CN1200099C - Method for increasing yield of superoxide dismutase of radioresistant coccus by radiation induction - Google Patents
Method for increasing yield of superoxide dismutase of radioresistant coccus by radiation induction Download PDFInfo
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- CN1200099C CN1200099C CN 03116366 CN03116366A CN1200099C CN 1200099 C CN1200099 C CN 1200099C CN 03116366 CN03116366 CN 03116366 CN 03116366 A CN03116366 A CN 03116366A CN 1200099 C CN1200099 C CN 1200099C
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Abstract
The present invention discloses a method for improving yield of superoxide dismutase of a radioresistant coccus by utilizing radiation induction. The radioresistant coccus fermented and cultured to a stable early stage induces the SOD synthesis of the superoxide dismutase of a thallus to improve the yield of a thallus enzyme through gamma-ray radiation and post culture. The SOD enzyme activity obtained by separating, ultrasonically treating and extracting thallus cells of the present invention is improved more than 75 % as compared with that before radiation treatment and is three times higher than that of the enzyme activity of SOD producing yeast. Simultaneously, the present invention has the advantages of simple process flow and low cost.
Description
Technical field
The present invention relates to a kind of method of radiation-induced raising radioresistant cocci superoxide-dismutase output.
Background technology
Superoxide-dismutase (SOD) is a kind of important antioxidase in the organism, and the physiological action of SOD is that ultra-oxygen anion free radical is generated hydrogen peroxide and oxygen molecule by disproportionation reaction, prevents ultra-oxygen anion free radical o
2 -Oxidative damage to biomacromolecule in the body.SOD is mainly used in delaying human body caducity, prevents the pigmentation that ultraviolet radiation causes, removes medicine, healthcare products, makeup aspects such as local inflammation.。
At present, the preparation of SOD mainly contains three kinds of sources: the 1. superoxide-dismutase in the animal (oxen and horses blood); 2. the SOD in plant (as tealeaves, wild cabbage) source; 3. microbe-derived SOD.How SOD in the market is the preparation raw material with animal blood, and the SOD in animality source may have the danger of pathogenic agent, as European popular mad cow disease.Compare with animals and plants SOD preparation method, it is short that the preparation of microorganism SOD has the raw material biology growing cycle, and fermentative production is larger, the advantage that extraction cost is lower.Therefore, microbial fermentation prepares SOD and has important application prospects and practical significance.Domestic and international application is yeast, milk-acid bacteria and genus bacillus in the microorganism of SOD research at present.
Summary of the invention
The method that the purpose of this invention is to provide a kind of radiation-induced raising radioresistant cocci superoxide-dismutase output.The strain classification of radioresistant cocci is Deinococcus radiodurans among the present invention, and preserving number is: ATCC NO.13939, bacterium numbering are R1.
Its step is as follows:
1) activation of bacterial classification:
On the TGY solid medium, substratum consists of peptone 5g/L with the radioresistant cocci streak inoculation, yeast extract 3g/L, glucose 1g/L cultivates 45-48h for 30-32 ℃, picking list colony inoculation in triangular flask shake-flask culture to logarithmic phase;
2) fermentation culture conditions:
Liquid seeds is pressed the 3-5% inoculum size, insert in the fermentation broth 30-32 ℃ of cultivation;
3) the radiation-induced condition of SOD:
1. the selection in thalline radiation-induced period: thalli growth is to 38-42h, and is promptly stable early stage, bacteria concentration＞10
8/ ml, the thalli morphology majority is diad, is at this moment to carry out radiation-induced best period;
2. illuminate condition: the yeast culture thing is placed Co
60The radiation platform, according to dosage rate 1000Gy/h carries out gammairradiation, and the total dose of radiation treatment that thalline is accepted is 200-1000Gy;
4) culture condition behind the thalline irradiation:
Thalline after radiation-induced need be cultivated 30-60min under 30-32 ℃ of condition, purpose is to make thalline after accepting external stimulus, synthetic in a large number SOD zymoprotein;
5) extraction of crude enzyme liquid:
1. the centrifugal 5000-10000rpm of bacterium liquid, 0-4 ℃, 10-15min collects the somatic cells precipitation.Use 50-60mM, the phosphate buffered saline buffer washing of pH7.0 three times, centrifugal collection somatic cells.
2. add three times of damping fluids by every gram thalline and make bacteria suspension, ultrasonication smudge cells 8-10min under the 600-700w power, every processing 2-3 second is 4-6 second at interval, and the bacterial cell disruption rate can reach more than 85%.
3. 10000-12000rpm, centrifugal 10-20min under the 0-4 ℃ of condition collects supernatant liquor, is the SOD crude enzyme liquid.Enzymic activity adopts pyrogallol autoxidation method to measure, and unit representation is every milligram of enzyme units U/mg alive that dry cell weight is contained.
The invention has the advantages that:
1) cultivate under the 30min condition at radiation-induced (1000Gy dosage) and recovery, the SOD enzymic activity of radioresistant cocci has reached 149.69U/mg, than having improved before the radiation treatment more than 75%, is 3 times of product SOD yeast enzymic activity;
2) this technical process is fairly simple, and cost is low;
3) radioresistant cocci belongs to prokaryotic organism, and the bacterial strain breeding is fast, and the fermentation culture cycle is shorter, is suitable for large-scale production.
Description of drawings
Accompanying drawing is the process flow sheet that radiation-induced method improves radioresistant cocci SOD content.
Embodiment
Used microorganism is radioresistant cocci (Deinococcus radiodurans R1) among the present invention, and the biological property of this bacterium is that with other species comparisons, it has superpower radiation hardness ability and DNA repair ability.Radioresistant cocci exceeds more than 200 times than colibacillary capability of resistance to radiation.The one of the main reasons of the superpower radiation hardness ability of this bacterium is high-load antioxidase SOD and Catalase.The active oxygen radical that the water radiolysis produces in the radiation-induced cell can be attacked biomacromolecule, causes the body oxidative damage.Therefore the superpower radiation hardness ability of radioresistant cocci and its SOD content have close getting in touch.Our SOD activity in the radioresistant cocci discovered is 4.8 times of intestinal bacteria (E.colli), is 2.3 times of yeast (Saccharomycescerevisiae).
Embodiment 1: with the radiation-induced 1L yeast culture of 1000Gy dosage thing, recovering to cultivate 30min is example.
1 with radioresistant cocci D.radiodurans R1 streak inoculation on the TGY solid medium, cultivate 45h for 32 ℃, the single colony inoculation of picking is in the 100ml nutrient solution, shake-flask culture 30h, thalli growth reach logarithmic phase (OD
600＞1.0).Liquid seeds by 5% inoculum size, is inserted in the 1L fermention medium, and revolution 200rpm is stirred in 32 ℃ of cultivations.Cultivate 42h to enumeration＞10
8/ ml.
2 are sub-packed in 1L yeast culture thing in the polyethylene tube (gamma-rays is penetrable), place Co
60Under the radiation irradiation, rate 1000Gy/h irradiation according to dosage 1 hour, the total dose that thalline is accepted radiation treatment is 1000Gy.
3 thalline after radiation-induced place and recover under 32 ℃ of conditions to cultivate 30min, and condition is the same.
4 with bacterium liquid centrifugal (5000rpm, 4 ℃) 10min, collects the somatic cells precipitation.With 50mM phosphoric acid buffer (pH7.0) washing three times, centrifugal collection somatic cells.Add 50mM in thalline and 1: 3 ratio of damping fluid, the phosphoric acid buffer of pH7.0 is made bacteria suspension, under low temperature (0-4 ℃) condition, and ultrasonication smudge cells under the 600w power (treatment time 8min), every processing 2 seconds 4 seconds at interval, the bacterial cell disruption rate reaches more than 85%.Then with bacterium liquid at 4 ℃, centrifugal 20min under the 12000rpm collects supernatant liquor, is the SOD crude enzyme liquid.The work of SOD enzyme reaches 149.69U/mg, and comparison has improved more than 75% according to (without radiation-induced group), is 3 times of yeast enzymic activity.
Embodiment 2: with the radiation-induced 1L yeast culture of 200Gy dosage thing, recovering to cultivate 30min is example.
1 with radioresistant cocci D.radiodurans R1 streak inoculation on the TGY solid medium, cultivate 48h for 30 ℃, the single colony inoculation of picking is in the triangular flask that the 100ml nutrient solution is housed, shake-flask culture 30h, thalli growth reach logarithmic phase (OD
600＞1.0).Liquid seeds by 5% inoculum size, is inserted in the 1L fermention medium, and revolution 200rpm is stirred in 32 ℃ of cultivations.Cultivate 40h to enumeration＞10
8/ ml.
2 are sub-packed in 1L yeast culture thing in the polyethylene tube (gamma-rays is penetrable), place Co
60Under the radiation irradiation, rate 1000Gy/h irradiation according to dosage 12 minutes, the total dose that thalline is accepted radiation treatment is 200Gy.
3 thalline after radiation-induced place and recover under 32 ℃ of conditions to cultivate 30min, and condition is the same.
4 with bacterium liquid centrifugal (10000rpm, 0-4 ℃) 10min, collects the somatic cells precipitation.With 60mM phosphoric acid buffer (pH7.0) washing three times, centrifugal collection somatic cells.Add 60mM in thalline and 1: 3 ratio of damping fluid, the phosphoric acid buffer of pH7.0 is made bacteria suspension, under low temperature (0-4 ℃) condition, and ultrasonication smudge cells under the 600w power (treatment time 8min), every processing 2 seconds 4 seconds at interval, the bacterial cell disruption rate reaches more than 85%.Then with bacterium liquid at 4 ℃, centrifugal 20min under the 12000rpm collects supernatant liquor, is the SOD crude enzyme liquid.The work of SOD enzyme reaches 122.41U/mg, and comparison has improved 43.59% according to (without radiation-induced group).
Embodiment 3: with the radiation-induced 1L yeast culture of 500Gy dosage thing, recovering to cultivate 60min is example.
1 with radioresistant cocci D.radiodurans R1 streak inoculation on the TGY solid medium, cultivate 48h for 32 ℃, the single colony inoculation of picking is in the triangular flask that contains the 100ml nutrient solution, shake-flask culture 30h, thalli growth reach logarithmic phase (OD
600＞1.0).Liquid seeds by 3% inoculum size, is inserted in the 1L fermention medium, and revolution 220rpm is stirred in 32 ℃ of cultivations.Cultivate 38h to enumeration＞10
8/ ml.
2 are sub-packed in 1L yeast culture thing in the polyethylene tube (gamma-rays is penetrable), place Co
60Under the radiation irradiation, rate 1000Gy/h irradiation according to dosage 30 minutes, the total dose that thalline is accepted radiation treatment is 500Gy.
3 thalline after radiation-induced place and recover under 32 ℃ of conditions to cultivate 60min, and condition is the same.
4 with bacterium liquid centrifugal (5000rpm, 0-4 ℃) 10min, collects the somatic cells precipitation.With 50mM phosphoric acid buffer (pH7.0) washing three times, centrifugal collection somatic cells.Add 50mM in thalline and 1: 3 ratio of damping fluid, the phosphoric acid buffer of pH7.0 is made bacteria suspension, under low temperature (0-4 ℃) condition, and ultrasonication smudge cells under the 700w power (treatment time 10min), every processing 3 seconds 6 seconds at interval, the bacterial cell disruption rate reaches more than 85%.Then with bacterium liquid at 4 ℃, centrifugal 20min under the 12000rpm collects supernatant liquor, is the SOD crude enzyme liquid.The work of SOD enzyme reaches 121.82U/mg, and comparison has improved 42.89% according to (without radiation-induced group).
Claims (1)
1. the method for a radiation-induced raising radioresistant cocci (Deinococcus radiodurans) ATCC NO.13939 superoxide-dismutase output is characterized in that its step is as follows:
1) activation of bacterial classification:
On the TGY solid medium, substratum consists of peptone 5g/L with the radioresistant cocci streak inoculation, yeast extract 3g/L, glucose 1g/L cultivates 45-48h for 30-32 ℃, picking list colony inoculation in triangular flask shake-flask culture to logarithmic phase;
2) fermentation culture conditions:
The inoculum size of above-mentioned seed liquor being pressed the 3-5% volume inserts in the fermentation broth, cultivates 38-42h for 30-32 ℃;
3) the radiation-induced condition of SOD:
1. the selection in thalline radiation-induced period: thalli growth is to 38-42h, and is promptly stable early stage, bacteria concentration＞10
8/ ml, the thalli morphology majority is diad, is at this moment to carry out radiation-induced best period;
2. illuminate condition: the yeast culture thing is placed Co
60The radiation platform, rate 1000Gy/h irradiation according to dosage, the total dose of radiation treatment that thalline is accepted is 200-1000Gy;
4) culture condition behind the thalline irradiation:
Thalline after radiation-induced need be cultivated 30-60min under 30-32 ℃ of condition, purpose is to make thalline after accepting external stimulus, synthetic in a large number SOD enzyme;
5) extraction of crude enzyme liquid:
1. the centrifugal 5000-10000rpm of bacterium liquid, 0-4 ℃, 10-15min collects the somatic cells precipitation, use 50-60mM, and the phosphate buffered saline buffer of pH7.0 washs three times, centrifugal collection somatic cells;
2. add three times of damping fluids by every gram thalline and make bacteria suspension, ultrasonication mycetocyte 8-10min under the 600-700w power, every processing 2-3 second is 4-6 second at interval, and the bacterial cell disruption rate can reach more than 85%;
3. 10000-12000rpm, centrifugal 10-20min under the 0-4 ℃ of condition collects supernatant liquor, is the SOD crude enzyme liquid.
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| CN 03116366 CN1200099C (en) | 2003-04-09 | 2003-04-09 | Method for increasing yield of superoxide dismutase of radioresistant coccus by radiation induction |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101245330B (en) * | 2008-02-03 | 2011-10-26 | 中国农业科学院生物技术研究所 | Gobi abnormal cocci |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101326960B (en) * | 2008-07-10 | 2010-12-15 | 浙江大学 | Use of radioresistant cocci in preparing feedstuff additive for improving fertility of sow |
| CN101671679B (en) * | 2009-04-30 | 2013-09-18 | 浙江大学 | Resistance-relevant gene and applications of radiation-resistant cocci |
| CN102277372A (en) * | 2011-07-22 | 2011-12-14 | 浙江大学 | Construction method and use of expression plasmid of canine gene CaIFN-alpha |
| CN108372307B (en) * | 2018-02-02 | 2021-01-08 | 浙江大学 | Preparation method of nanogold, nanogold and application |
| WO2020048836A1 (en) * | 2018-09-03 | 2020-03-12 | Arterra Bioscience S.R.L. | Industrial applications of plant cell extracts comprising sod enzymes of extremophilic micro-organisms |
| CN109294932A (en) * | 2018-10-22 | 2019-02-01 | 南京航空航天大学 | A kind of radiation hardness S. cervisiae and its abductive approach |
| CN109486681A (en) * | 2018-10-26 | 2019-03-19 | 深圳市太空科技南方研究院 | The preparation method and applications of bacterial lysate and D. radiodurans extract |
| CN117581993A (en) * | 2020-12-28 | 2024-02-23 | 郑州大学 | Lactic acid bacteria extract, preparation and antioxidation and anti-radiation application thereof |
| CN113025525A (en) * | 2021-03-22 | 2021-06-25 | 浙江大学 | Anti-oxidation protective agent based on deinococcus radiodurans and preparation method thereof |
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2003
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101245330B (en) * | 2008-02-03 | 2011-10-26 | 中国农业科学院生物技术研究所 | Gobi abnormal cocci |
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