CN101732819B - Organic phosphorus degrading enzyme preparation and preparation method thereof - Google Patents
Organic phosphorus degrading enzyme preparation and preparation method thereof Download PDFInfo
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- CN101732819B CN101732819B CN 200810228798 CN200810228798A CN101732819B CN 101732819 B CN101732819 B CN 101732819B CN 200810228798 CN200810228798 CN 200810228798 CN 200810228798 A CN200810228798 A CN 200810228798A CN 101732819 B CN101732819 B CN 101732819B
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- organic phosphorus
- degrading enzyme
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Abstract
The invention relates to microbe technology, in particular to a degrading enzyme preparation for degrading residual organic phosphorus on vegetables and fruits and a preparation method thereof. The invention particularly relates to organic phosphorus degrading enzyme preparation obtained by fermenting Methylobacterium. The preparation process comprises the following steps: after fermenting the inoculated strain, centrifugally collecting thalli, and breaking thalli to obtain the organic phosphorus degrading enzyme preparation. The organic phosphorus pesticide degrading enzyme preparation can degrade residual organic phosphorus pesticides on vegetables and fruits.
Description
Technical field
The present invention relates to microbial technique, residual organic phosphorus degrading enzyme preparation and preparation method thereof on a kind of specifically vegetable and fruit that is used to degrade.
Background technology
Organophosphorus pesticide is the primary categories in the agricultural chemicals, in agricultural production, has played important function.But it kills and wounds nontarget organism and problem of environment pollution caused can not be ignored equally.The low dosage organophosphor that remains in the food such as vegetables, fruit can produce chronic toxicity to the people, brings out DPN, apoplexy etc.In addition, some organophosphor has very high toxicity to honeybee, fish.The residual toxicity problem of organophosphorus pesticide more and more receives people's attention.The degraded of organophosphor in environment mainly contains present photochemical degradating, chemical degradation, 3 kinds of forms of microbial degradation.Utilize at present microorganism with and metabolite organophosphor is degraded is to be used gradually.Organophosphor contains three phosphide keys usually, and a phosphoric acid ester bond is hydrolyzed reducing its toxicity greatly.Opd degradable organophosphorus pesticide molecule destroys the phosphate bond of organophosphor and makes its detoxification.Because various organophosphorus pesticides all have similar structure, just substituting group is different, the multiple organophosphorus pesticide so a kind of opd often can be degraded.
Summary of the invention
The purpose of this invention is to provide simple organic phosphorus degrading enzyme preparation of a kind of technology and preparation method thereof.
For realizing above-mentioned purpose, the technical scheme that the present invention adopts is:
The organic phosphorus degrading enzyme preparation: the methyl bacillus promptly gets the organic phosphorus degrading enzyme preparation after fermentation.
Said organic phosphorus degrading enzyme preparation also comprises N-oleoyl contract amino acid sodium, sodium acid carbonate and sodium carboxymethylcellulose more, counts by weight and counts 5-20 part organic phosphorus degrading enzyme preparation, 2-8 part N-oleoyl contract amino acid sodium, 18-50 part sodium acid carbonate and 0.5-1 part sodium carboxymethylcellulose more by weight.Said methyl bacillus is inoculated in beef-protein medium in 30-35 ℃ through sweat for percentage 5-10% methyl bacillus at first by volume, and shaken cultivation 48-60h is for use; Then by volume the inoculum concentration of percentage 5-8% with the bacterial classification seed tank culture of above-mentioned cultivation with 25-30 ℃, fermentation time 48-60 hour, for use; At last with the sucrose solution of EDTA that adds the 1/30-1/40 zymotic fluid in the zymotic fluid centrifuged deposit and 1/10-1/15 fermentating liquid volume in the 30-60min ice bath; The frozen water that adds the 1/30-1/40 fermentating liquid volume in the centrifugation again is again at the 30-60min ice bath; Centrifugation adding ammonium sulfate to saturation degree is 100% once more, waits to saltout and spends the night; Centrifugal, deposition is gained.
The preparation method of organic phosphorus degrading enzyme preparation: at first by volume percentage 5-10% methyl bacillus is inoculated in beef-protein medium in 30-35 ℃, and shaken cultivation 48-60h is for use; Then by volume the inoculum concentration of percentage 5-8% with the bacterial classification seed tank culture of above-mentioned cultivation with 25-30 ℃, fermentation time 48-60 hour, for use; At last with the sucrose solution of EDTA that adds the 1/30-1/40 zymotic fluid in the zymotic fluid centrifuged deposit and 1/10-1/15 fermentating liquid volume in the 30-60min ice bath; The frozen water that adds the 1/30-1/40 fermentating liquid volume in the centrifugation again is again at the 30-60min ice bath; Centrifugation adding ammonium sulfate to saturation degree is 100% once more, waits to saltout and spends the night; Centrifugal, deposition is the organic phosphorus degrading enzyme preparation, behind above-mentioned mixing.
The freeze drying of said organic phosphorus degrading enzyme preparation is pulverized and is added water and mixes, and in comminutor, in 15-35 ℃, is made into 20-40 purpose particle.Said organic phosphorus degrading enzyme preparation also comprises N-oleoyl contract amino acid sodium, sodium acid carbonate and sodium carboxymethylcellulose more; Count by weight and count 5-20 part organic phosphorus degrading enzyme preparation, 2-8 part N-oleoyl contract amino acid sodium, 18-50 part sodium acid carbonate and 0.5-1 part sodium carboxymethylcellulose more by weight; By behind the above-mentioned mixing in tablet press machine compressing tablet, be pressed into thickness 1.0-1.5cm, diameter 1.0-1.5cm; The tablet of weight 2.0-4.0g obtains organophosphorus insecticide degradation agent effervescent tablet.Said centrifugal all under 4 ℃, with 6000-8000r/min, centrifugal 30-40min.300 rev/mins of tank pressure 0.02-0.05MPa during said seed tank culture, throughput 1:1.0-1.5v/v/min, mixing speed.
The invention has the advantages that:
1. preparation of the present invention has broad spectrum activity degraded, can degrade DDVP, phoxim, acephatemet, parathion etc. to organophosphorus insecticide.
2. preparation of the present invention soaks with common employing clear water, washing agent or cleans and compare, and effect is better, can degrade more than 75% to organophosphorus insecticide residual on the vegetables and fruits.
The specific embodiment
Embodiment 1
1) seed liquor fermentation: beef-protein medium, the methyl bacillus is inserted with the inoculum concentration of percent by volume 10% in the sterilization back, and 30 ℃, 180r/min cultivates 60h.
2) ferment tank: by volume the inoculum concentration of percentage 8% moves into seeding tank with seed liquor, carries out fermented and cultured, and condition is: jar 30 ℃ of temperature, tank pressure 0.05MPa, throughput 1:1.5v/v/min, 300 rev/mins of mixing speeds, fermentation time 60 hours.
3) with zymotic fluid in 4 ℃, the centrifugal 30min of 7000r/min. abandons supernatant, in bacterium mud, adds the pH7.5 of 1/40 fermentating liquid volume; 0.05mol/L EDTA and the sucrose solution of 1/15 fermentating liquid volume, shake up gently, thalline is fully suspended, ice bath 60min; 4 ℃, the centrifugal 30min of 7000r/min abandons high sepage, in bacterium mud, adds the frozen water of 1/40 fermentating liquid volume; Shake up ice bath 60min, 4 ℃, the centrifugal 30min of 7000r/min gently; Supernatant, add fine ground ammonium sulfate to saturation degree while stirring and be 100%, 4 ℃ and saltout and spend the night; Centrifugal, collect albumen precipitation, be the organic phosphorus degrading enzyme preparation.
The organic phosphorus degrading enzyme preparation of 8 parts of parts by weight of above-mentioned gained is added water mix, in comminutor, be made into 20 purpose particles, for use;
Meter by weight then, 20 parts of sodium acid carbonates, 5 parts of N-oleoyls amino acid sodium that contracts more, 0.5 part of sodium carboxymethylcellulose sodium acid carbonate mixes, and compressing tablet in tablet press machine is pressed into thickness 1.0cm, diameter 1.2cm, the tablet of weight 3.0g.
Above-mentioned organic phosphorus degrading enzyme preparation for use is mixed with above-mentioned tablet, compressing tablet in tablet press machine, 32 ℃ of compressing tablet temperature are pressed into thickness 1.0cm, diameter 1.2cm, the tablet of weight 3.0g obtains organophosphorus insecticide digestive enzyme effervescent tablet.
With the clean clear and clear water of above-mentioned organic phosphorus degrading enzyme preparation and organophosphorus insecticide digestive enzyme effervescent tablet and washing of same concentration to phoxim residual on the rape test (referring to table 1) of degrading.
The agent of table 1 organic phosphorus degrading enzyme is to the degradation effect of phoxim residual on the rape
Embodiment 2
Be with embodiment 1 difference:
1) seed liquor fermentation: beef-protein medium, the methyl bacillus is inserted with the inoculum concentration of percent by volume 5% in the sterilization back, and 35 ℃, 180r/min cultivates 48h.
2) ferment tank: by volume the inoculum concentration of percentage 5% moves into seeding tank with seed liquor, carries out fermented and cultured, and condition is: jar 25 ℃ of temperature, tank pressure 0.02MPa, throughput 1:1.0v/v/min, 300 rev/mins of mixing speeds, fermentation time 48 hours.
3) with zymotic fluid in 4 ℃, 6000r/min. is centrifugal, and 40min. abandons supernatant, in bacterium mud, adds the pH7.5 of 1/30 fermentating liquid volume, the sucrose solution of the EDTA of 0.05mol/L and 1/10 fermentating liquid volume; Shake up gently, thalline is fully suspended, ice bath 30min, 4 ℃; The centrifugal 40min of 6000r/min abandons high sepage, in bacterium mud, adds the frozen water of 1/30 fermentating liquid volume, shakes up gently; Ice bath 30min, 4 ℃, the centrifugal 40min of 6000r/min; Supernatant, add fine ground ammonium sulfate to saturation degree while stirring and be 100%, 4 ℃ and saltout and spend the night; Centrifugal, collect albumen precipitation, be the organic phosphorus degrading enzyme preparation.
The organic phosphorus degrading enzyme preparation of 5 parts of parts by weight of above-mentioned gained is added water mix, in comminutor, be made into 30 purpose particles in 15 ℃, for use;
Meter by weight then, 50 parts of sodium acid carbonates, 2 parts of N-oleoyls amino acid sodium that contracts more, 1 part of sodium carboxymethylcellulose sodium acid carbonate mixes, and compressing tablet in tablet press machine is pressed into thickness 1.5cm, diameter 1.5cm, the tablet of weight 2.0g.
Above-mentioned organic phosphorus degrading enzyme preparation for use is mixed with above-mentioned tablet, compressing tablet in tablet press machine, 25 ℃ of compressing tablet temperature are pressed into thickness 1.5cm, diameter 1.5cm, the tablet of weight 2.0g obtains organophosphorus insecticide digestive enzyme effervescent tablet.
With the clean clear and clear water of above-mentioned organic phosphorus degrading enzyme preparation and organophosphorus insecticide digestive enzyme effervescent tablet and washing of same concentration to phoxim residual on the rape test (referring to table 2) of degrading.
The agent of table 2 organic phosphorus degrading enzyme is to the degradation effect of phoxim residual on the rape
Embodiment 3
Be with embodiment 1 difference:
1) seed liquor fermentation: beef-protein medium, the methyl bacillus is inserted with the inoculum concentration of percent by volume 5% in the sterilization back, and 32 ℃, 180r/min cultivates 52h.
2) ferment tank: by volume the inoculum concentration of percentage 5% moves into seeding tank with seed liquor, carries out fermented and cultured, and condition is: jar 28 ℃ of temperature, tank pressure 0.04MPa, throughput 1:1.2v/v/min, 300 rev/mins of mixing speeds, fermentation time 52 hours.
3) with zymotic fluid in 4 ℃, 8000r/min. is centrifugal, and 35min. abandons supernatant, in bacterium mud, adds the pH7.5 of 1/30 fermentating liquid volume, the sucrose solution of the EDTA of 0.05mol/L and 1/10 fermentating liquid volume; Shake up gently, thalline is fully suspended, ice bath 50min, 4 ℃; The centrifugal 35min of 8000r/min abandons high sepage, in bacterium mud, adds the frozen water of 1/30 fermentating liquid volume, shakes up gently; Ice bath 50min, 4 ℃, the centrifugal 35min of 8000r/min; Supernatant, add fine ground ammonium sulfate to saturation degree while stirring and be 100%, 4 ℃ and saltout and spend the night; Centrifugal, collect albumen precipitation, be the organic phosphorus degrading enzyme preparation.
The organic phosphorus degrading enzyme preparation of 15 parts of parts by weight is added water mix, in comminutor, be made into 35 purpose particles, for use.
Meter by weight then, 20 parts of sodium acid carbonates, 5 parts of N-oleoyls contract amino acid sodium and 1.0 parts of sodium carboxymethylcellulose sodium acid carbonates mix more, and compressing tablet in tablet press machine is pressed into thickness 1.2cm, diameter 1.4cm, the tablet of weight 3.0g.
Above-mentioned organic phosphorus degrading enzyme preparation for use is mixed with above-mentioned tablet, compressing tablet in tablet press machine, 32 ℃ of compressing tablet temperature are pressed into thickness 1.2cm, diameter 1.4cm, the tablet of weight 4.0g obtains the special-purpose organophosphorus insecticide degradation agent of vegetables and fruits effervescent tablet.
With the clean clear and clear water of above-mentioned organophosphorus insecticide digestive enzyme effervescent tablet and washing of same concentration to DDVP residual on the rape test (referring to table 3) of degrading.
The agent of table 3 organic phosphorus degrading enzyme is to the degradation effect of DDVP residual on the rape
Claims (7)
1. organic phosphorus degrading enzyme preparation is characterized in that: the methyl bacillus promptly gets the organic phosphorus degrading enzyme preparation after fermentation;
Said methyl bacillus is inoculated in beef-protein medium in 30-35 ℃ through sweat for percentage 5-10% methyl bacillus at first by volume, and shaken cultivation 48-60h is for use; Then by volume the inoculum concentration of percentage 5-8% with the bacterial classification seed tank culture of above-mentioned cultivation with 25-30 ℃, fermentation time 48-60 hour, for use; At last with the sucrose solution of EDTA that adds the 1/30-1/40 zymotic fluid in the zymotic fluid centrifuged deposit and 1/10-1/15 fermentating liquid volume in the 30-60min ice bath; The frozen water that adds the 1/30-1/40 fermentating liquid volume in the centrifugation again is again at the 30-60min ice bath; Centrifugation adding ammonium sulfate to saturation degree is 100% once more, waits to saltout and spends the night; Centrifugal, deposition is gained.
2. by the described organic phosphorus degrading enzyme preparation of claim 1; It is characterized in that: said organic phosphorus degrading enzyme preparation also comprises N-oleoyl contract amino acid sodium, sodium acid carbonate and sodium carboxymethylcellulose more, counts 5-20 part organic phosphorus degrading enzyme preparation, 2-8 part N-oleoyl contract amino acid sodium, 18-50 part sodium acid carbonate and 0.5-1 part sodium carboxymethylcellulose by weight more.
3. preparation method by the described organic phosphorus degrading enzyme preparation of claim 1, it is characterized in that: at first by volume percentage 5-10% methyl bacillus is inoculated in beef-protein medium in 30-35 ℃, and shaken cultivation 48-60h is for use; Then by volume the inoculum concentration of percentage 5-8% with the bacterial classification seed tank culture of above-mentioned cultivation with 25-30 ℃, fermentation time 48-60 hour, for use; At last with the sucrose solution of EDTA that adds the 1/30-1/40 zymotic fluid in the zymotic fluid centrifuged deposit and 1/10-1/15 fermentating liquid volume in the 30-60min ice bath; The frozen water that adds the 1/30-1/40 fermentating liquid volume in the centrifugation again is again at the 30-60min ice bath; Centrifugation adding ammonium sulfate to saturation degree is 100% once more, waits to saltout and spends the night; Centrifugal, deposition is the organic phosphorus degrading enzyme preparation.
4. by the preparation method of the described organic phosphorus degrading enzyme preparation of claim 3, it is characterized in that: the freeze drying of said organic phosphorus degrading enzyme preparation, pulverize and add water and mix, in comminutor,, be made into 20-40 purpose particle in 15-35 ℃.
5. press the preparation method of the described organic phosphorus degrading enzyme preparation of claim 3; It is characterized in that: said organic phosphorus degrading enzyme preparation also comprises N-oleoyl contract amino acid sodium, sodium acid carbonate and sodium carboxymethylcellulose more; Count by weight and count 5-20 part organic phosphorus degrading enzyme preparation, 2-8 part N-oleoyl contract amino acid sodium, 18-50 part sodium acid carbonate and 0.5-1 part sodium carboxymethylcellulose more by weight; By behind the above-mentioned mixing in tablet press machine compressing tablet, be pressed into thickness 1.0-1.5cm, diameter 1.0-1.5cm; The tablet of weight 2.0-4.0g obtains organophosphorus insecticide degradation agent effervescent tablet.
6. by the preparation method of the described organic phosphorus degrading enzyme preparation of claim 3, it is characterized in that: said centrifugal all under 4 ℃, with 6000-8000r/min, centrifugal 30-40min.
7. by the preparation method of the described organic phosphorus degrading enzyme preparation of claim 3, it is characterized in that: 300 rev/mins of tank pressure 0.02-0.05MPa during said seed tank culture, throughput 1: 1.0-1.5v/v/min, mixing speed.
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| CN 200810228798 CN101732819B (en) | 2008-11-14 | 2008-11-14 | Organic phosphorus degrading enzyme preparation and preparation method thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146664A (en) * | 2013-02-04 | 2013-06-12 | 辽宁中科生物工程有限公司 | Random mutation reconstructed organophosphorus pesticide degradation enzyme and coding genes thereof |
| CN103146665A (en) * | 2013-02-04 | 2013-06-12 | 中国科学院沈阳应用生态研究所 | Organophosphorus pesticide degrading enzyme transformed by random mutation and encoding gene thereof |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101993839B (en) * | 2010-07-23 | 2012-06-27 | 浙江工业大学 | Methylobacterium rhodesianum H13 capable of efficiently degrading dichloromethane and application thereof |
| CN102199550A (en) * | 2011-03-24 | 2011-09-28 | 深圳西德赛科技有限公司 | Recombinant strain expressing organophosphorus hydrolase, complex enzyme preparation technology, complex enzyme preparation and fruit and vegetable detergent |
| CN103146658B (en) * | 2013-02-04 | 2014-07-23 | 中国科学院沈阳应用生态研究所 | Organophosphorus pesticide degrading enzyme transformed through mutation and encoding gene thereof |
| CN103834595B (en) * | 2014-03-07 | 2015-10-28 | 江苏农林职业技术学院 | A kind of Methylobacterium and application thereof |
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| CN1059668A (en) * | 1991-10-12 | 1992-03-25 | 徐谦 | Vegetable residual pesticide scavenger |
| US5453375A (en) * | 1993-08-18 | 1995-09-26 | Lockheed Idaho Technologies Company | Method and compositions for the degradation of tributyl phosphate in chemical waste mixtures |
| US7001758B1 (en) * | 1998-12-07 | 2006-02-21 | The United States Of America As Represented By The Secretary Of The Army | Microbial biodegradation of phosphonates |
| CN101293131A (en) * | 2007-04-29 | 2008-10-29 | 上海普天欣生物技术有限公司 | Complex enzyme preparation for eliminating residual pesticides and application thereof |
-
2008
- 2008-11-14 CN CN 200810228798 patent/CN101732819B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1059668A (en) * | 1991-10-12 | 1992-03-25 | 徐谦 | Vegetable residual pesticide scavenger |
| US5453375A (en) * | 1993-08-18 | 1995-09-26 | Lockheed Idaho Technologies Company | Method and compositions for the degradation of tributyl phosphate in chemical waste mixtures |
| US7001758B1 (en) * | 1998-12-07 | 2006-02-21 | The United States Of America As Represented By The Secretary Of The Army | Microbial biodegradation of phosphonates |
| CN101293131A (en) * | 2007-04-29 | 2008-10-29 | 上海普天欣生物技术有限公司 | Complex enzyme preparation for eliminating residual pesticides and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146664A (en) * | 2013-02-04 | 2013-06-12 | 辽宁中科生物工程有限公司 | Random mutation reconstructed organophosphorus pesticide degradation enzyme and coding genes thereof |
| CN103146665A (en) * | 2013-02-04 | 2013-06-12 | 中国科学院沈阳应用生态研究所 | Organophosphorus pesticide degrading enzyme transformed by random mutation and encoding gene thereof |
| CN103146665B (en) * | 2013-02-04 | 2014-05-14 | 中国科学院沈阳应用生态研究所 | Organophosphorus pesticide degrading enzyme transformed by random mutation and encoding gene thereof |
| CN103146664B (en) * | 2013-02-04 | 2014-07-02 | 辽宁中科生物工程有限公司 | Random mutation reconstructed organophosphorus pesticide degradation enzyme and coding genes thereof |
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| CN101732819A (en) | 2010-06-16 |
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