CN103146665A - Organophosphorus pesticide degrading enzyme transformed by random mutation and encoding gene thereof - Google Patents
Organophosphorus pesticide degrading enzyme transformed by random mutation and encoding gene thereof Download PDFInfo
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- CN103146665A CN103146665A CN2013100445477A CN201310044547A CN103146665A CN 103146665 A CN103146665 A CN 103146665A CN 2013100445477 A CN2013100445477 A CN 2013100445477A CN 201310044547 A CN201310044547 A CN 201310044547A CN 103146665 A CN103146665 A CN 103146665A
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Abstract
The invention relates to an organophosphorus pesticide degrading enzyme obtained by genetic engineering transformation. The organophosphorus pesticide degrading enzyme transformed by random mutation is generated by replacing an amino acid site of the organophosphorus pesticide degrading enzyme from pseudomonas stutzeri, and the replacing amino acid site is located at the 71st site. The organophosphorus pesticide degrading enzyme is excellent in degrading activity of chlorpyrifos and methyl to ethion and excellent in heat stability.
Description
Technical field
The invention belongs to the genetically engineered field, relate to by genetic engineering modified opd.
Background technology
Pesticide residue serious harm ecotope, the harm people's is healthy, is that each state all needs the Tough questions faced.Research is found, the pollution that has been subjected to pesticide residue all of the underground water of different areas, farm crop and soil.China is large agricultural country, and annual agricultural chemicals usage quantity is huge, and consequent Pesticide Residue also is on the rise.Related data shows that the pesticide residue of agricultural products in China exceed standard seriously, and all kinds of agricultural chemicals intakes in the daily meals of resident are higher than developed country's decades of times.In recent years, the removal technology of pesticide residue becomes research focus, seek a kind of safe, method that remove efficiently pesticide residue is significant.
Pesticide degradable enzyme is the effective technology means of removing pesticide residue, has the characteristics such as degraded is rapid, use safety, environmental friendliness.Had been found that at present the Multiple Pesticides degrading enzyme, such as opd OPH, opd MPH, pyrethrin degrading enzyme B1 etc.Wherein, domestic and international the most deep for opd research.Opd OPH is at first at Flavobacterium sp.ATCC27551(Mulbry and Karns, 1989) and Pseudomonas diminuta MG(McDaniel et al., 1988) separate in and obtain, at first opd MPH is found (Cui Zhongli etc., 2001) in Plesiomonas sp.strain M6 by Chinese scholar.Opd OPH and MPH have degraded spectrum widely, and the agricultural chemicals such as parathion-methyl, Chlorpyrifos 94, thiophos, fenitrothion 95, Malathion, SD-1750 of can degrading are the key enzymes in pesticide degradable enzyme field.it is more deep that the genetic modification work of opd OPH is carried out, the researchist has successfully improved 25 times of (Catherine Mee-Hie Cho et al. with opd OPH to the degrading activity of parathion-methyl by DNA shuffling technology, 2002), degrading activity to Chlorpyrifos 94 has improved more than 700 times of (Catherine Mee-Hie Cho et al., 2004), these successful genetic modifications make OPH have more outstanding zymologic property, be conducive to it in the application in chemical residual degradation field, has important actual application value.
With respect to opd OPH, the genetic modification of opd MPH does not obtain so significant achievement.The researchist is by rite-directed mutagenesis, makes opd MPH obtain more outstanding thermostability and ph stability (Jian Tian et al., 2010; Yidan Su et al., 2011; Lu Huang et al., 2012); By the random mutation means, having obtained a plant mutant site is the mutant strain of K173R, and its degradation efficiency to Chlorpyrifos 94 has improved 41.55%(Li Ya nanmu etc., 2012 than wild-type enzyme).The research of opd MPH aspect genetic modification awaits to further investigate and exploitation, transform opd MPH by genetic engineering means, the mutant enzyme that obtains the raising of enzyme activity and thermostability is significant for solving Pesticide Residue.
Summary of the invention
The object of the invention is to utilize genetic engineering means transformation opd MPH, makes it obtain more outstanding enzyme activity and thermostability.
For achieving the above object, the technical solution used in the present invention is:
35 amino acid of N end of opd MPH are signal peptide sequence, and the object of random mutation of the present invention is not for containing the ripe mpd gene of signal coding sequence.
Adopt the method for fallibility PCR to opd MPH encoding gene (mpd, the GenBank accession number: JQ686087) carry out random mutation in Pseudomonas stutzeri strain ZK-5 source.
Fallibility PCR reaction system (50 μ L) is: (the GenBank accession number: JQ686087) 50ng is as template for the mpd gene, each 0.3 μ M of upstream primer and downstream primer, each 200 μ M of dNTPs, 10 μ L sudden change damping fluids, 5U Taq archaeal dna polymerase, 5 μ LPCR damping fluids.Fallibility PCR reaction conditions is: 94 ℃ of thermally denature 5min, and 94 ℃ of thermally denature 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, 30 circulations.
Upstream primer: 5 '-GAATTCTGCAGCACCGCAGGTGCGCACC-3 '
Downstream primer: 5 '-TCCGGAAGCTTTCATCATCACTTGGGGTTGACGACCG-3 '
The sudden change damping fluid consists of: dTTP4mM, dCTP4mM, MgCl
227.5mM, MnCl
22.5mM solvent is deionized water.
The PCR damping fluid consists of: 100mM Tris-HCl pH8.3,500mM KCl and15mMMgCl
2, solvent is deionized water.
The mpd mutator gene that random mutation obtains is expressed, the purified mutant enzyme, the mutant enzyme that screening enzyme activity and thermostability improve is through screening in a large number, obtain the opd of 1 strain random mutation transformation, it has enzyme activity and the thermostability more outstanding than wild-type enzyme.Through order-checking, opd of the present invention is that to have aminoacid sequence by replacement be that 1 amino acid sites of the opd MPH that derives from Pseudomonas stutzeri strain ZK-5 of SEQ ID No.3 produces, described amino acid sites is to be positioned at the replacement of the 71st, is to replace Serine with Threonine the replacement of the 71st.
Opd three-dimensional structure of the present invention is as shown in Figure 5: opd of the present invention is with the subunit dimer, the active centre is two zine ion metal actives centers, the N-terminal of subunit is combined with the groove on another one subunit surface by hydrogen bond, and the stability of dimeric structure has been played keying action.Opd of the present invention occurs on the loop ring the replacement of the 71st, and this replaces the site near the N-terminal of another one subunit, by the combination of remote effect subunit, and then affects enzyme activity and three-dimensional structure stability.Opd of the present invention has obtained enzyme activity and the thermostability more outstanding than wild-type enzyme by the replacement in above-mentioned site.
The below be random mutation can referring to document:
（1）Cadwell?RC,Joyce?GF(1992)Randomization?of?Genes?by?PCR?Mutagenesis.Genome?Res2:28-33.
（2）Rasila?TS,Pajunen?MI,Savilahti?H(2009)Critical?evaluation?of?random?mutagenesis?by?error-prone?polymerase?chain?reaction?protocols,Escherichia?coli?mutator?strain,and?hydroxylamine?treatment.Analytical?Biochemistry388:71-80.
The present invention has advantages of
The opd of the random mutation transformation that the present invention obtains has by replacement the 71st amino acids site that aminoacid sequence is the opd MPH that derives from Pseudomonas stutzeri strain ZK-5 of SEQ ID No.3, has obtained enzyme activity and the thermostability more outstanding than wild-type enzyme.
Raising due to enzyme activity and thermostability, opd degradation efficiency of the present invention is high, be difficult for inactivation under high temperature, obtained larger actual application value, can be widely used in the degraded of agricultural-food and physical environment Pesticide Residues, provide a kind of effective way for thoroughly solving Pesticide Residue.
Description of drawings
Fig. 1 opd of the present invention and the wild-type enzyme ratio vigor to Chlorpyrifos 94 and parathion-methyl;
Fig. 2 opd of the present invention and wild-type Thermostability;
Fig. 3 p-NP typical curve;
Fig. 4 Chlorpyrifos 94 typical curve;
Fig. 5 opd tomograph of the present invention.
Embodiment
Embodiment 1: opd Expression in Escherichia coli of the present invention
(1) pcr amplification: with upstream primer 5'-ATTCATATGGCCGCACCGCAGGTG-3' and downstream primer 5'-TAACTCGAGCTTGGGGTTGACGACCG-3'PCR amplification opd gene of the present invention.
PCR(50 μ L system) reaction conditions is: opd gene of the present invention (SEQ ID No.1) 50ng is as template, each 0.3 μ M of upstream primer and downstream primer, each 200 μ M of dNTPs, 5U Taq archaeal dna polymerase, 5 μ LPCR damping fluids, 30 circulations, 94 ℃ of sex change 5min, 94 ℃ of sex change 1min, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, 72 ℃ of extension 10min of last circulation.The PCR damping fluid consists of: 100mM Tris-HCl pH8.3,500mM KCl and15mMMgCl
2, solvent is deionized water.
(2) expression vector establishment: above-mentioned PCR product is through PCR product purification test kit (QIAGEN) purifying (it is SEQ ID No.1 to purified product through Beijing Liuhe Huada Genomics Technology Co., Ltd's order-checking proof), adopt NdeI and XhoI double digestion, be connected in equally by the pET-30a plasmid vector of NdeI and XhoI double digestion, build the restructuring pET-30a expression vector that is connected with opd encoding gene of the present invention.
(3) transformed host cell: the restructuring pET-30a expression vector Transformed E .coli BL21 (DE3) with previous step obtains, obtain genetic engineering bacterium, concrete operations are according to molecular biology experiment guide (Sambrook J, Russell DW, the third edition).
(4) expression of opd of the present invention: with the said gene engineering bacteria in the LB liquid nutrient medium that contains kantlex (50 μ g/ml), 37 ℃, 180rpm is cultured to logarithmic phase, then 1% inoculum size is inoculated in nutrient solution the LB liquid nutrient medium that contains kantlex (50 μ g/ml) by volume, 37 ℃, 180rpm cultivates that to add wherein final concentration after 3 hours be that the IPTG of 1mM induces, and culture temperature is dropped to 25 ℃, abduction delivering 10 hours.
(5) purifying of opd of the present invention: the above-mentioned thalline of end of inducing is passed through broken also back dissolving in the phosphate buffered saline buffer of the 0.1mol/L of original fermented solution volume 1/10 pH7.0, the centrifugal supernatant liquor that obtains is crude enzyme liquid, adopt Ni ion affinity chromatography purifying opd of the present invention, then remove impurity with the PBS damping fluid dialysis of pH7.4, and with the PBS damping fluid of pH7.4, concentration dilution to 100 μ g/ml(purified product is proved that through Beijing Liuhe Huada Genomics Technology Co., Ltd's order-checking it is SEQ ID No.2).Molecular biology test guide (Sambrook J, Russell DW, the third edition) is seen in concrete operations.
SEQ?ID?No.1
GCCGCACCGC?AGGTGCGCAC?CTCGGCCCCC?GGCTACTACC
GGATGCTGCT?GGGCGACTTTGAAATCACCG?CGCTGTCGGA
CGGCACGGTG?GCGCTGCCGG?TCGACAAGCG
GCTGAACCAGCCGGCCCCGA?AGACGCAGAG?CGCGCTGGCC
AAGTCCTTCC?AGAAAGCGCC?GCTCGAAACCTCGGTCACCG
GTTACCTCGT?CAACACCGGC?ACCAAGCTGG?TGCTGGTGGA
CACTGGCGCGGCCGGCCTGT?TCGGCCCCAC?CCTGGGCCGG
CTGGCGGCCA?ACCTCAAGGC?CGCAGGCTATCAGCCCGAGC
AGGTCGACGA?GATCTACATC?ACCCACATGC?ACCCCGACCA
CGTGGGCGGCTTGATGGTGG?GTGAGCAACT?GGCGTTCCCG
AACGCGGTGG?TGCGTGCGGA?CCAGAAAGAAGCCGATTTCT
GGCTCAGCCA?GACCAACCTC?GACAAGGCCC?CGGACGACGA
GAGCAAAGGCTTCTTCAAAG?GCGCCATGGC?CTCGCTGAAC
CCCTATGTGA?AGGCCGGCAA?GTTCAAGCCTTTCTCGGGGA
ACACCGACCT?GGTGCCCGGC?ATCAAAGCGC?TGGCCAGCCA
CGGCCACACCCCGGGCCACA?CCACCTACGT?GGTCGAAAGC
CAGGGGCAAA?AGCTCGCCCT?GCTCGGCGACCTGATACTCG
TCGCCGCGGT?GCAGTTCGAC?GACCCCAGCG?TCACGACCCA
GCTCGACAGCGACAGCAAGT?CCGTCGCGGT?GGAGCGCAAG
AAGGCCTTCG?CGGATGCCGC?CAAGGGCGGCTACCTGATCG
CGGCGTCCCA?CCTGTCGTTC?CCCGGCATCG?GCCACATCCG
CGCCGAAGGCAAGGGCTACC?GTTTCGTGCC?GGTGAACTAC
TCGGTCGTCA?ACCCCAAGTG?A
SEQ?ID?No.2
AAPQVRTSAP?GYYRMLLGDF?EITALSDGTV?ALPVDKRLNQ
PAPKTQSALA?KSFQKAPLET?SVTGYLVNTG?TKLVLVDTGA
AGLFGPTLGR?LAANLKAAGY?QPEQVDEIYI?THMHPDHVGG
LMVGEQLAFP?NAVVRADQKE?ADFWLSQTNL?DKAPDDESKG
FFKGAMASLN?PYVKAGKFKPF?SGNTDLVPG?IKALASHGHT
PGHTTYVVES?QGQKLALLGD?LILVAAVQFD?DPSVTTQLDS
DSKSVAVERK?KAFADAAKGG?YLIAASHLSF?PGIGHIRAEG
KGYRFVPVNY?SVVNPK
SEQ?ID?No.3
AAPQVRTSAP?GYYRMLLGDF?EITALSDGTV?ALPVDKRLNQ
PAPKTQSALA?KSFQKAPLET?SVTGYLVNTG?SKLVLVDTGA
AGLFGPTLGR?LAANLKAAGY?QPEQVDEIYI?THMHPDHVGG
LMVGEQLAFP?NAVVRADQKE?ADFWLSQTNL?DKAPDDESKG
FFKGAMASLN?PYVKAGKFKP?FSGNTDLVPG?IKALASHGHT
PGHTTYVVES?QGQKLALLGD?LILVAAVQFD?DPSVTTQLDS
DSKSVAVERK?KAFADAAKGG?YLIAASHLSF?PGIGHIRAEG
KGYRFVPVNY?SVVNPK
Embodiment 2: organic phosphorus pesticide degradation of the present invention is than the mensuration of vigor
Respectively with parathion-methyl and Chlorpyrifos 94 as substrate, measure the ratio vigor of opd of the present invention and wild-type enzyme.
The production method of wild-type enzyme is: with opd gene mpd(GenBank accession number: JQ686087) as pcr template, other operation stepss are carried out according to the method for embodiment 1 fully.
Take parathion-methyl as substrate, as follows than the measuring method of vigor:
(1) preparation of p-NP standardized solution: take 20 μ M Tris-HCl(pH8.0) damping fluid is as solvent, and preparing respectively p-NP concentration is the p-NP standardized solution of 10 μ M, 20 μ M, 30 μ M, 40 μ M, 50 μ M, 60 μ M.
(2) drafting of p-NP typical curve: the p-NP standardized solution of different concns is measured absorbancy under the 410nm wavelength, X-coordinate is p-NP concentration, and ordinate zou is absorbancy, the drawing standard curve.
(3) draw the Tris-HCl(pH8.0 of 980 μ L20 μ M) damping fluid, 10 μ L parathion-methyl solution (10mg/ml, solvent is ethanol), be placed in the 2.2mL centrifuge tube, adding 10 μ L after 30 ℃ of temperature bath 10min is pesticide degradable enzyme of the present invention or the wild-type enzyme of 100 μ g/ml through the concentration of embodiment 1 purifying, 1min is reacted in concussion under 30 ℃ immediately, reaction adds 20 μ L6mol/L hydrochloric acid termination reactions after finishing immediately, then get 0.5mL termination reaction liquid, glycine-the sodium hydrate buffer solution (pH10.0) that is 0.05mol/L with concentration is settled to 5mL, measure absorbancy under the 410nm wavelength, with the sample that first adds 6mol/L hydrochloric acid as blank, contrast p-NP typical curve (Fig. 3), calculate enzyme activity and compare vigor.Take parathion-methyl as substrate, enzyme activity (U) is defined as: at 30 ℃, under the condition of pH8.0, the 1min reaction generates the 1 needed enzyme amount of μ mol p-NP.
Than vigor=enzyme activity (U)/every mg zymoprotein
(4) than vigor calculation formula:
X=(A×10.2×0.001)/(k×0.1×0.01×1)
In formula: X---than vigor (U/mg)
A---absorbancy
10.2---the multiple that enzymatic reaction system is diluted
0.001---enzymatic reaction system volume (L)
K---p-NP slope of standard curve
0.1---enzyme liquid concentration (mg/ml)
0.01---enzyme liquid amasss (ml)
1---the reaction times (min)
Take Chlorpyrifos 94 as substrate, as follows than the measuring method of vigor:
(1) preparation of Chlorpyrifos 94 standardized solution: to contain the 0.1%(volume percent) 20 μ MTris-HCl(pH8.0 of tween 80) damping fluid is as solvent, and preparing respectively Chlorpyrifos 94 concentration is the Chlorpyrifos 94 standardized solution of 10 μ M, 20 μ M, 30 μ M, 40 μ M, 50 μ M, 60 μ M.
(2) drafting of Chlorpyrifos 94 typical curve: the Chlorpyrifos 94 standardized solution of different concns is measured absorbancy under the 291nm wavelength, X-coordinate is Chlorpyrifos 94 concentration, and ordinate zou is absorbancy, the drawing standard curve.(3) enzyme activity detects: the Tris-HCl(pH8.0 that draws 2.91ml20 μ M) damping fluid, 30 μ L Chlorpyrifos 94 solution (10mg/ml, solvent is ethanol), 30 μ L10%(volume percent) tween 80 solution (solvent is the Tris-HCl pH8.0 damping fluid of 20 μ M), 30 μ L are opd of the present invention or the wild-type enzyme of 100 μ g/ml through the concentration of embodiment 1 purifying, be placed in quartz cuvette, react 1min after mixing under 30 ℃, the variation of absorbancy before and after detection reaction under the 291nm wavelength, contrast Chlorpyrifos 94 typical curve (Fig. 4), calculate enzyme activity and compare vigor.Take Chlorpyrifos 94 as substrate, enzyme activity (U) is defined as: at 30 ℃, and under the condition of pH8.0, the 1min 1 needed enzyme amount of μ mol Chlorpyrifos 94 of degrading.
Than vigor=enzyme activity (U)/every mg zymoprotein.
(4) than vigor calculation formula:
X=(A
1-A
2)×0.003/(k×0.1×0.03×1)
In formula: X---than vigor (U/mg)
A
1---absorbancy before reaction
A
2---absorbancy after reaction
0.003---enzymatic reaction system volume (L)
K---Chlorpyrifos 94 slope of standard curve
0.1---enzyme liquid concentration (mg/ml)
0.03---enzyme liquid amasss (ml)
1---the reaction times (min)
Opd of the present invention and wild-type enzyme than vigor measurement result as shown in Figure 1, in the situation that with Chlorpyrifos 94 as substrate, the ratio vigor of opd of the present invention is 4.6 times of wild-type specific activity of enzyme; In the situation that with parathion-methyl as substrate, the ratio vigor of opd of the present invention is 2 times of wild-type specific activity of enzyme.Opd of the present invention has the enzyme activity more outstanding than wild-type enzyme.
Embodiment 3: the mensuration of opd thermostability of the present invention
Be the opd of the present invention of 100 μ g/ml and wild-type enzyme respectively at after thermal treatment 10min in 40 ℃, 42.5 ℃, 45 ℃, 47.5 ℃, 50 ℃, 52.5 ℃, 55 ℃, 57.5 ℃, 60 ℃ with the purified concentration that obtains in embodiment 1, be positioned over immediately and keep 30min on ice, take Chlorpyrifos 94 as substrate, measure enzyme activity, analyze thermostability.
The production method of wild-type enzyme is: with opd gene mpd(GenBank accession number: JQ686087) as pcr template, other operation stepss are carried out according to the method for embodiment 1 fully.
The measuring method of enzyme activity is as follows:
(1) preparation of Chlorpyrifos 94 standardized solution: to contain the 0.1%(volume percent) 20 μ MTris-HCl(pH8.0 of tween 80) damping fluid is as solvent, and preparing respectively Chlorpyrifos 94 concentration is the Chlorpyrifos 94 standardized solution of 10 μ M, 20 μ M, 30 μ M, 40 μ M, 50 μ M, 60 μ M.
(2) drafting of Chlorpyrifos 94 typical curve: the Chlorpyrifos 94 standardized solution of different concns is measured absorbancy under the 291nm wavelength, X-coordinate is Chlorpyrifos 94 concentration, and ordinate zou is absorbancy, the drawing standard curve.(3) enzyme activity detects: the Tris-HCl(pH8.0 that draws 2.91ml20 μ M) damping fluid, 30 μ L Chlorpyrifos 94 solution (10mg/ml, solvent is ethanol), 30 μ L10%(volume percent) tween 80 solution (solvent is the Tris-HCl pH8.0 damping fluid of 20 μ M), 30 μ L are opd of the present invention or the wild-type enzyme of 100 μ g/ml through above-mentioned heat treated concentration, be placed in quartz cuvette, react 1min after mixing under 30 ℃, the variation of absorbancy before and after detection reaction under the 291nm wavelength, contrast Chlorpyrifos 94 typical curve (Fig. 4), calculate enzyme activity.Take Chlorpyrifos 94 as substrate, enzyme activity (U) is defined as: at 30 ℃, and under the condition of pH8.0, the 1min 1 needed enzyme amount of μ mol Chlorpyrifos 94 of degrading.(4) enzyme activity calculation formula:
X=(A
1-A
2)×0.003/(k×0.03×1)
In formula: X---enzyme activity (U/ml)
A
1---absorbancy before reaction
A
2---absorbancy after reaction
0.003---enzymatic reaction system volume (L)
K---Chlorpyrifos 94 slope of standard curve
0.03---enzyme liquid amasss (ml)
1---the reaction times (min)
After above-mentioned thermal treatment, the enzyme activity determination result of opd of the present invention and wild-type enzyme as shown in Figure 2, the temperature of the T50(enzyme activity of opd of the present invention loss one half) it is 50 ℃, higher than T50(48.5 ℃ of wild-type enzyme).Opd of the present invention has the thermostability more outstanding than wild-type enzyme.
Claims (3)
1. the opd of random mutation transformation is characterized in that:
The opd of described random mutation transformation has the aminoacid sequence of SEQ ID No.2.
2. the opd of random mutation according to claim 1 transformation is characterized in that:
It is that to have aminoacid sequence by replacement be that 1 amino acid sites of the opd MPH that derives from Pseudomonas stutzeri strain ZK-5 of SEQ ID No.3 produces, and described amino acid sites is to be positioned at the replacement of the 71st; Replacement at the 71st is to replace Serine with Threonine;
The opd that described random mutation is transformed has the aminoacid sequence of SEQ ID No.2.
3. the encoding gene of the opd of a random mutation claimed in claim 1 transformation, the encoding gene DNA molecular, it has the nucleotide sequence of SEQ ID No.1.
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| CN104726426A (en) * | 2013-12-23 | 2015-06-24 | 北京百川飞虹生物科技有限公司 | High-activity methyl parathion hydrolase mutant and use thereof |
| CN108410779A (en) * | 2018-05-14 | 2018-08-17 | 中国科学院生态环境研究中心 | A kind of microbial inoculum including Meng Shi pseudomonads |
| CN109517814A (en) * | 2018-11-28 | 2019-03-26 | 硅羿科技（上海）有限公司 | The mutant of organic phosphorus degrading enzyme and its application |
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| CN101732819B (en) * | 2008-11-14 | 2012-07-25 | 中国科学院沈阳应用生态研究所 | Organic phosphorus degrading enzyme preparation and preparation method thereof |
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| CN101732819B (en) * | 2008-11-14 | 2012-07-25 | 中国科学院沈阳应用生态研究所 | Organic phosphorus degrading enzyme preparation and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726426A (en) * | 2013-12-23 | 2015-06-24 | 北京百川飞虹生物科技有限公司 | High-activity methyl parathion hydrolase mutant and use thereof |
| CN108410779A (en) * | 2018-05-14 | 2018-08-17 | 中国科学院生态环境研究中心 | A kind of microbial inoculum including Meng Shi pseudomonads |
| CN108410779B (en) * | 2018-05-14 | 2019-11-05 | 中国科学院生态环境研究中心 | A kind of microbial inoculum comprising Meng Shi pseudomonad |
| CN109517814A (en) * | 2018-11-28 | 2019-03-26 | 硅羿科技（上海）有限公司 | The mutant of organic phosphorus degrading enzyme and its application |
| CN109517814B (en) * | 2018-11-28 | 2021-06-01 | 硅羿科技（上海）有限公司 | Mutant of organophosphorus degrading enzyme and application thereof |
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