CN102134559B - Bacillus brevis, method for extracting bacillus brevis esterase and application of same - Google Patents
Bacillus brevis, method for extracting bacillus brevis esterase and application of same Download PDFInfo
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- CN102134559B CN102134559B CN 201010546319 CN201010546319A CN102134559B CN 102134559 B CN102134559 B CN 102134559B CN 201010546319 CN201010546319 CN 201010546319 CN 201010546319 A CN201010546319 A CN 201010546319A CN 102134559 B CN102134559 B CN 102134559B
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- genus bacillus
- esterase
- bacillus
- bacillus brevis
- gdxease8
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Abstract
The invention discloses bacillus brevis GDXEase8, a method for extracting bacillus brevis esterase and the application of same. The bacillus brevis is preserved in CCTCC (China Center for Type Culture Collection), the preservation date is 9th, Nov. 2010, and the serial number of preservation is CCTCC M2010295. The bacillus brevis has the advantages of low cost, high sensitivity and stable enzyme source.
Description
Technical field
The present invention relates to the process for extracting and the application of a kind of genus bacillus and esterase thereof.
Background technology
Organophosphorus pesticide is avoided disease, insect pest the protection farm crop; Improve the grain yield aspect and have huge effect; But its excessive use often causes agricultural-food pesticide residue such as vegetables and fruit to exceed standard; Directly the harm people's is healthy, and therefore effectively the pesticide residue of control agricultural-food are one of main tasks of the current solution food-safety problem of China.One of valid approach that reduces the harm of agricultural-food pesticide residue is to set up Detecting Pesticide method fast and effectively.The market of farm produce of most domestic is all using enzyme inhibition method to detect the organophosphorus pesticide of fruits and vegetables at present; This method has related to enzyme reagent, substrate and developer, and gordian technique wherein is extraction and the acquisition to the esterase of organophosphorus and carbamate chemicals for agriculture sensitivity.The domestic existing esterase that is used for scale prodn all extracts from animal at present, mainly also has following shortcoming and problem: (1) screening enzyme source workload is very big, cost is higher; (2) enzyme source is unstable, the differing greatly of the enzyme in different batches Different Individual source.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, the process for extracting of the genus bacillus esterase that a kind of cost is low, highly sensitive and enzyme source is stable is provided.
This bacterium called after genus bacillus (Bacillus brevis) GDXEase8 is numbered.
Confirming of the molecular classification status of genus bacillus of the present invention (Bacillus brevis) GDXEase8.
The 16SrDNA sequence of this bacterial strain is shown in SEQ ID NO:1.
The bacterium colony of this bacterial strain on flat board is rounded, light yellow, opaque, smooth surface, neat in edge.It is shaft-like that cell is, about diameter 0.8 μ m, and the about 2-3 μ of length m, gramstaining is positive, the gemma ovalize, near-end is given birth to.
Antibiotics resistance, catalase experimental result that this bacterial strain is carried out show that bacterial strain has resistance to nalidixic acid and spectinomycin, and the catalase experimental result is positive.
The present invention also provides the process for extracting of genus bacillus esterase, may further comprise the steps:
(1) slant strains: GDXEase8 is inoculated on the nutrient agar medium inclined-plane and cultivates with genus bacillus (Bacillus brevis);
(2) enlarging seed liquor cultivates: slant strains is inoculated in the triangular flask that seed culture medium is housed, and 37 ± 3 ℃, 100-300rpm, shaking table was cultivated 12-14 hour;
Said seed culture medium is a peptone 1%, yeast extract paste 0.5%, and NaCl 0.5%, pH7.0;
(3) fermentation culture: the enlarged culturing seed liquor is inoculated in the fermention medium, cultivated 24-26 hour in 37 ± 3 ℃;
Said fermention medium is: glucose 1%, peptone 2%, K
2HPO
40.1%, NaH
2PO
40.1%, pH7.0;
(4) extraction of genus bacillus esterase: with the centrifugal 10-20min of zymocyte liquid 3000-5000rpm; Collect thalline; With suspending with phosphoric acid buffer once more after the washing of 0.01-003mol/L pH7.0 phosphoric acid buffer; Carry enzyme with the supersonic wave wall breaking method, the centrifugal 10-20min of 3000-5000rpm, the gained supernatant is the genus bacillus esterase.
The present invention also provides this application of genus bacillus esterase aspect Detecting Pesticide.
Compared with prior art, the present invention has following beneficial effect:
1. the present invention is to extract enzyme liquid after starting strain ferments with a bacillus (Bacillus brevis) GDXEase8; This enzyme its character under the situation that bacterial classification does not morph is identical; Good reproducibility has overcome the problem that the individual esterase of originating of different plant-animal there are differences.
2. the present invention carries out fermented extracted enzyme liquid with genus bacillus (Bacillus brevis) GDXEase8, need not screen the enzyme source, does not also need purifying, has reduced the workload and the cost of enzyme extraction.
3. genus bacillus esterase provided by the invention is at SRA-5172, inhibiting rate all was higher than 50% when SD-1750 concentration was respectively 0.3ppm, 0.005ppm; Make detection limit far below relevant national standard; It is not high to the susceptibility of organophosphorus pesticide to have overcome present product, particularly to the low shortcoming of susceptibility of SRA-5172 etc.
Genus bacillus of the present invention (Bacillus brevis) GDXEase8 is preserved in specified depositary institution of State Intellectual Property Office China typical culture collection center (CCTCC) on November 9th, 2010, and deposit number is No.CCTCC M 2010295.
Embodiment
Embodiment 1
Genus bacillus provided by the invention (Bacillus brevis) GDXEase8 has property:
1. morphological specificity
The bacterium colony of bacterial strain on flat board is rounded, light yellow, opaque, smooth surface, neat in edge.It is shaft-like that cell is, about diameter 0.8 μ m, and the about 2-3 μ of length m, gramstaining is positive, the gemma ovalize, near-end is given birth to.
2. Physiology and biochemistry is identified
Antibiotics resistance, catalase experimental result that bacterial strain is carried out show that bacterial strain has resistance to nalidixic acid and spectinomycin, and the catalase experimental result is positive.
3.16SrDNA sequence
The 16SrDNA sequence of this bacterial strain is shown in SEQ ID NO:1.
Embodiment 2
The preparation method of embodiment 1 described genus bacillus esterase:
(1) slant strains: the bacterial classification inoculation of preservation is cultivated 0-4 ℃ of preservation to the nutrient agar medium inclined-plane.
(2) enlarging seed liquor cultivates: with slant strains be inoculated into be equipped with the 100ml seed culture medium (NaCl 0.5% for peptone 1%, yeast extract paste 0.5%, in 250ml triangular flask pH7.0), 37 ℃, 200rpm, shaking table was cultivated 12 hours.
(3) fermentation culture: 100ml enlarged culturing seed liquor is inoculated in the 10L fermentor tank, cultivates 24 hours (culture medium prescriptions: glucose 1%, peptone 2%, K in 37 ℃
2HPO
40.1%, NaH
2PO
40.1%, pH7.0).
(4) extraction of enzyme liquid: with zymocyte liquid centrifugal (4000rpm) 15min; Collect thalline,, carry enzyme with the supersonic wave wall breaking method with suspending with phosphoric acid buffer once more after 0.02mol/L phosphoric acid buffer (pH7.0) washing; Centrifugal (4000rpm) 15min, the gained supernatant is esterase liquid.
Embodiment 3
Embodiment 2 described genus bacillus esterases detect the susceptibility of organophosphorus pesticide:
Material: bacterium esterase, indophenols acetate solution and phosphoric acid buffer
Instrument: thermostat water bath, spectrophotometer
Esterase activity is measured: in test tube, add 4.7mL 0.02mol/L; The pH7.0 phosphoric acid buffer, 200 μ L2% indophenols acetic ester, mixing; 37 ℃ of water-baths 10 minutes; Add 100 μ L genus bacillus esterases, react adding 5mL absolute ethyl alcohol termination reaction (control tube adds the genus bacillus esterase again after adding absolute ethyl alcohol) after 5 minutes, survey OD605.
Inhibiting rate calculates: (OD0-OD1)/and OD0 * 100% (OD0: the OD value when not being suppressed; OD1: the OD value after the inhibition)
Measure the result:
(1) the genus bacillus esterase is to the susceptibility of SD-1750
When SD-1750 concentration was 0.005ppm, inhibiting rate was higher than 50%, was lower than relevant criterion [rapid detection of organophosphorus and carbamate pesticide residue amount in the GB/T 5009.199-2003 vegetables].
(2) the genus bacillus esterase is to the susceptibility of Trichlorphon
When Trichlorphon concentration was 0.1ppm, inhibiting rate was higher than 50%, was lower than relevant criterion [rapid detection of organophosphorus and carbamate pesticide residue amount in the GB/T5009.199-2003 vegetables].
(3) the genus bacillus esterase is to the susceptibility of SRA-5172
When SRA-5172 concentration was 0.5ppm, inhibiting rate was higher than 50%, was lower than relevant criterion [rapid detection of organophosphorus and carbamate pesticide residue amount in the GB/T5009.199-2003 vegetables].
(4) the genus bacillus esterase is to the susceptibility of Rogor
When Rogor concentration was 2ppm, inhibiting rate was higher than 50%, was lower than relevant criterion [rapid detection of organophosphorus and carbamate pesticide residue amount in the GB/T 5009.199-2003 vegetables].
(5) the genus bacillus esterase is to the susceptibility of acephate
When acephate concentration was 0.6ppm, inhibiting rate was higher than 50%, was lower than relevant criterion [rapid detection of organophosphorus and carbamate pesticide residue amount in the GB/T5009.199-2003 vegetables].
Claims (4)
1. a genus bacillus (Bacillus brevis) GDXEase8, it is characterized in that: its deposit number is CCTCC M 2010295.
2. genus bacillus according to claim 1 (Bacillus brevis) GDXEase8, it is characterized in that: the 16SrDNA sequence of said genus bacillus is shown in SEQ ID NO:1.
3. the process for extracting of claim 1 or 2 described genus bacillus esterase is characterized in that: may further comprise the steps:
(1) slant strains: GDXEase8 is inoculated on the nutrient agar medium inclined-plane and cultivates with genus bacillus (Bacillus brevis);
(2) enlarging seed liquor cultivates: slant strains is inoculated in the triangular flask that seed culture medium is housed, and 37 ± 3 ℃, 100-300rpm, shaking table was cultivated 12-14 hour;
Said seed culture medium is a peptone 1%, yeast extract paste 0.5%, and NaCl 0.5%, pH7.0;
(3) fermentation culture: the enlarged culturing seed liquor is inoculated in the fermention medium, cultivated 24-26 hour in 37 ± 3 ℃;
Said fermention medium is: glucose 1%, peptone 2%, K
2HPO
40.1%, NaH
2PO
40.1%, pH7.0;
(4) extraction of genus bacillus esterase: with the centrifugal 10-20min of zymocyte liquid 3000-5000rpm; Collect thalline; With suspending with phosphoric acid buffer once more after the washing of 0.01-0.03mol/L pH7.0 phosphoric acid buffer; Carry enzyme with the supersonic wave wall breaking method, the centrifugal 10-20min of 3000-5000rpm, the gained supernatant is the genus bacillus esterase.
4. the application of genus bacillus esterase as claimed in claim 3 aspect Detecting Pesticide.
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| CN107384834B (en) * | 2017-08-29 | 2020-05-22 | 侨康生物科技(广东)有限公司 | Lysinibacillus fusiformis strain, enzyme preparation and application of lysine bacillus fusiformis strain and enzyme preparation in degradation of pesticide residues |
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| WO2002089830A1 (en) * | 2001-05-04 | 2002-11-14 | North Carolina State University | Polymer conjugates of insecticidal peptides or nucleic acids and methods of use thereof |
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| CN1687756A (en) * | 2004-12-27 | 2005-10-26 | 南京农业大学 | Kit for testing pesticide residue rapidly and use method thereof |
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| CN101586100A (en) * | 2009-05-19 | 2009-11-25 | 浙江师范大学 | Immobilized cholinesterase and preparation and application thereof |
| CN101709282A (en) * | 2009-12-23 | 2010-05-19 | 河北科技大学 | Organophosphorus pesticide degrading bacterium and method for producing microbial inoculum thereof |
| CN101724580A (en) * | 2008-10-24 | 2010-06-09 | 中国科学院生态环境研究中心 | Bacillus for degrading pyrethroid insecticides and method for producing fungicide thereof |
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2010
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Patent Citations (7)
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| WO2002089830A1 (en) * | 2001-05-04 | 2002-11-14 | North Carolina State University | Polymer conjugates of insecticidal peptides or nucleic acids and methods of use thereof |
| CN1414371A (en) * | 2002-12-13 | 2003-04-30 | 江苏省农业科学院植物保护研究所 | Conventional pesticide residue quick detecting method |
| CN1687756A (en) * | 2004-12-27 | 2005-10-26 | 南京农业大学 | Kit for testing pesticide residue rapidly and use method thereof |
| CN101059423A (en) * | 2007-03-02 | 2007-10-24 | 内蒙古伊利实业集团股份有限公司 | Quick detection method for agricultural chemical residue in milk |
| CN101724580A (en) * | 2008-10-24 | 2010-06-09 | 中国科学院生态环境研究中心 | Bacillus for degrading pyrethroid insecticides and method for producing fungicide thereof |
| CN101586100A (en) * | 2009-05-19 | 2009-11-25 | 浙江师范大学 | Immobilized cholinesterase and preparation and application thereof |
| CN101709282A (en) * | 2009-12-23 | 2010-05-19 | 河北科技大学 | Organophosphorus pesticide degrading bacterium and method for producing microbial inoculum thereof |
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| T. Panda·B. S. Gowrishankar.Production and applications of esterases.《Appl Microbiol Biotechnol》.2005,第67卷160-169. * |
| 刘增柱.芽孢杆菌胞内酯酶的电泳分析.《微生物学杂志》.1989,第09卷(第03期),45-49,33. * |
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Address after: 510663 Guangzhou, China Hi tech Industrial Development Zone, Science City, science and technology road, No. 10, Sino German research and development building (F2) room 216 Applicant after: Guangzhou Jijiazhuang Biotechnology Co., Ltd. Address before: 510620, B2, building 36, 601 hi tech Road, Guangzhou, Guangdong, Tianhe District Applicant before: Guangzhou Jijiazhuang Biotechnology Co., Ltd. |
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