed culture is inoculated into fermentation medium, inoculum concentration is 5% (v/v), and 5L is sent out
Liquid amount is 2L in fermentation tank, and cultivation temperature is 37 DEG C, while is passed through air, to keep dissolved oxygen environment suitable in fermentation system,
Mixing speed controls zymotic fluid pH, fermentation time 62h in 200rpm, fermentation process using ammoniacal liquor, 2mol/L hydrochloric acid.Through SBA-
It is 73g/L that 40E type bio-sensing instrument, which measures 1B content in zymotic fluid,.
Embodiment 7：This example demonstrates that the method for Escherichia coli ZY0217 production 1Bs
Slant medium：LB culture mediums, mass percent component are：Tryptone 1%, yeast extract 0.5%, chlorination
Sodium 1%, remaining is water, pH 6.8-7.2.
Seed culture medium：Glucose 0.1%, sucrose 0.1%, peptone 2%, ammonium sulfate 1%, dipotassium hydrogen phosphate 0.2%,
Magnesium sulfate 0.03%, ferrous sulfate 0.15%, manganese sulfate 0.02%, sodium dihydrogen phosphate 0.05%, calcium carbonate 0.05%, remaining is
Water, pH 6.8-7.2.
Fermentation medium：Glucose 1.5%, sucrose 0.5%, yeast extract 0.5%, ammonium sulfate 1%, potassium dihydrogen phosphate
0.05%th, manganese sulfate 0.02%, magnesium sulfate 0.02%, sodium chloride 0.01%, remaining is water, pH 6.8-7.2.
1) inclined-plane culture
The Escherichia coli ZY0217 that embodiment 1 obtains is inoculated in slant medium, 1d is activated under the conditions of 40 DEG C；
2) seed culture
The bacterial strain ZY0217 of step 2) inclined-plane culture is inoculated in seed culture medium, liquid amount is in 500mL triangular flasks
20mL, cultivation temperature are 40 DEG C, and 10h is cultivated under 250rpm shaking speeds；
3) fermented and cultured
The bacterial strain ZY0217 of step 2) seed culture is inoculated into fermentation medium, inoculum concentration is 5% (v/v), and 5L is sent out
Liquid amount is 2L in fermentation tank, and cultivation temperature is 34 DEG C, while is passed through air, to keep dissolved oxygen environment suitable in fermentation system,
Mixing speed controls zymotic fluid pH, fermentation time 62h in 900rpm, fermentation process using ammoniacal liquor, 2mol/L hydrochloric acid.Through SBA-
It is 71g/L that 40E type bio-sensing instrument, which measures 1B content in zymotic fluid,.
Embodiment 8：This example demonstrates that the Escherichia coli ZY0217 production advantages
Inventor's early stage using E.col iA301 as starting strain, screening obtain E.coli NT1003 (Chen Kequan, what Xun,
Ying Hanxiao, Wang Zhen, Yuan Peipei, mono- plant of Ouyang Pingkai production 1B bacterial strain and its produce the method China of 1B specially
Sharp CN103361289B, applying date 2013.07.08).E.coli ZY0217 and E.coli NT1003 1B Fermented
It can compare and see the table below：
Compared with E.coli NT1003, E.coli ZY0217 1B output increased 17.7%, fermentation time reduction
10h。
Claims (5)
1. the bacterial strain of one plant of production 1B, it is characterised in that its Classification And Nomenclature is ETEC（Escherichia coli）ZY0217, it is preserved in China typical culture collection center, deposit number：CCTCC NO：M 2015079.
2. a kind of method that bacterial strain ZY0217 using described in claim 1 produces 1B, it is characterised in that including such as
Lower step：
1）Inclined-plane culture
ZY0217 is inoculated in slant medium, 34-40 DEG C of cultivation temperature, incubation time 1-2 d；
2）Seed culture
By step 1）The bacterial strain ZY0217 of inclined-plane culture is inoculated in seed culture medium, and cultivation temperature is 34-40 DEG C, stirring training
Support 7-10 h；
3）Fermented and cultured
By step 2）Bacterial strain ZY0217 after culture is inoculated into fermentation medium, and cultivation temperature is 34-40 DEG C；It is passed through simultaneously
Air, stirring fermentation 50-62 h.
3. according to the method for claim 2, it is characterised in that the step 1）In slant medium be LB culture mediums,
Include following mass percent component：Tryptone 1%, yeast extract 0.5%, sodium chloride 1%, remaining is water, pH 6.8-
7.2；
The step 2）In seed culture medium include following mass percent component：Carbon source 0.1%-0.2%, nitrogen source 3%-8%,
Inorganic salts 0.5%-1.3%, remaining is water, pH 6.8-7.2；Wherein, the carbon source is glucose and/or sucrose；The nitrogen source is
One or more of mixing in peptone, yeast extract or ammonium sulfate；The inorganic salts be sylvite, magnesium salts, molysite, manganese salt,
One or more of mixing in sodium salt or calcium salt；
The step 3）In fermentation medium include following mass percent component：Carbon source 1.0%-2%, nitrogen source 0.5%-2%,
Inorganic salts 0.1%-1%, remaining is water, pH 6.8-7.2；Wherein, the carbon source is glucose and/or sucrose；The nitrogen source is flower
One or more of mixing in raw cake powder, soybean cake powder, corn steep liquor, yeast extract or ammonium sulfate；The inorganic salts be sylvite,
One or more of mixing in magnesium salts, molysite, manganese salt, sodium salt or calcium salt.
4. according to the method for claim 2, it is characterised in that the step 2）In, mixing speed is 150-250 rpm；
The step 3）In, mixing speed is 200-900 rpm.
5. according to the method for claim 4, it is characterised in that the formula of the seed culture medium：Sucrose 0.2%, peptone
2%th, ammonium sulfate 2%, yeast extract 1%, dipotassium hydrogen phosphate 0.4%, magnesium sulfate 0.3%, ferrous sulfate 0.02%, manganese sulfate 0.07%,
Remaining is water, pH 6.8-7.2；
The formula of the fermentation medium：Glucose 1.5%, corn steep liquor 0.5%, ammonium sulfate 0.5%, dipotassium hydrogen phosphate 0. 05%, sulphur
Sour manganese 0. 02%, ferrous sulfate 0.04%, magnesium sulfate 0.05%, remaining is water, pH 6.8-7.2.
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