CN102660481A - Bacterium for stably producing 2,3-butanediol at high yield and method for compound mutation by using low-temperature plasma and diethyl sulfate - Google Patents
Bacterium for stably producing 2,3-butanediol at high yield and method for compound mutation by using low-temperature plasma and diethyl sulfate Download PDFInfo
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- 241000894006 Bacteria Species 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 28
- 230000035772 mutation Effects 0.000 title claims abstract description 24
- OWBTYPJTUOEWEK-UHFFFAOYSA-N butane-2,3-diol Chemical compound CC(O)C(C)O OWBTYPJTUOEWEK-UHFFFAOYSA-N 0.000 title abstract description 10
- DENRZWYUOJLTMF-UHFFFAOYSA-N diethyl sulfate Chemical compound CCOS(=O)(=O)OCC DENRZWYUOJLTMF-UHFFFAOYSA-N 0.000 title abstract 4
- 229940008406 diethyl sulfate Drugs 0.000 title abstract 4
- 150000001875 compounds Chemical class 0.000 title abstract 3
- 241000588697 Enterobacter cloacae Species 0.000 claims abstract description 17
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses enterobacter cloacae for stably producing 2,3-butanediol at high yield and a method for compound mutation by using low-temperature plasma and diethyl sulfate. The enterobacter cloacae is classified and named as Enterobacter cloacae DLM and has the preservation number of CGMCC (China General Microbiological Culture Collection Center) 6053. The mutation method is characterized in that bacterium is suspended into physiological saline and pretreatment of diethyl sulfate and compound mutation of the low-temperature plasma and the diethyl sulfate are sequentially carried out. The strain can be used for producing the 2,3-butanediol by fermenting different effectively-utilized carbon sources, wherein the conversion rate of sugar is high and the concentration of the 2,3-butanediol is high; and when glucose or synanthrin is hydrolyzed into a carbon source, the concentrations of the 2,3-butanediol in a 5L fermentation tank respectively reach 125.2g/l and 120.2g/l. The mutation method used in the invention has the characteristics of simpleness and feasibility in operation, short treatment time in mutation and the like and provides a reliable method for mutation breeding of microorganisms.
Description
Technical field
The invention belongs to the microorganism mutation breeding technical field, relate to and use low-temperature plasma and ethyl sulfate complex mutation to produce plant height product 2, the bacterial strain of 3-butyleneglycol and application thereof.
Background technology
2, and the 3-butyleneglycol (2,3-Butanediol, 2, be a kind of very important industrial chemicals and fuel 3-BD), in fields such as medicine, food, fine chemistry industries extensive use is arranged.Because 2; The 3-butyleneglycol contains two chiral centres, and chemosynthesis difficulty is so be raw material with the biomass resource, produce 2 through microbial method; The 3-butyleneglycol has very big industrial prospect; Wherein how to obtain high yield 2, the bacterial strain of 3-butyleneglycol produces 2 for biological process, and the industriallization of 3-butyleneglycol is significant.
Present fermentative prodn 2 is if the microbial host bacterium class of 3-butyleneglycol comprises Klebsiella (Klebsiella), genus bacillus (Bacillus), enterobacter (Enterobacter), false monospore Bacillaceae (Pseudomonad) etc.2 of the acquisition of klepsiella pneumoniae fermentation at present, 3-butyleneglycol production concentration is the highest, also is the maximum bacterial classification of research.But this Pseudomonas has significant limitation and insecurity in conditioned pathogen as a kind of industrial application bacterial classification.
In recent years, both at home and abroad to 2, the Research on Fermentation of 3-butyleneglycol is a lot, mainly round the mutagenesis screening of bacterial classification and genetic engineering modified etc.The most frequently used mutafacient system has ultraviolet mutagenesis, chemomorphosis or both combinations.Though ultraviolet mutagenesis safety, complicated operation, and also efficiency of inducing mutation is low.The effect of chemomorphosis is better, but the general action time is long, and toxic side effect is big.
Low-temperature plasma is a kind of low temperature nonequilibrium plasma; Be meant and under atmospheric pressure receive extraneous high-energy (high-voltage, strong-electromagnetic field, radiation etc.) by gas to make the time spent gas molecule breakdown; Produce charged particle, radical, active substance and ultraviolet ray etc., and whole system presents low-temperature condition.Low-temperature plasma can destroy cell walls when handling cell; Can make a large amount of charged particles, active substance and ultraviolet ray can get into cell and directly act on nucleic acid in the born of the same parents; This also makes chemical molecular more effectively directly act on the DNA simultaneously, has reduced the treatment time.
The present invention uses the method for low-temperature plasma and ethyl sulfate complex mutation that mikrobe is carried out selection by mutation.The ethyl sulfate consumption that uses in this method is low, and exposure duration lacks in air, has not only guaranteed the high efficiency of chemomorphosis, has increased safety of operators simultaneously.Operation is simple for the composite mutagenesis method that adopts, efficient is high, and complex mutation greatly reduces the response rate of mutant bacteria, has guaranteed the stability of mutant bacteria.
Summary of the invention
Content of the present invention is to use a kind of novel mutafacient system-low-temperature plasma and ethyl sulfate complex mutation method to handle mikrobe, and high yield 2 is stablized in seed selection one strain, and the mutant strain of 3-butyleneglycol and this bacterial strain are in fermentative prodn 2; Application in the 3-butyleneglycol; Solve production safety and fermented liquid 2 in the industrial production, the problem that 3-butyleneglycol concentration is low is 2; The industrial production and the application of 3-butyleneglycol lay the foundation, and also for the mutagenic and breeding of other mikrobes a kind of new mutafacient system are provided simultaneously.
The invention provides a kind of effective selection by mutation new technology.With Enterobacter cloacae (enterobacter cloacae) or other bacteriums is original strain; At first use ethyl sulfate pre-treatment original strain; Use the bacteria suspension that Low Temperature Plasma Treating contains ethyl sulfate to carry out complex mutation then, obtain stablizing the mutant strain of high yield at last through multiplex screening.
Technical scheme of the present invention is following:
High yield 2 is stablized in one strain, the bacterium of 3-butyleneglycol, and its classification called after Enterobacter cloacae DLM, its preserving number is: CGMCC 6053.
Above-mentioned bacterium utilizes the method for low-temperature plasma and ethyl sulfate complex mutation, comprises the steps:
(1) original strain is carried out the ethyl sulfate pre-treatment
Bacterial classification is inserted in the seed culture medium with the 1-2% inoculum size, and behind the cultivation 12-16h, it is centrifugal to get bacterium liquid, abandons supernatant, and it is 10 that residual thalline uses SPSS to be diluted to number of cells
7-10
9/ mL.The ethyl sulfate that in bacteria suspension, adds 1-3% (v/v) mixing of vibrating, the treatment time is 3-10min.
(2) complex mutation bacterial strain
Adopt dielectric barrier discharge (DBD) device.Get the pretreated bacteria suspension of 500uL ethyl sulfate and be added drop-wise to the central authorities of the sterilization cooling back glass disk of diameter 60mm, place the plasma inducing platform that flattens.Regulate the high voltage terminal on glass disk top and the gap of bacterium liquid liquid level, regulate electric current and voltage, make gas produce discharge, obtain uniform air dielectric barrier discharge plasma, discharge process 15-160s.This bacteria suspension dilution is applied on the plate culture medium, places 25 ~ 37 ° of C constant incubators to cultivate flat board, incubation time is 1 ~ 2 day.Colony count on the counting flat board draws lethality rate, thereby confirms best mutation time, and mutation time generally selects lethality rate to be about the time point about 70%.
(3) bacterial screening
Carry out primary dcreening operation in bigger bacterium colony to the seed culture medium of picking well-grown, bacterium colony on flat board, behind the shake-flask culture 24-48h, detect production concentration; The mutant bacteria that then production concentration is obviously improved is forwarded to and carries out multiple sieve in the fermention medium, and 3 bottles of parallel cultivation 24-48h detect production concentration; Choose the bacterial strain of high production concentration and in fermentor tank, cultivate, verify its leavening property; Go down to posterity through shaking bottle, relatively more different fermentor cultivation experimental results for bacterial strain are verified its genetic stability.
Through the stable high yield 2 that aforesaid method obtains, the bacterial strain of 3-butyleneglycol is used in the 3-butyleneglycol in fermentative prodn 2, and the substratum that its fermentation is used comprises following composition: carbon source 2-14%, nitrogenous source 0.5-3%, inorganic salt 0.2-0.5%, and all the other are water; Wherein carbon source is that one or both reach above mixing in glucose, starch hydrolyzate, the hydrolysis of inulin liquid; Said nitrogenous source is inorganic or nitrogen-containing organic compound, and wherein inorganic nitrogen-sourced is one or both mixing in Secondary ammonium phosphate and the urea, and the organic nitrogen-containing source is one or both mixing in yeast powder and the steeping water; Said inorganic salt are sodium salt, sylvite, magnesium salts, phosphoric acid salt, Hydrocerol A, ferrous, manganese, zinc and EDTA.
Embodiment
Embodiment 1
Plasma body and ethyl sulfate complex mutation method of microorganism are used in the present embodiment explanation.
Experimental strain is Enterobacter cloacae (enterobacter cloacae), from soil, screens to obtain.The plasma discharge apparatus that uses in the experiment is dielectric barrier discharge (DBD) device that uses among patent 200610047213.5 embodiment one.
The seed culture medium that present embodiment uses is: glucose 40g/L, (NH
4)
2HPO
46g/L, KCl 1.8g/L, EDTA 0.51g/L, MgSO
40.88g/L, FeSO
47H
2O 0.0225g/L, ZnSO
47H
2O 0.0075g/L, MnSO
4H
2O 0.0023g/L, Hydrocerol A 0.21g/L, Trisodium Citrate 0.294g/L, yeast powder 1g/L.
Fermention medium is: glucose 120g/L, (NH
4)
2HPO
418g/L, KCl 1.8g/L, EDTA 0.51g/L, MgSO
40.88g/L, ZnSO
47H
2O 0.0075g/L, FeSO
47H
2O 0.0225g/L, MnSO
4H
2O 0.0023g/L, Hydrocerol A 0.21g/L, Trisodium Citrate 0.294g/L, yeast powder 1g/L.
Culture condition is: shaking bottled liquid measure is volume 1/3,37 ℃ of temperature, shaking speed 200rpm.
The ethyl sulfate pre-treatment
Original strain is inoculated in the 100mL seed culture medium with the 1-2% inoculum size, under 37 ℃, 200rpm condition, cultivates 12-16h, obtain being in the bacterium liquid of logarithmic phase.Get the bacterium liquid of 1ml, centrifugal 2 minutes of 12000rpm abandons supernatant, and residual thalline is diluted to number of cells with SPSS and is about 10
7-10
9/ mL.The bacteria suspension of getting after 1mL dilutes places centrifuge tube, in suspension, adds the ethyl sulfate (about 20uL) of 2% (v/v), places on the vortex oscillation device and handles 5min.
Mutagenic processes
Get the pretreated bacteria suspension of 500uL ethyl sulfate and be added drop-wise to the central authorities of glass disk after alcohol disinfecting sterilization of diameter 60mm; Place the plasma inducing platform that flattens; Distance between adjustment top electrode and bacterium liquid liquid level is to 3mm, discharge process 100s under the condition of voltage 25v, electric current 1.2A.
Embodiment 2
Present embodiment explanation screening high yield 2, the method for the mutant strain of 3-butyleneglycol
Shake a bottle primary dcreening operation
The dilution of bacterium liquid after the mutagenesis is coated with flat board; Place 37 ℃ of constant incubators to cultivate 24h; The colony inoculation that colonial morphology is good on the picking flat board is cultivated 36h, vapor detection 2 under 37 ℃, 200rpm condition in the 50mL seed culture medium; 3-butyleneglycol concentration is chosen production concentration and is higher than the bacterial strain of original bacterium more than 20%.
Shake the multiple sieve of bottle
To transfer in the 200mL fermention medium through shaking the bacterial strain that bottle primary dcreening operation obtains, each sample do three parallel, place 37 ℃ of constant temperature shaking tables, 200rpm shaking culture 36h.Vapor detection 2,3-butyleneglycol concentration is chosen production concentration and still is higher than the bacterial strain of original bacterium more than 20%.
The fermentor cultivation checking
With Biotech 5 L automatic fermenters, the fermention medium volume is 1.5 L, and temperature is 37 ℃; Mixing speed is 250 rpm, and using 5 mol/L NaOH control pH is 5.9, keeps the pressurized air of logical 200ml/ (Lmin) in the fermenting process; The glucose initial concentration is 120 g/L, and 10% inoculation uses the peristaltic pump even flow to add the dried glucose syrup of 800 g/L in the fermenting process; Glucose concn is about 50 g/L in the fermented liquid in the maintenance fermenting process, and fermenting experiment repeats 3 times at least.In the fermented liquid 2,3-butyleneglycol concentration has improved about 26% than original strain about 110 g/L.
Embodiment 3
The genetic stability of present embodiment explanation mutant bacteria Enterobacter cloacae DLM.
Continuous passage in the seed culture medium that with glucose is carbon source is carried out fermentor tank with the first-generation, the third generation and the 7th generation and is criticized formula stream and add experiment, the mitotic stability of inspection mutant strain DLM.Experimental result is as shown in table 1.
Table 1
| Strain | Fermentation?time(h) | 2,3-BD(g/L) | 2,3-BD?yield(g/g) |
| Enterobacter?cloacae | 56 | 87.29 | 0.29 |
| E.cloacae?DLM?<1st>; | 56 | 109.32 | 0.40 |
| E.cloacae?DLM?<3rd>; | 56 | 110.92 | 0.41 |
| E.cloacae?DLM?<7th>; | 56 | 111.14 | 0.39 |
Can find out that from experimental result through going down to posterity of continuous 7 generations, this mutant bacteria leavening property is highly stable.
Embodiment 4
Present embodiment explanation Enterobacter cloacae DLM glucose fermentation in the 5L fermentor tank produces 2, the technology of 3-butyleneglycol.
The seed culture medium that present embodiment adopts is the seed culture medium of embodiment 1.
The fermention medium that present embodiment adopts is the fermention medium of embodiment 1.
Enterobacter cloacae DLM is seeded to the 500mL that contains the 150mL seed culture medium to be shaken in the bottle and cultivates 37 ℃ of culture temperature, shaking speed 200rpm, incubation time 12-16h.Again seed culture medium is inoculated in the 5L fermentor tank that contains the 1.5L fermention medium; Inoculum size is 10%, and leavening temperature is 37 ℃, and mixing speed is 250rpm; The initial pH value of fermenting is 7; Control pH value is 5.9 in the fermenting process, and (the initial oxygen dissolving value of fermention medium is 9.4ppm, and the oxygen dissolving value in the fermenting process remains on 0.4 ~ 0.5ppm) to feed the pressurized air of 150ml/ (Lmin) continuously; And even flow adds the dried glucose syrup of 800g/L during the fermentation, and glucose concn is about 50g/L in the maintenance substratum.Behind the fermentation culture 56h, vapor detection 2,3-butyleneglycol output reaches 125.2g/L, and sugared transformation efficiency is 0.42g/g.
Embodiment 5
Present embodiment explanation Enterobacter cloacae DLM inulin simultaneous saccharification and fermentation in the 5L fermentor tank produces 2, the technology of 3-butyleneglycol.
The seed culture medium that present embodiment adopts is the seed culture medium of embodiment 1.
Fermention medium: inulin 120g/L, (NH
4)
2HPO
418g/L, KCl 1.8g/L, EDTA 0.51g/L, MgSO
40.88g/L, ZnSO
47H
2O 0.0075g/L, FeSO
47H
2O 0.0225g/L, MnSO
4H
2O 0.0023g/L, Hydrocerol A 0.21g/L, Trisodium Citrate 0.294g/L, yeast powder 1g/L.
Enterobacter cloacae DLM is seeded to the 500mL that contains the 150mL seed culture medium to be shaken in the bottle and cultivates 37 ℃ of culture temperature, shaking speed 200rpm, incubation time 12-16h.The 5L fermentor tank contains the 1.5L fermention medium, and temperature is 37 ℃, and mixing speed is 250rpm, and the pH value is 5.9, and the inoculum size by 10% behind the inulinase hydrolysis 1h of adding 500U inserts above-mentioned bacterium liquid.Control pH value is 5.9 in the fermenting process, feeds the pressurized air of 150ml/ (Lmin) continuously.Mend 40g inulin and 150U inulinase at fermenting process since the every 4h of 11h, mend altogether 11 times.Behind the fermentation culture 63h, vapor detection 2,3-butyleneglycol output reaches 120.2g/L.
Claims (3)
1. high yield 2 is stablized in a strain, the bacterium of 3-butyleneglycol, and its classification called after Enterobacter cloacae DLM, its preserving number is: CGMCC 6053.
2. use the method for low-temperature plasma and the described bacterium of ethyl sulfate complex mutation claim 1, it is characterized in that:
(1) original strain is carried out the ethyl sulfate pre-treatment: bacterial classification is inserted in the seed culture medium with the 1-2% inoculum size, and behind the cultivation 12-16h, it is centrifugal to get bacterium liquid, abandons supernatant, and it is 10 that residual thalline uses SPSS to be diluted to number of cells
7-10
9/ mL; The ethyl sulfate that in bacteria suspension, adds 1 ~ 3% (v/v) mixing of vibrating, the treatment time is 3-10min;
(2) complex mutation bacterial strain: adopt dielectric barrier discharge (DBD) device, get the sterilization that bacteria suspension that 500uL handled is added drop-wise to diameter 60mm and cool off the central authorities of glass disk afterwards, place the plasma inducing platform that flattens; Regulate the high voltage terminal on glass disk top and the gap of bacterium liquid liquid level, regulate electric current and voltage, make gas produce discharge, obtain uniform air dielectric barrier discharge plasma, discharge process 15-160s; This bacteria suspension dilution is applied on the plate culture medium, places 25 ~ 37 ° of C constant incubators to cultivate flat board, incubation time is 1 ~ 2 day;
(3) bacterial screening: picking well-grown bacterium colony on flat board carries out seed culture medium successively and shakes a bottle primary dcreening operation, fermention medium and shake the multiple sieve of bottle, obtains the mutant strain that production concentration obviously improves, and carries out its leavening property of fermentor cultivation checking then; Go down to posterity through shaking bottle, relatively more different fermentor cultivation experimental results for bacterial strain are verified its genetic stability.
3. method according to claim 2 is characterized in that, the substratum that fermentation is used comprises following composition: carbon source 2-14%, nitrogenous source 0.5-3%, inorganic salt 0.2-0.5%, and all the other are water; Wherein carbon source is that one or both reach above mixing in glucose, starch hydrolyzate, the hydrolysis of inulin liquid; Said nitrogenous source is inorganic or organic nitrogen compound, and wherein inorganic nitrogen-sourced is one or both mixing in Secondary ammonium phosphate and the urea, and organic nitrogen source is one or both mixing in yeast powder and the steeping water; Said inorganic salt are sodium salt, sylvite, magnesium salts, phosphoric acid salt, Hydrocerol A, ferrous, manganese, zinc and EDTA.
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| CN201310080237.0A CN103275886B (en) | 2012-05-16 | 2013-03-14 | Bacterium for stable and high yielding of 2,3-butylene glycol, and method for utilizing low-temperature plasma and diethyl sulfate compound mutation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651348A (en) * | 2015-01-06 | 2015-05-27 | 唐山拓普生物科技有限公司 | Induced mutation method of ribonucleic acid-enriched saccharomyces cerevisiae |
| CN108484285A (en) * | 2018-06-01 | 2018-09-04 | 葛江丽 | A kind of dedicated disease-resistant multi-functional bacterial manure of fixed nitrogen of the Northeast's rice |
| CN109880765A (en) * | 2019-03-14 | 2019-06-14 | 清华大学 | Produce the bacterial strain and method of 2,3- butanediol and organic acid |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101709281A (en) * | 2009-12-15 | 2010-05-19 | 西北大学 | Method for producing 2,3-butanediol by fermentation of enterobacter sp.Y5 |
| CN102226159B (en) * | 2010-11-29 | 2014-08-27 | 山东大学 | Strain of Enterobacter cloacae and its application in the preparation of 2,3-butylene glycol |
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2012
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104651348A (en) * | 2015-01-06 | 2015-05-27 | 唐山拓普生物科技有限公司 | Induced mutation method of ribonucleic acid-enriched saccharomyces cerevisiae |
| CN104651348B (en) * | 2015-01-06 | 2017-12-15 | 唐山拓普生物科技有限公司 | The method of mutagenesis of rich ribonucleic acid saccharomyces cerevisiae |
| CN108484285A (en) * | 2018-06-01 | 2018-09-04 | 葛江丽 | A kind of dedicated disease-resistant multi-functional bacterial manure of fixed nitrogen of the Northeast's rice |
| CN109880765A (en) * | 2019-03-14 | 2019-06-14 | 清华大学 | Produce the bacterial strain and method of 2,3- butanediol and organic acid |
| CN109880765B (en) * | 2019-03-14 | 2020-12-01 | 清华大学 | Strains and methods for producing 2, 3-butanediol and organic acids |
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| CN103275886A (en) | 2013-09-04 |
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