CN1082605A - A kind of genus bacillus and production method thereof that produces superoxide-dismutase - Google Patents
A kind of genus bacillus and production method thereof that produces superoxide-dismutase Download PDFInfo
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- CN1082605A CN1082605A CN93103774A CN93103774A CN1082605A CN 1082605 A CN1082605 A CN 1082605A CN 93103774 A CN93103774 A CN 93103774A CN 93103774 A CN93103774 A CN 93103774A CN 1082605 A CN1082605 A CN 1082605A
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Abstract
A kind of genus bacillus and production method thereof that produces superoxide-dismutase selected genus bacillus to contain the high new bacterial strain of SOD through mutagenic treatment, strain expanded culture, and zymocyte liquid separates extraction process.Mycetocyte of the present invention is except also using the osmotic pressure fragmentation through N,O-Diacetylmuramidase and ultrasonic disruption, and obtains the SOD product with 30% and 90% ammonium sulfate precipitation, SephadexG-25 desalination.The present invention compares with prior art, has that SOD content height, enzyme stability are good, technical process is simplified, has reduced loss, saves the energy, advantage such as reduce cost.
Description
The present invention relates to produce the new bacterial strain screening and the production technique thereof of superoxide-dismutase (SOD), is improvement and raising to application number 91108549.1.
Patent applicant of the present invention is " utilizing genus bacillus to produce the method for superoxide-dismutase ", number of patent application 91108549.1 in the denomination of invention of application on September 2nd, 91.The applicant find that the genus bacillus CGMCC 0137 bacterial strain SOD content of this application utilization is low, and screening method is outmoded in the experimental study process, its production technique link is many, and the time is long, and the loss of enzyme is big, and output capacity is low.
The objective of the invention is: genus bacillus is carried out mutagenic treatment, therefrom screen the high new bacterial strain of SOD enzyme content, and simplify the extraction separation production technique, thereby reach the purpose that reduces cost, improves the SOD production of enzyme.
The invention is characterized in: a kind of Bacillus strain that produces superoxide-dismutase, this strain classification are genus bacillus Bacilius cereus, and bacterium numbering is: M-22, preserving number are CGMCC No 0175.Genus bacillus obtains bacterial strain (preserving number CGMCC 0175) through mutagenic treatment, strain expanded culture, and zymocyte liquid, intermittently ventilation, the osmotic pressure fragmentation, 30% and 90% ammonium sulfate precipitation, Sephadex G-25 desalination separates extraction process.
(1) screening of high SOD content bacterial strain:
Bacterial strain SOD content by the screening of method shown in the 91108549.1 patent application specification page 4 is not high, has adopted the method for mutagenesis to screen the new bacterial strain that SOD content is the 0.420mg/g bacterium (preserving number CGMCC 0175) for this reason.Mutafacient system is as follows: 1. be inoculated on the beef broth protein culture medium separating the genus bacillus that obtains in the middle plant tissue, on Bechtop, shone 5 minutes apart from 20cm with 15% ultraviolet lamp, cultivate only 5% bacterium survival compared with the control in 18 hours in 25 30 ℃, select the survival strains single culture, measure the content of SOD, selecting content is the bacterial strain of 0.420mg/g bacterium, and preserving number is (CGMCC 0175).
The biological characteristics of bacterial strain of the present invention:
Genus bacillus (Bacillus cereus) thalline direct rod shape, chain is given birth to, and general size is 1.0-1.2X3.0-5.0 μ m, is measured as 0.9-1.2X2.0-4.0 μ m under the Electronic Speculum.The Gram-reaction positive.The gemma ellipse, middle life or nearly middle giving birth to are not expanded.Content colored particles not in the protoplasma.The bacterium colony circle is swelled tarnish, wax slightly.But anaerobic growth, the catalase positive, the V-P reacting positive, V-P nutrient solution PH is 4.3-5.6.35-45 ℃ of growth top temperature, minimum temperature 10-20 ℃, in N,O-Diacetylmuramidase 0.001%, can grow, 7%NaCl can grow, the PH5.7 growth, glucose produces acid, and hydrolyzed starch utilizes citric acid, and reducible NO3 is NO2, can decompose tyrosine.
(2) fermentation process:
1, actication of culture:
The streak inoculation of high SOD content bacterial strain on the beef broth protein culture medium, was cultivated 24 hours down, is made into spore suspension then for 28 30 ℃.The time of cultivating can not surpass 24 hours, because be in the logarithmic phase that genus bacillus is cultivated this moment, the bacterium vigor is the highest.
2, fermentation culture conditions:
1. fermentor tank inoculation back incubation time should not surpass 18～20 hours, otherwise generates gemma in a large number, is difficult to fragmentation, and production of enzyme is reduced.
2. in the fermentation, the fermentation air flow is 1-2Vol./Vol/min, changes continuous ventilation and is intermittently ventilation, and every ventilation stopped to ventilate 40-60 minute after 2 hours, thereby induced the raising of SOD content in the genus bacillus.
3. fermented liquid requires:
Zymocyte liquid content is 6,000,000,000/ml, cultivates to put jar in 18～20 hours, and this moment, the bacterium amount reached the highest and seldom gemma generation.
(3) fragmentation of mycetocyte:
Because the bacterial strain that is adopted is waxy Bacillus (Bacillus cereus), its cell walls tool wax, extremely difficult broken, every liter of bacterium is stuck with paste and adds the 50mg N,O-Diacetylmuramidase, ultrasonication still had 30～40% bacterium to fail fragmentation in 10 minutes under the 250W power, therefore, after N,O-Diacetylmuramidase and ultrasonication, adopt the osmotic pressure crush method again.Concrete grammar is: the Tris damping fluid that every gram thalline adds 20ml 0.0005M EDTA, 0.5M glucose, 0.03M pH8 vibrated 10 minutes down in 23 ℃.Percentage of damage is reached more than 95%.Can also reduce the consumption of N,O-Diacetylmuramidase with the osmotic pressure fragmentation.
(4) thick enzyme production technique:
Thick enzyme production technique comprises 30% ammonium sulfate precipitation, 90% ammonium sulfate precipitation and three steps of Sephadex G-100 column chromatography.
1,30% ammonium sulfate precipitation:
91108549.1 precipitation concentration is 55% in the patent application specification, through repetition test, a large amount of enzyme losses is arranged in the precipitation behind 55% ammonium sulfate precipitation, selects 30% ammonium sulfate precipitation, the free of losses of SOD enzyme.After crude extract added 30% ammonium sulfate, churning time was 60 minutes, leaves standstill can carry out centrifugal in 2 hours.
2,90% ammonium sulfate precipitation:
Adopt 75% ammonium sulfate precipitation in the former patent, still have a large amount of SOD enzymic activitys in the supernatant liquor, the centrifugal supernatant liquor adds 90% ammonium sulfate precipitation SOD again, and enzyme is all precipitated.
3, Sephadex G-25 desalination:
Slough the method for the salt ion employing dynamic dialysis in the precipitation in the former patent application specification, the time is 24～48 hours.Now change the desalination of Sephadex G-25 column chromatography into, only need 2～3 hours, saved the time greatly, concrete grammar is: will be dissolved in through the enzyme sample of 90% ammonium sulfate precipitation in the phosphoric acid buffer of 5mM pH7.8 of minimum, through Sephadex G-25 column chromatography, part is collected.The G-25 post is used the abundant balance of the phosphoric acid buffer of 5mM pH7.8 in advance.Detect the SOD enzyme liquid of collecting desalination with Nessler's reagent.
4, Sephadex G-100 column chromatography:
Use 5mM pH7.8 phosphoric acid buffer balance Sephadex G-100 post in advance, the crude enzyme liquid after the desalination is added in the G-100 post, full-automatic substep is collected.The part that the SOD vigor is good is collected.Survey the SOD vigor, use electrophoresis detection purity simultaneously.
5, crude zyme preparation specification of quality:
Thick zyme extract reaches more than 1200 than living after Sephadex G-100, and productive rate reaches 65%.Electrophoresis detection is mainly SOD enzyme band.
(5) production technique of refining enzyme: referring to 91108549.1 patent application specification 8-9 pages or leaves.
The present invention compared with prior art its advantage is:
(1) bacterial strain SOD content height:
The refining SOD content of the new bacterial strain that method by mutagenesis obtains is up to the 0.42mg/g bacterium, and is higher by 57% than former patent bacterial strain uses therefor 0137.
(2) this bacterial strain contains Cu, Zn-SOD, Fe-SOD and Mn-SOD, and wherein Cu, Zn-SOD account for 25%, and Mn-SOD accounts for 40%, and Fe-SOD accounts for 55%.
(3) enzyme stability that extracts with this bacterial strain is very high.90 ℃ following 5 minutes to not influence of activity, 90 ℃, 50 minutes complete deactivations; Active neither influenced in the 3-9 scope at pH.
(4) the SOD extraction process that the present invention founded can obtain the zymin of different purity, medicine, veterinary drug, crop reverse-resisting yield-increasing had both been can be used for, daily cosmetics, nourishing drink etc., also can be used for the research such as character, structure, amino-acid sequence mensuration of enzyme, have development prospect widely.
(5) this technical process is simplified, and has reduced loss, and save energy, has reduced cost.
(6) this bacterial strain breeding is fast, and convenient sources is cheap, is suitable for batch production production.
Description of drawings:
Fig. 1: the strain selection figure that is high SOD content
Fig. 2: the zymotechnique schema of producing SOD
Fig. 3: SOD crude extract process flow sheet
Fig. 4: SOD crude zyme preparation technological process of production figure
Fig. 5: refining SOD preparation production technique schema
Example: sticking with paste production 8.6 gram SOD with 100 kilograms of genus bacillus bacterium is example.
1, from the plant materials tissue, filter out genus bacillus, under the 15W UV-light, apart from 20cm, mutagenesis 5 minutes, the wax system genus bacillus strain (Bacillus cereus) that obtains SOD content and be 0.42mg/g is numbered M-22, preserving number CGMCC NO0175.
2, produce 14 tons of zymocyte liquids with 15 tons of fermentor tanks.With fermentor tank sterilization earlier, load onto behind the nutrient solution in sterilization.High pressure steam sterilization is adopted in sterilization, promptly at 121 ℃, pressure 0.5Kg/cm
2Under the condition, sterilized 30 minutes fermentor cultivation liquid sterilization stirring velocity 250rpm.Be cooled to 28 ℃ then, the inoculum size by 2% inserts fermentor tank with the seeding tank zymocyte liquid.After the fermentor tank inoculation, under 30 ℃ of conditions, cultivated 24 hours.Air flow 2Vols/Vol/min, oxygen content 40% 60 minutes at interval, was ventilated 2 hours.
The fermentor cultivation liquid formula is: beef broth 0.5%, and peptone 0.5%, NaCl0.5%, soya-bean oil 0.5%, glucose 0.2%, starch 0.4%, sal epsom 0.008%, potassium primary phosphate 0.04%, Cu, Zn, Mn, the Fe of lime carbonate 0.5%, 800 μ M stir.PH value sterilization is preceding 7.6, sterilization afterwards 7.4.
The zymocyte liquid bacteria containing amount is put jar at every milliliter more than 6,000,000,000 before the gemma maturation, and pH is value 7.5, no living contaminants.
3, mycetocyte is emanated.Fermentor tank bacterium liquid injects whizzer at dominant discharge 400L/h, and whizzer is stuck with paste mycetocyte segregation precipitation automatically with bacterium with 4000rpm/min speed and flowed out, and enters the physiological saline groove, through three washings, collects 100 kilograms of bacterium after centrifugal and sticks with paste.
4, with the mycetocyte fragmentation.Stick with paste and the potassium phosphate buffer of damping fluid by bacterium, stir into bacterium liquid with 1: 3 volume ratio adding 50mM, pH7.8.Stick with paste adding 50mg N,O-Diacetylmuramidase by every liter of bacterium, under 4 ℃ of cold condition, stirred 30 minutes.Under 0～4 ℃ of cold condition, bacterium liquid was handled 10 minutes every processing 1 minute 50 seconds at interval with 250W power ultrasonic cell pulverization machine.After the ultrasonication to bacterium liquid microscopy cytoclasis situation.Looking percentage of damage continues broken with osmometry.Percentage of damage reaches more than 95%.Then that bacterium liquid is centrifugal with 4000rpm.Collect the crude extract of 130Kg supernatant liquor as SOD.
5, extract thick enzyme:
(1) 30% ammonium sulfate precipitation.Crude extract is added ammonium sulfate, make that ammonium sulfate reaches 30% saturation ratio in the crude extract.Under 22 ℃, utilize agitator to stir 60 minutes, adopt membrane filtration system to filter and obtain filtrate.
(2) 90% ammonium sulfate precipitations.In the filtrate behind 30% ammonium sulfate precipitation, adding ammonium sulfate makes its saturation ratio reach 90%, at room temperature utilize agitator to stir 60 minutes, centrifugal 13000rpm, precipitation is dissolved among the 5mM, pH7.8 potassium phosphate buffer of minimum (including 2mM EDTA), use above-mentioned damping fluid and Sephadex G-25 desalination then, obtain the thick zyme extract of 108Kg.
(3) Sephadex G-100 column chromatography.Sephadex G-100 post is used the potassium phosphate buffer balance of pH7.8,5mM in advance; Thick zyme extract 80Kg after the desalination is joined in the post, and with above-mentioned buffering buffer solution elution.Adopt full-automatic substep gathering system to collect elutriant, merge the high collection liquid part of SOD vigor.
6, the preparation of the refining enzyme of different sorts SOD:
(1) Cu, Zn-SOD: will be through the crude enzyme liquid behind the Sephadex G-100 column chromatography by using the potassium phosphate buffer equilibrated DE-52 post of pH7.8,5mM in advance, with above-mentioned 0.005-0.3mM buffer solution for gradient elution, the collection liquid that enzyme activity is high merges, and obtains the pure enzyme liquid of Cu, Zn-SOD.The pure enzyme liquid of Cu, Zn-SOD is concentrated, lyophilize can obtain pure enzyme pulvis 1.30 grams of Cu, Zn-SOD;
(2) Mn-SOD: above-mentioned leacheate before DE-52 post gradient elution is collected, be heated to 65 ℃, be placed on cooling rapidly under the low temperature in 5 minutes, repeat according to this 3 times, isolate filtrate, filtrate is concentrated by pressure filtration.
Concentrated solution is passed through to use the Potassium ethanoate damping fluid equilibrated CM-52 post of pH5.5,40mM, and carry out gradient elution with the Potassium ethanoate damping fluid of pH5.5,0.04-0.20M, collect simultaneously, the collection liquid that enzymic activity is high merges together, obtain the pure enzyme liquid of Mn-SOD, obtain pure enzyme powder 3.44 grams of Mn-SOD through concentrated, lyophilize again.
(3) Fe-SOD: will through the ammonium sulfate precipitation of 90% saturation ratio, and the thick enzyme 50Kg of desalination by using pH5.5,0.01M Potassium ethanoate damping fluid equilibrated CM-52 post in advance, above buffer solution elution, collect the high elutriant of enzyme activity, with pH7.8, the dialysis of 50mM potassium phosphate buffer.With dialyzate by using the potassium phosphate buffer equilibrated DE-52 post of pH7.8,5mM in advance, wash post with same damping fluid, and carry out gradient elution with the potassium phosphate buffer of 5-50mM, pH7.8, the collection liquid that will meet pure enzyme standard mixes, adding ammonium sulfate makes and reaches 80% saturation ratio, that obtain after filtration is pure Fe-SOD again, filtrate is obtained the pure enzyme liquid of Fe-SOD with the dialysis of pH7.8,50mM potassium phosphate buffer or by pressure filtration, again through concentrate, lyophilize obtains pure enzyme pulvis 3.87 grams of Fe-SOD.
Obtain pure enzyme pulvis 8.6 grams of SOD altogether.
Claims (7)
1, a kind of genus bacillus that produces superoxide-dismutase is characterized in that: this bacterial strain is waxy Bacillus Bacillus Cereus, bacterium numbering: M-22, preserving number: CGMCC NO 0175.
2, a kind of method of utilizing bacillus to produce superoxide-dismutase comprises that genus bacillus obtains bacterial strain (preserving number is CGMCC 0175) through mutagenic treatment.Strain expanded culture, zymocyte liquid separates, and extraction process is characterized in that, also has intermittently ventilation, osmotic pressure fragmentation, 30% and 90% ammonium sulfate precipitation, SephadexG-25 desalination.
3,, it is characterized in that mutagenic treatment is: utilize genus bacillus, at ultraviolet lamp 15W according to the described method of claim 2, apart from 20cm, shone 5 minutes, select survival strains to join foster separately, measure the content of SOD, selecting content is the bacterial strain of 0.420mg/g bacterium, and preserving number is: CGMCC 0175.
4, method according to claim 2, it is characterized in that intermittently ventilation is: in fermentation, every ventilation stopped to ventilate 40 60 minutes after 2 hours, thereby SOD content is improved.
5, method according to claim 2, it is characterized in that osmotic pressure is broken for: the thin spore of bacterium is after molten bacterium and ultrasonication, every gram thalline adds the Tris buffered soln of 20ml0.0005MEDTA, 0.5M glucose, 0.03M PH8, vibrated 10 minutes down in 23 ℃, the mycetocyte percentage of damage is reached more than 95%.
6, method according to claim 2 is characterized in that 30% and 90% ammonium sulfate precipitation is: crude extract is added 30% ammonium sulfate, stirred 60 minutes, carry out centrifugally, feelings liquid adds 90% ammonium sulfate precipitation SOD again on the centrifugal.
7, method according to claim 2, it is characterized in that Sephadex G 25 desalinations are: will be dissolved in through the enzyme sample of 90% ammonium sulfate precipitation in the phosphoric acid buffer of 5mM PH7.8 of minimum, through Sepadex G-25 column chromatography, part is collected, and detects the SOD enzyme liquid of collecting desalination with Nessler's reagent.
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| CN93103774A CN1054398C (en) | 1993-04-21 | 1993-04-21 | Bacillus capable of producing super oxide dismutase and its production method |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1297190C (en) * | 2004-03-08 | 2007-01-31 | 三门峡市农业科技咨询服务中心 | Superoxide dismutase apple, its prodn. method and special use biological prepn. therefrom |
| CN1308440C (en) * | 2004-12-21 | 2007-04-04 | 中国水产科学研究院黄海水产研究所 | New type marine microorganism of lysozyme and bacillus 5-12 of producing lysozyme in high yield |
| CN103088093A (en) * | 2013-01-08 | 2013-05-08 | 南京工业大学 | Method for purifying antibacterial protein in bacillus cereus fermentation broth |
| CN103992971A (en) * | 2014-05-15 | 2014-08-20 | 郝之奎 | Bacillus cereus MBRH3 strain as well as screening method and application thereof |
| CN106987565A (en) * | 2017-05-22 | 2017-07-28 | 大连大学 | A kind of marine low temperature superoxide dismutase extraction and separation process |
| CN110100668A (en) * | 2019-06-17 | 2019-08-09 | 郑州市农林科学研究所 | The implantation methods of cereal crop rich in SOD |
| CN110484597A (en) * | 2019-09-02 | 2019-11-22 | 河南大学 | A kind of method of intuitive measurement SOD enzyme activity |
| CN115196999A (en) * | 2018-06-20 | 2022-10-18 | 五月阳光生物科技(浙江)有限公司 | Bacteria, fertilizer and method for cultivating peach rich in SOD (superoxide dismutase) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1924010B (en) * | 2006-09-13 | 2010-05-12 | 山西众成生物工程有限公司 | Extraction technology of edible fungus chaff superoxide dismutase |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1058614A (en) * | 1991-09-02 | 1992-02-12 | 北京农业大学 | Utilize genus bacillus to produce the method for superoxide dismutase |
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1993
- 1993-04-21 CN CN93103774A patent/CN1054398C/en not_active Expired - Lifetime
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1297190C (en) * | 2004-03-08 | 2007-01-31 | 三门峡市农业科技咨询服务中心 | Superoxide dismutase apple, its prodn. method and special use biological prepn. therefrom |
| CN1308440C (en) * | 2004-12-21 | 2007-04-04 | 中国水产科学研究院黄海水产研究所 | New type marine microorganism of lysozyme and bacillus 5-12 of producing lysozyme in high yield |
| CN103088093A (en) * | 2013-01-08 | 2013-05-08 | 南京工业大学 | Method for purifying antibacterial protein in bacillus cereus fermentation broth |
| CN103088093B (en) * | 2013-01-08 | 2014-10-29 | 南京工业大学 | Method for purifying antibacterial protein in bacillus cereus fermentation broth |
| CN103992971A (en) * | 2014-05-15 | 2014-08-20 | 郝之奎 | Bacillus cereus MBRH3 strain as well as screening method and application thereof |
| CN106987565A (en) * | 2017-05-22 | 2017-07-28 | 大连大学 | A kind of marine low temperature superoxide dismutase extraction and separation process |
| CN115196999A (en) * | 2018-06-20 | 2022-10-18 | 五月阳光生物科技(浙江)有限公司 | Bacteria, fertilizer and method for cultivating peach rich in SOD (superoxide dismutase) |
| CN110100668A (en) * | 2019-06-17 | 2019-08-09 | 郑州市农林科学研究所 | The implantation methods of cereal crop rich in SOD |
| CN110484597A (en) * | 2019-09-02 | 2019-11-22 | 河南大学 | A kind of method of intuitive measurement SOD enzyme activity |
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