CN101230337A - Preparation of nitrite reductase and method for preparing nitrite reductase preparation - Google Patents
Preparation of nitrite reductase and method for preparing nitrite reductase preparation Download PDFInfo
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- CN101230337A CN101230337A CNA2008101010838A CN200810101083A CN101230337A CN 101230337 A CN101230337 A CN 101230337A CN A2008101010838 A CNA2008101010838 A CN A2008101010838A CN 200810101083 A CN200810101083 A CN 200810101083A CN 101230337 A CN101230337 A CN 101230337A
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- nitrite reductase
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Abstract
The invention discloses a preparation method of preparing nitrite reductase and preparing the preparation of the nitrite reductase, which belongs to the preparing technology field of biologic enzyme and enzyme preparation. Bacillus megatherium which can produce nitrite reductase is activated and prepared into seed, the obtained seed culture fluid is inoculate into a liquid ferment substrate by 1-10 percent of inoculation dosage; the initial pH value is 5.0-7.5, at 25-40 DEG C and 100-240r/min of rotation speed, after 20-36 hours of constant temperature cultivation, bacterial suspension is produced. Re-suspending the bacterial suspension by phosphate buffer can produce thalli. After the thalli is smashed by ultrasonic, the supernatant fluid is frozen and centrifugated, and then the rough enzyme solution is obtained. Nitrite reductases of different consistences and purities can be obtained after the rough enzyme solution is separated and purified. Liquid enzyme preparation can be prepared after enzyme protecting solute is added in the nitrite reductase. The enzyme activity of nitrite reductase preparation produced by using the invention is 4000-8000U/mL. The preparing method is simple, the production period is short, and public food safety can be maintained.
Description
Technical field
The invention belongs to the preparing technical field of biological enzyme and zymin, in particular, adopt fermentation technique to prepare the preparation of a kind of nitrite reductase that can reduce nitrite and the preparation method of nitrite reductase preparation.
Background technology
The nitrite foodstuff additive that permission is used as the state food assanation are extensively used in meat product processing.It and myohaemoglobin react, and generate nitrosomyoglobin, and pinkiness gives meat product tempting color and luster, i.e. color development effect; Can suppress simultaneously the breeding of microorganism, especially suppress the breeding of meat poison shuttle shape bud pole bacterium, this bacterium can produce botulinus toxin, causes food poisoning, i.e. preservative activity; Can also make meat product have elasticity, good mouthfeel, the peculiar smell of elimination raw meat improves product quality.But nitrite also is the carcinogenic substance of generally acknowledging, human body is had very big injury.Countries in the world all are being devoted to the nitrite surrogate and are being reduced its residual research now.But also do not find a kind of ideal so far, can substitute the material of nitrite fully, the measure that reduces nitrate residue in the meat product all is being devoted to study now in countries in the world.The nitrite reductase that utilizes microbial fermentation to produce can well degrading nitrite, reduces that it is residual, is processed with very application prospects at meat product.
Summary of the invention
The purpose of this invention is to provide a kind of preparation of nitrite reductase and the preparation method of nitrite reductase preparation.
The preparation of described nitrite reductase is to adopt the fermentation technique preparation, and the preparation method may further comprise the steps:
(1) bacillus megaterium (the Bacillus megaterium MPF-906) dry bacterial powder that will produce nitrite reductase is dissolved in the physiological saline, be inoculated in the inclined-plane seed culture medium, obtain seed culture fluid, wherein bacillus megaterium dry bacterial powder: physiological saline=1: 2-5 (weight ratio);
(2) seed culture fluid that step (1) is obtained is inoculated in the liquid fermentation medium with the inoculum size of 1-10%, and initial pH is 5.0-7.5, and constant temperature culture 20-36h under 25-40 ℃, rotating speed 100-240r/min obtains bacteria suspension;
(3) collection of somatic cells, the bacteria suspension shake flask fermentation liquid that step (2) is obtained is 10000-20000 * g, 1-4 ℃ of frozen centrifugation 10-20min in eccentricity, the precipitation thalline suspends again with phosphoric acid buffer, and then the centrifugal thalline that obtains;
(4) ultrasonic disruption of somatic cells with the somatic cells that extracts, is pressed 1000mL nutrient solution somatic cells and is added 50-100mM, the 100mL phosphoric acid buffer of pH6.5-7.5 (is to prepare with dipotassium hydrogen phosphate and potassium primary phosphate, ratio is controlled with the pH value, and people native to a place is all known in the industry), ultrasonic disruption, the ultrasonic wave condition is 180-220w, ultrasonic 2-10s, 9-15s intermittently, 60-120 time, in eccentricity is 26000 * g, 4 ℃ of frozen centrifugation 15min, and supernatant liquor is crude enzyme liquid;
(5) with the crude enzyme liquid separation and Extraction, obtain nitrite reductase.
Described crude enzyme liquid separation and Extraction also comprises following two steps:
1. ammonium sulfate precipitation: accurately measuring crude enzyme liquid 100mL, add ammonium sulfate to the 10-30% saturation ratio, slowly stir thorough mixing, leave standstill 2-6h under 4 ℃, is 20000-30000 * g in eccentricity, and 4 ℃ of centrifugal 10-30min down discard precipitation, leave and take supernatant liquor; In supernatant liquor, continue to add the ammonium sulfate powder to saturation ratio 50-80%, slowly stir fully mixed, 4 ℃ of following standing over night, in eccentricity is 20000-30000 * g, behind 4 ℃ of following centrifugal 10-30min, and collecting precipitation, be dissolved in 50mM, in the 20mL phosphoric acid buffer of pH6.5, deposit under 4 ℃, it is standby to do dialysis;
2. enzyme liquid dialysis and concentrating:, every 6-8h exchange buffering liquid once, after the dialysis, be concentrated into 4000-8000IU/mL with the polyoxyethylene glycol powder with the ammonium sulfate precipitation gained enzyme liquid 24-36h that in the phosphoric acid buffer of pH6.5-7.5,50-100mM, dialyses.
Described inclined-plane seed culture medium is nutrient broth powder 1-2wt%, agar 1.8-2wt%, pH6.5-7.5; Or glucose 15g, NaNO
21g, K
2HPO
41.35g, KH
2PO
40.7g, MgSO
47H
2O 0.2g,, agar 20g is settled to 1L, and regulating pH is 7,121 ℃ of sterilization 20min.
Described fermentative medium formula: glucose 1-2wt%, peptone 0.1wt%, sodium-chlor 0.1-0.5wt%, dipotassium hydrogen phosphate 0.1-0.3wt%, potassium primary phosphate 0.05-0.2wt%, MgSO
47H
2O 0.02-0.06wt%, MnSO
4H
2O 0.001-0.003wt%, CaCl
22H
2O 0.002-0.004wt%, pH 6.5-7.5.
The preparation of described nitrite reductase zymin
Add sodium-chlor and sugar in concentrated nitrite reductase liquid, make its final quality concentration sodium-chlor 10-20%, sucrose 5-20% makes the nitrite reductase liquid enzyme formulation.
The active mensuration of described nitrite reductase
(1) foundation of nitrite typical curve
According to GB/T 5009.33-2003 standard test, measure with the general UV-1900 model twin-beam ultraviolet spectrophotometer of analysing instrument plant's production in Beijing, be X-coordinate with concentration, with the optical density value ordinate zou drawing standard curve.
(2) the active mensuration of nitrite reductase
With reference to the method for Rosa (Rosa M, et al, 2001), make up the activity determination method of nitrite reductase, measure enzyme reaction system alive totally 250 μ L:0.1M phosphate buffered saline buffers (pH6.5) 125 μ L, 0.1MNaCl 15 μ L, 0.1M NO
212.5 μ L, 0.1M MV 7.5 μ L, 0.1M Na
2S
2O
440 μ L, enzyme liquid 50 μ L.React 10min in the 20-36 ℃ of water-bath, the thermal agitation termination reaction is got 10 μ L and is added the Griess reagent colour developing, in the variation of 480-620nm colorimetric method for determining nitrite.
According to typical curve, the enzyme that can draw nitrite reductase is lived.
The enzyme definition of living: per minute reduces the needed enzyme amount of 1nmoL nitrite and is defined as an enzyme activity unit.Enzyme unit alive: U/mL nutrient solution.
The bacterial classification that uses among the present invention derives from China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation, commercially available acquisition.Activation and growth conditions are undertaken by the explanation that culture presevation unit provides.
Effect intentionally of the present invention is:
1. use the crude enzyme liquid or the purification liquid of the nitrite reductase of the present invention's acquisition, draw the enzyme 4000-8000U of being alive, enzymatic activity recovery reaches 70-95%.
2. because the nitrite reductase that the used bacterial classification of the present invention produces has high-temperature stability, under 70-80 ℃, still keep very high enzyme and live.Therefore might use it for meat products processing, greatly reduce the residual quantity of nitrite, guarantee the edible safety of meat product, also will promote meat product processing enterprise to improve the quality, have important social benefit.The present invention is with common microorganism culturing device and Experiment on Microbiology means production zymin, and method is easy, (4-5 days) with short production cycle.Be used for reducing the harm of nitrite simultaneously, reduce human diseases and produce, safeguard public food safety, guaranteed people's physical and mental health, have important social benefit human body.
Embodiment
The invention provides a kind of preparation of nitrite reductase and the preparation method of nitrite reductase preparation, further specify the present invention with example below, but content of the present invention is not limited thereto.
Embodiment 1
1. medium preparation
(1) inclined-plane seed culture based formulas: nutrient broth powder 1-2wt%, agar 1.8-2wt%, pH6.5-7.5.
(2) fermentative medium formula: glucose 1-2wt%, peptone 0.1wt%, sodium-chlor 0.1-0.5wt%, dipotassium hydrogen phosphate 0.1-0.3wt%, potassium primary phosphate 0.05-0.2wt%, MgSO
47H
2O 0.02-0.06wt%, MnSO
4H
2O 0.001-0.003wt%, CaCl
22H
2O 0.002-0.004wt%, pH6.5-7.5.
2. the weight ratio of bacillus megaterium lyophilized powder with 1: 4 is dissolved in the physiological saline,, is inoculated on the inclined-plane seed culture medium, cultivate 20h for 37 ℃, obtain activated inclined plane with transfering loop picking one ring; The activatory slant strains is washed under aseptic condition with the 10mL fermention medium, insert 250mL and shake bottle, every bottled 100mL seed culture medium, every inclined-plane inoculation 2-4 is individual, constant temperature culture 20-24h under 25～30 ℃, rotating speed 100-240r/min obtains liquid seeds liquid.
3. the aforesaid liquid seed liquor is transferred to 250mL respectively and shakes in the bottle, every bottled fermention medium 100mL, the perhaps bottle of 500mL dress 200mL substratum, constant temperature culture 20-24h under 25～30 ℃, rotating speed 100-240r/min obtains the nutrient solution somatic cells.
4. the collection of somatic cells, (20000 * g), the precipitation thalline suspends with phosphoric acid buffer the bacteria suspension shake flask fermentation liquid that step 3 is obtained again, and then the centrifugal thalline that obtains at 1-4 ℃ of frozen centrifugation 10min.
5. the somatic cells that step 4 is extracted, add 100mL, 50-70mM by 1000mL nutrient solution somatic cells, sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of pH6.5, ultrasonic disruption, ultrasonic wave condition are 180-220w, ultrasonic 2-10s, 9-15s intermittently, 60-120 time, 4 ℃ of frozen centrifugation 15min, 26000 * g, supernatant liquor is crude enzyme liquid.
6. accurately measure the crude enzyme liquid 100mL that step 5 is obtained, add ammonium sulfate, slowly stir thorough mixing, leave standstill 2-6h under 4 ℃ to the 10-30% saturation ratio, 30000 * g, 4 ℃ of centrifugal 10min down discard precipitation and leave and take supernatant liquor.In supernatant liquor, continue to add the ammonium sulfate powder to saturation ratio 50-80%, slowly stir fully mixed, 4 ℃ of following standing over night, 20000 * g, behind 4 ℃ of centrifugal 20min, collecting precipitation is dissolved in 20mL, 50mM, in sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of pH6.5, deposit under 4 ℃, it is standby to do dialysis.
With an amount of enzyme liquid dialysis tubing of packing into, the 24-28h that dialyses in the 50-70mM of pH6.5-7.5 phosphoric acid buffer changes a damping fluid every 6-8h.Enzyme liquid in the dialysis tubing is concentrated to the 4000-8000IU/mL of institute.
7. add sodium-chlor and sugar in concentrated nitrite reductase liquid, make its final quality concentration sodium-chlor 10%, sucrose 10% is made the nitrite reductase liquid enzyme formulation.
Above-mentioned nitrite reductase liquid is carried out determination of activity, draw the enzyme 4000-8000U of being alive, enzymatic activity recovery reaches 70-95%.
Embodiment 2
1. obtain the thalline preparation
(1) inclined-plane seed culture based formulas: glucose 15.0g, NaNO
21.0g, K
2HPO
41.35g, KH
2PO
40.70g, MgSO
47H
2O 0.20g, agar 20.0g is settled to 1L, and regulating pH is 7.0,121 ℃ of sterilization 20min.
(2). fermentative medium formula: glucose 15.0, peptone 2.0g, Na
2HPO
41.35g, KH
2PO
40.70g, MgSO
47H
2O 0.10g, NaCl 1.0g, CaCl
22H
2O 0.02g, MnSO
4H
2O 0.01g, NaNO
2, 0.138g is settled to 1L, and regulating pH is 7.0,121 ℃ of sterilization 20min.
2. the weight ratio of bacillus megaterium lyophilized powder with 1: 2 is dissolved in the physiological saline,, is inoculated on the slant medium, cultivate 20h for 37 ℃, obtain activated inclined plane with transfering loop picking one ring.Be inoculated in 250mL with transfering loop picking 3-4 ring again and shake in the bottle, every bottled seed culture medium 50mL, at 30 ~ 35 ℃, constant temperature culture 24h under the rotating speed 100-240r/min obtains seed culture fluid.
3. seed culture fluid is transferred in the 250mL liquid fermentation medium, every bottled 100mL, the perhaps bottle of 500mL dress 200mL substratum, constant temperature culture 24-30h under 30 ~ 35 ℃, rotating speed 100-240r/min obtains the nutrient solution somatic cells.
4. the collection of somatic cells, (10000 * g), the precipitation thalline suspends with phosphoric acid buffer the bacteria suspension shake flask fermentation liquid that step 3 is obtained again, and then the centrifugal thalline that obtains at 1-4 ℃ of frozen centrifugation 20min.
5. the somatic cells that step 4 is extracted adds 100mL, 70-90mM by 1000mL nutrient solution somatic cells, sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of pH6.5-7.0, ultrasonic disruption, ultrasonic wave condition are 180-220w, ultrasonic 2-10s, 9-15s intermittently, 60-120 time.4 ℃ of frozen centrifugation 15min, 26000 * g, supernatant liquor is crude enzyme liquid.
6. accurately measure the crude enzyme liquid 100mL that step 5 is obtained, add ammonium sulfate, slowly stir thorough mixing, leave standstill 2-6h under 4 ℃ to the 10-30% saturation ratio, 30000 * g, 4 ℃ of centrifugal 10min down,, discard precipitation and leave and take supernatant liquor.In supernatant liquor, continue to add the ammonium sulfate powder to saturation ratio 50-80%, slowly stir fully mixed, 4 ℃ of following standing over night, 20000 * g, behind 4 ℃ of centrifugal 20min, collecting precipitation is dissolved in 20mL, 50mM, in sodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution of pH6.5, deposit under 4 ℃, it is standby to do dialysis.
With an amount of enzyme liquid dialysis tubing of packing into, the 28-32h that dialyses in the 70-80mM of pH6.5-7.5 phosphoric acid buffer changes a damping fluid every 6-8h.Enzyme liquid in the dialysis tubing is concentrated to needed concentration.
7. add sodium-chlor in concentrated nitrite reductase enzyme liquid, make its final quality concentration sodium-chlor 20%, sucrose 5% is made the nitrite reductase liquid enzyme formulation.
Above-mentioned nitrite reductase liquid is carried out determination of activity, draw the enzyme 4000-8000U of being alive, enzymatic activity recovery reaches 70-95%.
It should be noted that at last, above embodiment only is used to illustrate technical scheme of the present invention and is unrestricted, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can make amendment or replace technical scheme of the present invention, and not breaking away from the spirit and scope of the present invention, it all should be encompassed within the claim scope of the present invention.
Claims (5)
1. the preparation method of a nitrite reductase is characterized in that, the preparation of described nitrite reductase is to adopt the fermentation technique preparation, and the preparation method may further comprise the steps:
(1) bacillus megaterium (Bacillus megateriumMPF-906) dry bacterial powder that will produce nitrite reductase is dissolved in the physiological saline, be inoculated in the inclined-plane seed culture medium, obtain activated spawn, wherein bacillus megaterium dry bacterial powder: physiological saline=1: 2-5 (weight ratio); Again this activated spawn is inoculated in and obtains seed culture fluid in the seed culture medium;
(2) seed culture fluid that step (1) is obtained is inoculated in the liquid fermentation medium with the inoculum size of 1-10%, and initial pH is 5.0-7.5, and constant temperature culture 20-36h under 25-40 ℃, rotating speed 100-240r/min obtains bacteria suspension;
(3) collection of somatic cells, the bacteria suspension shake flask fermentation liquid that step (2) is obtained is 10000-20000 * g, 1-4 ℃ of frozen centrifugation 10-20min in eccentricity, the precipitation thalline suspends again with phosphoric acid buffer, and then the centrifugal thalline that obtains;
(4) ultrasonic disruption of somatic cells, with the somatic cells that extracts, press 1000mL nutrient solution somatic cells and add 50-100mM, the 100mL phosphoric acid buffer of pH6.5-7.5, ultrasonic disruption, the ultrasonic wave condition is 180-220w, ultrasonic 2-10s, 9-15s intermittently, 60-120 time, in eccentricity is 26000 * g, 4 ℃ of frozen centrifugation 15min, and supernatant liquor is crude enzyme liquid;
(5) with the crude enzyme liquid separation and Extraction, obtain nitrite reductase.
2. according to the preparation method of the described nitrite reductase of claim 1, it is characterized in that described crude enzyme liquid separation and Extraction also comprises following two steps:
1. ammonium sulfate precipitation: accurately measure crude enzyme liquid 100mL, add ammonium sulfate, slowly stir thorough mixing to the 10-30wt% saturation ratio, leaving standstill 2-6h under 4 ℃, is 20000-30000 * g in eccentricity, 4 ℃ of following centrifugal 10-30min, discard precipitation, leave and take supernatant liquor; In supernatant liquor, continue to add the ammonium sulfate powder to saturation ratio 50-80%, slowly stir fully mixed, 4 ℃ of following standing over night, in eccentricity is 20000-30000 * g, behind 4 ℃ of following centrifugal 10-30min, and collecting precipitation, be dissolved in 50mM, in the 20mL phosphoric acid buffer of pH6.5, deposit under 4 ℃, it is standby to do dialysis;
2. enzyme liquid dialysis and concentrating:, every 6-8h exchange buffering liquid once, after the dialysis, be concentrated to 4000-8000IU/mL with the polyoxyethylene glycol powder with the ammonium sulfate precipitation gained enzyme liquid 24-36h that in the phosphoric acid buffer of pH6.5-7.5,50-100mM, dialyses.
3. according to the preparation method of the described nitrite reductase of claim 1, it is characterized in that described slant medium is nutrient broth powder 1-2wt%, agar 1.8-2wt%, pH6.5-7.5; Or be glucose 15.0g, NaNO
21.0g, K
2HPO
41.35g, KH
2PO
40.70g, MgSO
47H
2O 0.20g, agar 20.0g is settled to 1L, and regulating pH is 7.0,121 ℃ of sterilization 20min.
4. according to the preparation method of the described nitrite reductase of claim 1, it is characterized in that described fermentative medium formula: glucose 1-2wt%, peptone 0.1wt%, sodium-chlor 0.1-0.5wt%, dipotassium hydrogen phosphate 0.1-0.3wt%, potassium primary phosphate 0.05-0.2wt%, MgSO
47H
2O 0.02-0.06wt%, MnSO
4H
2O0.001-0.003wt%, CaCl
22H
2O 0.002-0.004wt%, pH6.5-7.5.
5. the preparation method of a nitrite reductase zymin, it is characterized in that, sodium-chlor and sucrose are added in being prepared as of described nitrite reductase zymin in concentrated nitrite reductase liquid, make its final quality concentration sodium-chlor 10-20%, sucrose 5-20% makes the nitrite reductase liquid enzyme formulation.
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| CNA2008101010838A CN101230337A (en) | 2008-02-28 | 2008-02-28 | Preparation of nitrite reductase and method for preparing nitrite reductase preparation |
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| CNA2008101010838A CN101230337A (en) | 2008-02-28 | 2008-02-28 | Preparation of nitrite reductase and method for preparing nitrite reductase preparation |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914505A (en) * | 2010-06-24 | 2010-12-15 | 中国农业大学 | Method for producing nitrite reductase through fermentation |
| CN101979651A (en) * | 2010-11-08 | 2011-02-23 | 中国农业大学 | Method for synthesizing nitrosohaemoglobin by enzyme method |
| CN102495011A (en) * | 2011-11-24 | 2012-06-13 | 上海应用技术学院 | Method for determining activity of bacterial nitrite reductase |
| CN106467907A (en) * | 2016-10-11 | 2017-03-01 | 南京财经大学 | A kind of method preparing nitrite reductase from Flammulina velutiper (Fr.) Sing |
| CN113637651A (en) * | 2021-08-16 | 2021-11-12 | 武汉科技大学 | Preparation method and application of nitrite reductase |
-
2008
- 2008-02-28 CN CNA2008101010838A patent/CN101230337A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101914505A (en) * | 2010-06-24 | 2010-12-15 | 中国农业大学 | Method for producing nitrite reductase through fermentation |
| CN101914505B (en) * | 2010-06-24 | 2013-01-23 | 中国农业大学 | Method for producing nitrite reductase through fermentation |
| CN101979651A (en) * | 2010-11-08 | 2011-02-23 | 中国农业大学 | Method for synthesizing nitrosohaemoglobin by enzyme method |
| CN101979651B (en) * | 2010-11-08 | 2012-11-28 | 中国农业大学 | Method for synthesizing nitrosohaemoglobin by enzyme method |
| CN102495011A (en) * | 2011-11-24 | 2012-06-13 | 上海应用技术学院 | Method for determining activity of bacterial nitrite reductase |
| CN102495011B (en) * | 2011-11-24 | 2013-08-14 | 上海应用技术学院 | Method for determining activity of bacterial nitrite reductase |
| CN106467907A (en) * | 2016-10-11 | 2017-03-01 | 南京财经大学 | A kind of method preparing nitrite reductase from Flammulina velutiper (Fr.) Sing |
| CN113637651A (en) * | 2021-08-16 | 2021-11-12 | 武汉科技大学 | Preparation method and application of nitrite reductase |
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Open date: 20080730 |