CN101597598A - Preparation method of nitrite reductase and enzyme preparation thereof - Google Patents
Preparation method of nitrite reductase and enzyme preparation thereof Download PDFInfo
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- CN101597598A CN101597598A CNA2009100551200A CN200910055120A CN101597598A CN 101597598 A CN101597598 A CN 101597598A CN A2009100551200 A CNA2009100551200 A CN A2009100551200A CN 200910055120 A CN200910055120 A CN 200910055120A CN 101597598 A CN101597598 A CN 101597598A
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Abstract
The invention discloses a kind of preparation method of nitrite reductase, comprise the following steps: lactobacillus inoculum in the MRS substratum, cultivate and to win for bacterial classification, cultivate again s-generation bacterial classification; S-generation bacterial classification is received the MRS fermention medium, cultivate 30-48h; Above-mentioned fermented liquid is added sodium nitrite solution, cultivate 16-32h; Collecting precipitation; Add damping fluid in the precipitation, the fermented liquid precipitation is suspended fully, add lysozyme soln and ultrasonication again and obtain smudge cells liquid, collect supernatant liquor; Supernatant liquid filtering is collected filtered solution; Filtered solution is separated with the sephadex chromatography post, and gained is precipitated as nitrite reductase, adds 10-50% starch and promptly obtain described enzyme preparation in the gained precipitation.The zymin of utilizing this method to obtain can be used for removing the remnant nitrite in food, feed and the environment, improves the food safety quality, and the food poisoning that causes of prevention nitrite and cancer etc.
Description
Technical field
The present invention relates to a kind of separation purification method of milk-acid bacteria nitrite reductase, and the zymin of utilizing this method to obtain.
Background technology
Nitrite is one of main chemical virulence factor of China recent years food poisoning.After nitrite is absorbed and enters blood, can make Fe in the normal oxyphorase
2+Be oxidized to Fe
3+, make oxyphorase lose function of carrying oxygen, histanoxia occurs, even cause anoxia asphyxia.Nitrite still is the precursor substance of strong carcinogens nitrosamine, potential carcinogenic harm.It and protein decompose the reaction of intermediate product secondary amine and form nitrosamine, and the latter can bring out multiple cancer such as liver cancer, cancer of the stomach, esophagus cancer and pharynx cancer
At present, because trade effluent and irrigation of domestic sewage are used in a large amount of nitrogenous fertilizer and some areas of using, cause water Central Asia azotate pollution serious; Nitrite exceeds standard also more general in vegetables, grain, the feed.In addition, there is the nitrite problem that exceeds standard in traditional pickled vegetable, the various meat enema always.Totally 13013 parts of commercially available meat product samples of 21 provinces and cities of 1990 to 2003 pairs of China of Hu Pingcong find that average annual recall rate of nitrite and exceeding standard rate reach 52.6% and 26.2% unexpectedly.Average recall rate in various places and exceeding standard rate reach 61.4% and 27.3% unexpectedly over 14 years, always monitor disqualification rate near 30%.Illustrate that nitrate residue maintains quite high level in China's meat product.Detecting with exceeding standard of nitrite relates to 60% province, the whole nation in the meat product.On the other hand, find that nitrite content is higher than the low district of sending out in the DRINKING WATER of cancer district occurred frequently according to epidemiology survey.
Serious in view of the harm of nitrite in the food, people are striving to find the approach that reduces the food nitrite.Nitrite reductase is a catalysis nitrite reductive class of enzymes, and it can be converted into NH4 and H2O by the catalysis nitrite, thereby also can be used for nitrite in the decomposing food.From the food safety angle, the milk-acid bacteria nitrite reductase has broad application prospects aspect nitrite content exceeds standard in reducing food.
Summary of the invention
Technical problem to be solved by this invention provides a kind of separation purification method of milk-acid bacteria nitrite reductase, and the zymin of utilizing this method to obtain, this nitrite reductase preparation can be used for removing the remnant nitrite in food, feed and the environment, improve the food safety quality, and the food poisoning that causes of prevention nitrite and cancer etc.
Technical solution of the present invention: a kind of preparation method of nitrite reductase comprises the following steps:
(1) milk-acid bacteria is inoculated in the MRS substratum by the 2-5% inoculum size, under 25-30 ℃ of condition, cultivates 20-24h and win, again generation bacterial classification is received the MRS substratum with the 2-5% inoculum size, cultivate 20-24h at 25-30 ℃ and get s-generation bacterial classification for bacterial classification;
(2) s-generation bacterial classification is received the MRS fermention medium according to fermention medium volume 3%-8% inoculum size, cultivate 30-48h at 25-30 ℃;
(3) above-mentioned fermented liquid is regulated the pH value to 6.2-6.4 with the sodium hydroxide of 1mol/L, press 0.2--0.45mg/ml and add sodium nitrite solution.Cultivate 16--32h for 30 ℃;
(4) with above-mentioned fermented liquid through 4000-8000r/min, 4-10 ℃, centrifugal 5-20min collecting precipitation, the phosphate buffered saline buffer that in precipitation, adds pH6.5～7.5 of 4-5 times of volume, treat that precipitation suspends the back fully at 4-10 ℃, 4000-8000r/min, centrifugal 5-20min, collecting precipitation, and repeat twice with same procedure and regather precipitation;
(5) phosphate buffered saline buffer that adds 1-5ml, pH6.5-7.5 during every 50-100mg precipitates suspends the fermented liquid precipitation fully, and every again 100ml suspension adds 2ml-5ml, 0.05g/ml lysozyme soln, 35-50 ℃, 80-100r/m, handle 10-15h in the shaking table, use ultrasonic wave addi-tional crusher cell again, 4-10 ℃, 100-150W, 5-10min, mode is work 2-5s, intermittently 2-5s obtains smudge cells liquid, with 3000-8000r/min, 4-10 ℃ of centrifugal 5-30min collects supernatant liquor;
(6) supernatant liquor is collected filtered solution with the filtering with microporous membrane of 0.22-0.45um;
(7) filtered solution is separated with the sephadex chromatography post, wherein gel chromatography column is G-75, G-100 or G-150, with pH6.5-7.5 phosphate buffered saline buffer wash-out, collection has the active component of nitrite reductase, with this component dialysis tubing of packing into, handle 24h with PEG-20000 for 4-10 ℃ and promptly obtain nitrite reductase.
Wherein, the active detection of nitrite reductase is: get a small test tube, pack into the chromatography column parting liquid of smudge cells supernatant liquor of 3-5ml, add 20-25ul sodium nitrite solution (sodium nitrite solution concentration is 0.02g-0.05g/ml), mixing, detect content of sodium nitrite behind 30 ℃ of reaction 10-12h and change, content of sodium nitrite reduces 20-100% for active ingredient is arranged.
The measuring method of nitrite content adopts the hydrochloric acid-naphthalene-ethylenediamine method among the GB/T 5009.33-2003 that nitrite is detected.
Wherein, antalzyme activity is 10000-20000u/mg
Milk-acid bacteria of the present invention comprises plant lactobacillus (Lactobacillus plantarun), lactobacillus leichmannii (Lactobacillusleichmannii), pentose sheet ball (Pediococcuspentosaceus).Streptococcus uberis (Streptococcuslastic), butterfat hammer (Streptococcuscremoris), lactobacterium casei (Lactobacillus casei), streptococcus thermophilus (Streptococcus thermophilus), lactobacterium casei rhamnosyl subspecies milk-acid bacterias such as (Lactobacillus caseisubsp rhamnosus).
A kind of nitrite reductase goods prepare through the following steps and get:
(1) milk-acid bacteria is inoculated in the MRS substratum by the 2-5% inoculum size, under 25-30 ℃ of condition, cultivates 20-24h and win, again generation bacterial classification is received the MRS substratum with the 2-5% inoculum size, cultivate 20-24h at 25-30 ℃ and get s-generation bacterial classification for bacterial classification;
(2) s-generation bacterial classification is received the MRS fermention medium according to fermention medium volume 3%-8% inoculum size, cultivate 30-48h at 25-30 ℃;
(3) above-mentioned fermented liquid is regulated the pH value to 6.2-6.4 with the sodium hydroxide of 1mol/L, press 0.2--0.45mg/ml and add sodium nitrite solution.Cultivate 16--32h for 30 ℃;
(4) with above-mentioned fermented liquid through 4000-8000r/min, 4-10 ℃, centrifugal 5-20min collecting precipitation, the phosphate buffered saline buffer that in precipitation, adds pH6.5～7.5 of 4-5 times of volume, treat that precipitation suspends the back fully at 4-10 ℃, 4000-8000r/min, centrifugal 5-20min, collecting precipitation, and repeat twice with same procedure and regather precipitation;
(5) phosphate buffered saline buffer that adds 1-5ml, pH6.5-7.5 during every 50-100mg precipitates suspends the fermented liquid precipitation fully, and every again 100ml suspension adds 2ml-5ml, 0.05g/ml lysozyme soln, 35-50 ℃, 80-100r/m, handle 10-15h in the shaking table, use ultrasonic wave addi-tional crusher cell again, 4-10 ℃, 100-150W, 5-10min, mode is work 2-5s, intermittently 2-5s obtains smudge cells liquid, with 3000-8000r/min, 4-10 ℃ of centrifugal 5-30min collects supernatant liquor;
(6) supernatant liquor is collected filtered solution with the filtering with microporous membrane of 0.22-0.45um;
(7) filtered solution is separated with the sephadex chromatography post, wherein gel chromatography column is G-75, G-100 or G-150, with pH6.5-7.5 phosphate buffered saline buffer wash-out, collection has the active component of nitrite reductase, with this component dialysis tubing of packing into, handle 24h with PEG-20000 for 4-10 ℃ and promptly obtain nitrite reductase;
(8) in the gained precipitation, add 10-50% starch and promptly obtain described enzyme preparation.
Wherein, the active detection of nitrite reductase is: get a small test tube, pack into the chromatography column parting liquid of smudge cells supernatant liquor of 3-5ml, add 20-25ul sodium nitrite solution (sodium nitrite solution concentration is 0.02g-0.05g/ml), mixing, detect content of sodium nitrite behind 30 ℃ of reaction 10-12h and change, content of sodium nitrite reduces 20-100% for active ingredient is arranged.Active ingredient of the present invention is meant and reduces content of sodium nitrite 20-100%.
The measuring method of nitrite content adopts the hydrochloric acid-naphthalene-ethylenediamine method among the GB/T 5009.33-2003 that nitrite is detected.
Beneficial effect of the present invention: the enzyme preparation of isolating the nitrite reductase preparation by chromatography column has stability preferably.In low temperature environment, the activity of enzyme can be preserved the long period, and the zymin of utilizing this method to obtain can be used for removing the remnant nitrite in food, feed and the environment, improve the food safety quality, and the food poisoning that causes of prevention nitrite and cancer etc.
Embodiment
Below by embodiment the present invention is described in further detail, a kind of preparation method of nitrite reductase comprises the following steps:
(1) milk-acid bacteria is inoculated in the MRS substratum by the 2-5% inoculum size, under 25-30 ℃ of condition, cultivates 20-24h and win, again generation bacterial classification is received the MRS substratum with the 2-5% inoculum size, cultivate 20-24h at 25-30 ℃ and get s-generation bacterial classification for bacterial classification;
(2) s-generation bacterial classification is received the MRS fermention medium according to fermention medium volume 3%-8% inoculum size, cultivate 30-48h at 25-30 ℃;
(3) above-mentioned fermented liquid is regulated the pH value to 6.2-6.4 with the sodium hydroxide of 1mol/L, press 0.2--0.45mg/ml and add sodium nitrite solution.Cultivate 16--32h for 30 ℃;
(4) with above-mentioned fermented liquid through 4000-8000r/min, 4-10 ℃, centrifugal 5-20min collecting precipitation, the phosphate buffered saline buffer that in precipitation, adds pH6.5～7.5 of 4-5 times of volume, treat that precipitation suspends the back fully at 4-10 ℃, 4000-8000r/min, centrifugal 5-20min, collecting precipitation, and repeat twice with same procedure and regather precipitation;
(5) phosphate buffered saline buffer of adding 1-5ml pH6.5-7.5 in every 50-100mg precipitation suspends the fermented liquid precipitation fully, and every again 100ml suspension adds 2ml-5ml, 0.05g/ml lysozyme soln, 35-50 ℃, 80-100r/m handles 10-15h in the shaking table, use ultrasonic wave addi-tional crusher cell again, 4-10 ℃, 100-150W, 5-10min (work 2-5s, 2-5s intermittently).Obtain smudge cells liquid, with 3000-8000r/min, 4-10 ℃ of centrifugal 5-30min collects supernatant liquor;
(6) supernatant liquor is collected filtered solution with the filtering with microporous membrane of 0.22-0.45um;
(7) filtered solution is separated with the sephadex chromatography post, (gel chromatography column is G-75, G-100 or G-150), with pH6.5-7.5 phosphate buffered saline buffer wash-out, collection has the active component of nitrite reductase, with this component dialysis tubing of packing into, handle 24h. with PEG-20000 for 4-10 ℃ and promptly obtain nitrite reductase.
2. nitrite reductase goods is characterized in that preparing through the following steps and get:
(1) milk-acid bacteria is inoculated in the MRS substratum by the 2-5% inoculum size, under 25-30 ℃ of condition, cultivates 20-24h and win, again generation bacterial classification is received the MRS substratum with the 2-5% inoculum size, cultivate 20-24h at 25-30 ℃ and get s-generation bacterial classification for bacterial classification;
(2) s-generation bacterial classification is received the MRS fermention medium according to fermention medium volume 3%-8% inoculum size, cultivate 30-48h at 25-30 ℃;
(3) above-mentioned fermented liquid is regulated the pH value to 6.2-6.4 with the sodium hydroxide of 1mol/L, press 0.2--0.45mg/ml and add sodium nitrite solution.Cultivate 16--32h for 30 ℃;
(4) with above-mentioned fermented liquid through 4000-8000r/min, 4-10 ℃, centrifugal 5-20min collecting precipitation, the phosphate buffered saline buffer that in precipitation, adds pH6.5～7.5 of 4-5 times of volume, treat that precipitation suspends the back fully at 4-10 ℃, 4000-8000r/min, centrifugal 5-20min, collecting precipitation, and repeat twice with same procedure and regather precipitation;
(5) phosphate buffered saline buffer of adding 1-5ml pH6.5-7.5 in every 50-100mg precipitation suspends the fermented liquid precipitation fully, and every again 100ml suspension adds 2ml-5ml, 0.05g/ml lysozyme soln, 35-50 ℃, 80-100r/m handles 10-15h in the shaking table, use ultrasonic wave addi-tional crusher cell again, 4-10 ℃, 100-150W, 5-10min (work 2-5s, 2-5s intermittently).Obtain smudge cells liquid, with 3000-8000r/min, 4-10 ℃ of centrifugal 5-30min collects supernatant liquor;
(6) supernatant liquor is collected filtered solution with the filtering with microporous membrane of 0.22-0.45um;
(7) filtered solution is separated with the sephadex chromatography post, (gel chromatography column is G-75, G-100 or G-150), with pH6.5-7.5 phosphate buffered saline buffer wash-out, collection has the active component of nitrite reductase, with this component dialysis tubing of packing into, handle 24h. with PEG-20000 for 4-10 ℃ and promptly obtain nitrite reductase.
(8) in the gained precipitation, add 10-50% starch and promptly obtain described enzyme preparation.
Wherein, the active detection of nitrite reductase is: get a small test tube, pack into the chromatography column parting liquid of smudge cells supernatant liquor of 3-5ml, add 20-25ul sodium nitrite solution (sodium nitrite solution concentration is 0.02g-0.05g/ml), mixing, detect content of sodium nitrite behind 30 ℃ of reaction 10-12h and change, content of sodium nitrite reduces 20-100% for active ingredient is arranged.
The substratum that embodiment 1,2 adopts
(1) seed liquid nutrient medium: the old 10g of Trypsin, extractum carnis 10g, yeast extract paste 5g, ammonium citrate 2g, glucose 20g, KH2PO4 2g, sodium acetate 5g, tween 80 1mL, sal epsom 0.5g, manganous sulfate 0.2g, water 1000mL, 6.5,121 ℃ of 25min sterilizations of pH.
(2) liquid fermentation and culture: plant soybean protein 20g, ammonium citrate 2g, glucose 20g, KH
2PO
42g, sodium acetate 5g, tween 80 mL, sal epsom 0.5g, manganous sulfate 0.2g, 0.4-0.80.6mol/LKH
2PO
4Water 1000mL,
Embodiment 1
(1) milk-acid bacteria is inoculated in the MRS substratum by 3% inoculum size, under 30 ℃ of conditions, cultivates 20h and win, again generation bacterial classification is received the MRS substratum with 5% inoculum size, cultivate 20h at 30 ℃ and get s-generation bacterial classification for bacterial classification.
(2) s-generation bacterial classification is received the MRS fermention medium according to fermention medium volume 5% inoculum size, regulate medium pH value to 6.2, cultivate 30h at 30 ℃.
(3) above-mentioned fermented liquid pH value is adjusted to 6.2.Add sodium nitrite solution 0.2mg/ml, cultivate 16h for 30 ℃.
(4) with above-mentioned fermented liquid through 8000r/min, 4 ℃, centrifugal 5min collecting precipitation;
(5) after the phosphate buffered liquid precipitate of the pH6.5 of 4 times of volumes of adding suspended fully in precipitation, 4 ℃, the centrifugal 5min of 8000r/min, collecting precipitation repeated twice with same procedure again, regather precipitation.
(5) press the phosphate buffered saline buffer that adds 2mlpH6.5 in the 100mg precipitation, fermented liquid is precipitated complete suspension, add the lysozyme soln of 0.1g/ml again, 35 ℃, shaking table training 80r/m, 12h uses ultrasonic wave addi-tional crusher cell again, at temperature 1-10 ℃ 100W, 5, the 5s that wherein works, 5s intermittently.
(6) (5) are obtained smudge cells liquid, with 8000r/min, 1-10 ℃ of centrifugal 10min collects supernatant liquor.
(7) supernatant liquor is collected filtered solution with the filtering with microporous membrane of 0.45um,
(8) filtered solution is separated with the sephadex g-100 chromatography column,, collect and have the active component of nitrite reductase,, handle 24h. with PEG-20000 for 4 ℃ and promptly obtain nitrite reductase this component dialysis tubing of packing into pH6.5 phosphate buffered saline buffer wash-out.
(9) in the nitrite reductase that (8) obtain, add 30% starch and promptly obtain enzyme preparation.
Embodiment 2
(1) actication of culture: milk-acid bacteria is inoculated in the MRS substratum by 5% inoculum size, under 27 ℃ of conditions, cultivates 22h and win, again generation bacterial classification is received the MRS substratum with 5% inoculum size, cultivate 20h at 27 ℃ and get s-generation bacterial classification for bacterial classification.
(2) s-generation bacterial classification is received the MRS fermention medium according to fermention medium volume 3% inoculum size, regulate medium pH value to 6.8, cultivate 36h at 25 ℃.
(3) above-mentioned fermented liquid pH value is adjusted to 6.2.Add sodium nitrite solution 0.40mg/ml, cultivate 16h for 30 ℃.
(4) with above-mentioned fermented liquid through 4000r/min, 4 ℃, centrifugal 20min collecting precipitation;
(5) after the phosphate buffered saline buffer of the pH7.5 of 5 times of volumes of adding suspended precipitation fully in precipitation, 4 ℃, the centrifugal 20min of 4000r/min, collecting precipitation repeated twice with same procedure again, regather precipitation.
(5) press the phosphate buffered saline buffer that adds 3ml adding pH7.2 in the 100mg precipitation, the fermented liquid precipitation is suspended fully, add the lysozyme soln of 0.1g/ml again, 35 ℃, 100r/m, 12h in the shaking table uses ultrasonic wave at 10 ℃ of temperature, 150W again, 10min, (5s that wherein works, intermittently 5s) smudge cells.
(6) (5) are obtained smudge cells liquid, with 3000-8000r/min, 1-10 ℃ of centrifugal 5-30min collects supernatant liquor.
(7) supernatant liquor is collected filtered solution with the filtering with microporous membrane of 0.22-0.45um,
(8) filtered solution is separated with sephadex G-150 chromatography column, with pH6.5 phosphate buffered saline buffer wash-out, collection has the active component of nitrite reductase, with this component dialysis tubing of packing into, handles 24h. with PEG-20000 for 10 ℃ and promptly obtains nitrite reductase.
(9) in the nitrite reductase that (8) obtain, add 50% starch and promptly obtain enzyme preparation.
Described content only is the basic explanation of the present invention under conceiving, and according to any equivalent transformation that technical scheme of the present invention is done, all should belong to protection scope of the present invention.
Claims (2)
1. a preparation method of nitrite reductase comprises the following steps:
(1) milk-acid bacteria is inoculated in the MRS substratum by the 2-5% inoculum size, under 25-30 ℃ of condition, cultivates 20-24h and win, again generation bacterial classification is received the MRS substratum with the 2-5% inoculum size, cultivate 20-24h at 25-30 ℃ and get s-generation bacterial classification for bacterial classification;
(2) s-generation bacterial classification is received the MRS fermention medium according to fermention medium volume 3%-8% inoculum size, cultivate 30-48h at 25-30 ℃;
(3) above-mentioned fermented liquid is regulated the pH value to 6.2-6.4 with the sodium hydroxide of 1mol/L, press 0.2--0.45mg/ml and add sodium nitrite solution.Cultivate 16--32h for 30 ℃;
(4) with above-mentioned fermented liquid through 4000-8000r/min, 4-10 ℃, centrifugal 5-20min collecting precipitation, the phosphate buffered saline buffer that in precipitation, adds pH6.5～7.5 of 4-5 times of volume, treat that precipitation suspends the back fully at 4-10 ℃, 4000-8000r/min, centrifugal 5-20min, collecting precipitation, and repeat twice with same procedure and regather precipitation;
(5) phosphate buffered saline buffer that adds 1-5ml, pH6.5-7.5 during every 50-100mg precipitates suspends the fermented liquid precipitation fully, and every again 100ml suspension adds 2ml-5ml, 0.05g/ml lysozyme soln, 35-50 ℃, 80-100r/m, handle 10-15h in the shaking table, use ultrasonic wave addi-tional crusher cell again, 4-10 ℃, 100-150W, 5-10min, mode is work 2-5s, intermittently 2-5s obtains smudge cells liquid, with 3000-8000r/min, 4-10 ℃ of centrifugal 5-30min collects supernatant liquor;
(6) supernatant liquor is collected filtered solution with the filtering with microporous membrane of 0.22-0.45um;
(7) filtered solution is separated with the sephadex chromatography post, wherein gel chromatography column is G-75, G-100 or G-150, with pH6.5-7.5 phosphate buffered saline buffer wash-out, collection has the active component of nitrite reductase, with this component dialysis tubing of packing into, handle 24h with PEG-20000 for 4-10 ℃ and promptly obtain nitrite reductase.
2. nitrite reductase goods is characterized in that preparing through the following steps and get:
(1) milk-acid bacteria is inoculated in the MRS substratum by the 2-5% inoculum size, under 25-30 ℃ of condition, cultivates 20-24h and win, again generation bacterial classification is received the MRS substratum with the 2-5% inoculum size, cultivate 20-24h at 25-30 ℃ and get s-generation bacterial classification for bacterial classification;
(2) s-generation bacterial classification is received the MRS fermention medium according to fermention medium volume 3%-8% inoculum size, cultivate 30-48h at 25-30 ℃;
(3) above-mentioned fermented liquid is regulated the pH value to 6.2-6.4 with the sodium hydroxide of 1mol/L, press 0.2--0.45mg/ml and add sodium nitrite solution.Cultivate 16--32h for 30 ℃;
(4) with above-mentioned fermented liquid through 4000-8000r/min, 4-10 ℃, centrifugal 5-20min collecting precipitation, the phosphate buffered saline buffer that in precipitation, adds pH6.5～7.5 of 4-5 times of volume, treat that precipitation suspends the back fully at 4-10 ℃, 4000-8000r/min, centrifugal 5-20min, collecting precipitation, and repeat twice with same procedure and regather precipitation;
(5) phosphate buffered saline buffer that adds 1-5ml, pH6.5-7.5 during every 50-100mg precipitates suspends the fermented liquid precipitation fully, and every again 100ml suspension adds 2ml-5ml, 0.05g/ml lysozyme soln, 35-50 ℃, 80-100r/m, handle 10-15h in the shaking table, use ultrasonic wave addi-tional crusher cell again, 4-10 ℃, 100-150W, 5-10min, mode is work 2-5s, intermittently 2-5s obtains smudge cells liquid, with 3000-8000r/min, 4-10 ℃ of centrifugal 5-30min collects supernatant liquor;
(6) supernatant liquor is collected filtered solution with the filtering with microporous membrane of 0.22-0.45um;
(7) filtered solution is separated with the sephadex chromatography post, wherein gel chromatography column is G-75, G-100 or G-150, with pH6.5-7.5 phosphate buffered saline buffer wash-out, collection has the active component of nitrite reductase, with this component dialysis tubing of packing into, handle 24h with PEG-20000 for 4-10 ℃ and promptly obtain nitrite reductase;
(8) in the gained precipitation, add 10-50% starch and promptly obtain described enzyme preparation.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286438A (en) * | 2011-07-25 | 2011-12-21 | 上海应用技术学院 | Method for producing nitrite reductase from lactobacillus |
| CN102362637A (en) * | 2011-09-30 | 2012-02-29 | 邓毛程 | Method for removing nitrite in edible bird's nest by using microbial viable bacteria suspension and/or crude enzyme liquid and application |
| CN102742733A (en) * | 2012-07-19 | 2012-10-24 | 云南师范大学 | Application method of nitrite reductase in livestock and poultry farming |
| 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 |
| WO2017155471A1 (en) * | 2016-03-11 | 2017-09-14 | Lim Kah Meng | Edible bird's nest extract and method of extraction |
| CN109679930A (en) * | 2019-02-15 | 2019-04-26 | 大连大学 | A kind of fermentation process producing nitrite reductase |
-
2009
- 2009-07-21 CN CNA2009100551200A patent/CN101597598A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286438A (en) * | 2011-07-25 | 2011-12-21 | 上海应用技术学院 | Method for producing nitrite reductase from lactobacillus |
| CN102362637A (en) * | 2011-09-30 | 2012-02-29 | 邓毛程 | Method for removing nitrite in edible bird's nest by using microbial viable bacteria suspension and/or crude enzyme liquid and application |
| CN102742733A (en) * | 2012-07-19 | 2012-10-24 | 云南师范大学 | Application method of nitrite reductase in livestock and poultry farming |
| WO2017155471A1 (en) * | 2016-03-11 | 2017-09-14 | Lim Kah Meng | Edible bird's nest extract and method of extraction |
| US10918674B2 (en) | 2016-03-11 | 2021-02-16 | Kah Meng LIM | Edible bird's nest extract and method of extraction |
| US11672833B2 (en) | 2016-03-11 | 2023-06-13 | Kah Meng LIM | Edible bird's nest extract and method of extraction |
| 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 |
| CN109679930A (en) * | 2019-02-15 | 2019-04-26 | 大连大学 | A kind of fermentation process producing nitrite reductase |
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