CN117025462B - Enterococcus strain for degrading insecticide and application thereof - Google Patents
Enterococcus strain for degrading insecticide and application thereof Download PDFInfo
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- 241000194033 Enterococcus Species 0.000 title claims abstract description 19
- 230000000593 degrading effect Effects 0.000 title claims abstract description 15
- 239000002917 insecticide Substances 0.000 title claims description 5
- 239000005886 Chlorantraniliprole Substances 0.000 claims abstract description 54
- PSOVNZZNOMJUBI-UHFFFAOYSA-N chlorantraniliprole Chemical compound CNC(=O)C1=CC(Cl)=CC(C)=C1NC(=O)C1=CC(Br)=NN1C1=NC=CC=C1Cl PSOVNZZNOMJUBI-UHFFFAOYSA-N 0.000 claims abstract description 52
- 238000004321 preservation Methods 0.000 claims abstract description 8
- 238000006731 degradation reaction Methods 0.000 claims description 21
- 230000015556 catabolic process Effects 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 9
- 241001522957 Enterococcus casseliflavus Species 0.000 claims description 6
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- 230000007017 scission Effects 0.000 claims description 3
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- 238000009472 formulation Methods 0.000 claims 1
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- -1 pyridine ring compound Chemical class 0.000 claims 1
- 239000000575 pesticide Substances 0.000 abstract description 25
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
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- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
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- 241000894007 species Species 0.000 description 2
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- 241000256846 Apis cerana Species 0.000 description 1
- 241001124181 Bactrocera dorsalis Species 0.000 description 1
- 241000255789 Bombyx mori Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
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- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
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- 241001312569 Ribes nigrum Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 101000693622 Spodoptera frugiperda Allatotropin Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
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- 238000012544 monitoring process Methods 0.000 description 1
- FTQWRYSLUYAIRQ-UHFFFAOYSA-N n-[(octadecanoylamino)methyl]octadecanamide Chemical compound CCCCCCCCCCCCCCCCCC(=O)NCNC(=O)CCCCCCCCCCCCCCCCC FTQWRYSLUYAIRQ-UHFFFAOYSA-N 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
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Abstract
The invention discloses an enterococcus casei strain for degrading pesticides and application thereof, wherein the preservation name of the enterococcus casei strain is: EMBL-3, accession number: china center for type culture Collection, date of preservation: 2023, 17, 07, accession number: cctccc M20231305. The strain is identified by gene sequence analysis as enterococcus buttermidis of enterococcus, named EMBL-3, and belongs to gram-positive bacteria. The strain can be used for degrading chlorantraniliprole and has the prospect of further research and development and engineering application in the fields of agricultural pest control, novel green pesticide development, environmental pesticide pollutant restoration and the like.
Description
Technical Field
The invention relates to a bacterial strain, in particular to an enterococcus buttermidis strain and application thereof.
Background
Spodoptera frugiperda is an extremely destructive important pest which gives out global early warning by the grain and agriculture organization of the united nations, has rapidly spread and spread a plurality of provinces in China since the first invasion of the Burma into the south of China in 12 months of 2018, has a continuous north expansion trend, and forms a serious threat to the production of various important crops. In order to ensure the safe production of crops, the use of pesticides, especially bisamide pesticides represented by chlorantraniliprole, is the first recommended medicament for preventing and controlling spodoptera frugiperda at present, and plays a key role in the process of preventing and controlling spodoptera frugiperda. The environmental problems and drug resistance problems caused by the unreasonable and unscientific use of pesticides are increasingly serious, and according to the recent resistance monitoring data, the high-level resistance of spodoptera frugiperda to chlorantraniliprole is monitored in various parts of the United states, while the domestic spodoptera frugiperda field population is still at a low resistance level to chlorantraniliprole, but has a continuous rising trend, and the situation is not optimistic. The efficient control of spodoptera frugiperda, which severely pulls the development of new pesticide resistance, poses a great threat to the quality and quantity conservation production of crops, and the increased pesticide amount for successful pest control permeates into the environment in a large amount, so that the problem of environmental pollution is not negligible. Therefore, it is particularly important to excavate and develop pesticide degrading bacteria which can be used for 'gram-resistant' targets and restoration of pesticide polluted environments.
In recent years insect gut has been considered as a natural reactor containing various genes for nutritional synthesis and xenobiotic metabolic activity, which houses multifunctional, diverse microorganisms as an important resource pool in nature. An important reason for resistance development of pests is the enhanced ability of the pests to detoxify and metabolize pesticides, and there is growing evidence that symbiotic bacteria are important participants in detoxication and metabolism of pest pesticides. In insect pests such as Apis cerana, bactrocera dorsalis and the like, the colonization of a plurality of symbiotic bacteria can directly degrade the pesticide to enhance the drug resistance of a host, and in addition, a large number of pesticide degrading bacteria derived from insect intestinal tracts are found at present, and the bacterial isolates can be effectively developed as environmental remediation and heterologous substance degradation and transformation. However, there is no study reported to date that enterococcus strains are involved in the degradation and transformation of pesticides. We isolated an enterococcus strain Enterococcus casseliflavus EMBL-3 from Spodoptera frugiperda larva intestinal microorganisms and demonstrated the ability of the strain to degrade the pesticide chlorantraniliprole. The casseliflavus exists in almost all lepidopteran pests, so that the casseliflavus has great application prospect in the fields of pest control technology and environmental remediation and control.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an enterococcus strain and application thereof, which can provide a new strategy and a new method for solving the selection of pest drug resistance targets and the pollution of pesticide chlorantraniliprole in the environment.
The enterococcus strain is enterococcus buttermidis strain (Enterococcus casseliflavus), and the preservation information is as follows: the preservation name is EMBL-3, the preservation unit: china center for type culture Collection, date of preservation: 2023, 17, 07, accession number: cctccc M20231305.
The strain in the research is novel in source, is isolated from the intestinal tract of invasion of the caterpillar fungus spodoptera frugiperda larva, is a beneficial strain isolated from the intestinal tract of pests, and is proved to be applicable to the degradation of chlorantraniliprole by the research, and is an enterococcus member of which the first strain is found to be capable of degrading the chlorantraniliprole.
The strain is bacteria-enterococcus casei EMBL-3, and enterococcus casei is a common symbiotic bacterium of lepidoptera pest intestinal tract and is a very important tryptophan producer in silkworms. The above study shows that the strain is a beneficial symbiotic strain of most lepidopteran pests, however, no report on the degradation and conversion capability of the strain to pesticides is yet seen.
In addition, the invention also provides a pesticide pollution repairing agent, namely a preparation containing the strain or a culture solution or dry powder thereof, and the strain or the culture thereof can be used for degrading the pesticide. The invention can be based on a method for identifying the microbial degradation pesticide active protein, can primarily screen and identify the potential active protein for degrading chlorantraniliprole, and is beneficial to research on pest drug resistance target selection.
The beneficial effects of the invention are as follows:
the invention discovers that the strain can obviously degrade chlorantraniliprole under the condition of short-time liquid culture (3 days), and can reduce the concentration of the chlorantraniliprole by 25 percent. The potential active proteins for degradation of chlorantraniliprole can be identified by screening the proteome results of pre-exposure of the chlorantraniliprole for intracellular and extracellular proteins, and further speculated as the degradation mechanism, cleavage of the chemical bond of the chlorantraniliprole molecule, possibly involving amide bond cleavage and dehalogenation.
The specific liquid culture conditions are as follows: the tests were divided into four groups: (1) minimum inorganic salt liquid medium (MSM) was added with 10mg/L chlorantraniliprole as a control group; (2) adding 10mg/L chlorantraniliprole and EMBL-3 strain culture into minimum inorganic salt liquid culture medium (MSM); (3) minimum inorganic salt liquid Medium (MSM) was added with 100mg/L of intracellular protein extract of EMBL-3; (4) a minimum inorganic salt broth (MSM) was added with 100mg/L of the extracellular protein extract of EMBL-3. All treatments were incubated at 30℃in a temperature-controlled shaker at 150rpm/min for 3 days, during which time detection was performed at the set time points (1 d,2d and 3 d).
Drawings
FIG. 1 growth status of EMBL-3 strain on LB plates;
FIG. 2 strain growth curves for EMBL-3 strain and chlorantraniliprole during 3 days of culture;
FIG. 3EMBL-3 strain degradation efficiency of chlorantraniliprole;
FIG. 4 degradation efficiency of intracellular and extracellular proteins of EMBL-3 strain on chlorantraniliprole;
FIG. 5 response of intracellular and extracellular proteins of EMBL-3 strain to chlorantraniliprole;
FIG. 6EMBL-3 strain versus potential degradation protein of chlorantraniliprole;
FIG. 7 mass spectrum of EMBL-3 strain versus chlorantraniliprole degradation product;
FIG. 8 potential degradation process of chlorantraniliprole by EMBL-3 strain.
Detailed Description
The invention will be further described with reference to the accompanying drawings and specific examples.
Example 1, isolation culture and identification of enterococcus buttermidis EMBL-3 (Enterococcus casseliflavus EMBL-3):
the feed was fed with chlorantraniliprole sublethal concentration (LC 50 ) Spodoptera frugiperda larvae surviving the last 2 days were dissected under aseptic conditions to obtain their intestinal tracts. Combining 15 intestinal tracts into a group, adding normal saline, grinding and vibrating to obtain an intestinal tract content mixed solution, and carrying out gradient dilution on the intestinal tract content mixed solution for 10 -1 To 10 -7 Double-coating on solid inorganic salt culture medium plate containing chlorantraniliprole, culturing at 30deg.C, collecting single colony after single colony grows to a certain size, enlarging culturing on enriched LB liquid culture medium, further streaking, collecting single colony for purification, and subculturing again after visible colony grows on the plate until pure fungus is obtainedA strain. Then inoculating the pure strain into a liquid inorganic salt culture medium containing chlorantraniliprole again for culturing, wherein the strain can grow by taking the chlorantraniliprole as the only energy source substance, namely the chlorantraniliprole degradation strain, and is marked as an EMBL-3 strain.
Example 2 identification of Strain EMBL-3
After obtaining the EMBL-3 strain, the strain identification is carried out on the strain, and the species classification is judged. Thus, the species classification of the EMBL-3 strain was identified by 16S rDNA sequencing technology, the full length of the gene was amplified by using primers 27F and 1492R, and the strain was identified as enterococcus buttermidis (see additional material 1) after 16S rRNA sequencing and comparison with the National Center for Biotechnology Information (NCBI) ribosomes, designated EMBL-3, belonging to gram positive bacteria, which were off-white in colony, translucent in edge, smooth and moist in surface, regular in edge, round, no halo, central bulge, smooth in colony, and sticky (FIG. 1).
The strain is enterococcus cassis and is preserved under the following names: EMBL-3, accession number: china center for type culture Collection, date of preservation: 2023, 17, 07, accession number: cctccc M20231305.
EXAMPLE 3 degradation of Chlorantraniliprole by strain EMBL-3
The culture solution obtained by liquid culture of the EMBL-3 strain can be used for degrading chlorantraniliprole.
The liquid culture condition is that the EMBL-3 strain is inserted into a 50mL triangular flask filled with 20mL of inorganic salt liquid culture medium according to 10% inoculation amount, chlorantraniliprole is added into the culture medium, the culture is carried out at the temperature of 30 ℃ at 220rpm of a shaking table, meanwhile, a control group is arranged, namely, only chlorantraniliprole is added into the liquid inorganic salt culture medium, the EMBL-3 strain is not added, sampling is carried out on 1 st, 2 nd and 3 rd days of culture respectively, and the EMBL-3 can slowly grow in the inorganic salt culture medium taking the chlorantraniliprole as the only carbon source (figure 2). The chlorantraniliprole in the culture medium is extracted by ethyl acetate, and is detected by high performance liquid chromatography, and the concentration of the chlorantraniliprole can be obviously reduced by 25% after 3 days of culture, which shows that EMBL-3 can effectively degrade the chlorantraniliprole in a short time (figure 3).
EXAMPLE 4 identification of active protein of strain EMBL-3 for degradation of Chlorantraniliprole
Active proteins potentially associated with chlorantraniliprole degradation can be screened and identified by the pattern of response of the EMBL-3 intracellular and extracellular proteins to chlorantraniliprole and compared to controls.
Protein degradation experiments showed that extracellular secreted protein was about 18.6% better than intracellular protein degradation of chlorantraniliprole (fig. 4). By proteomic analysis of the response pattern of the intracellular proteins and extracellular secreted proteins of EMBL-3 during degradation of chlorantraniliprole, extracellular proteins were found to be more responsive to chlorantraniliprole than intracellular proteins, with up to 543 of the differentially expressed proteins (283 down-regulated expression, 260 up-regulated expression), more up-regulated expressed proteins were detected extracellular than intracellular proteins (30 down-regulated expressed proteins and 44 up-regulated expressed proteins) (fig. 5), with 18 proteins being selected, including four N-acetyltransferases, methyltransferases, dehalogenases, etc., defined as potential chlorantraniliprole degradation proteins (fig. 6). The potential degradation process of EMBL-3 p-chlorantraniliprole can be initially proposed to be the cleavage of amide bond and dehalogenation reaction through the function of protein and the mass spectrum result prediction of the combined degradation products (figure 7), and benzene ring derivatives and pyridine ring compounds are generated (figure 8).
The specific detection method comprises the following steps: intracellular and extracellular protein extraction: EMBL-3 is cultured in inorganic salt culture medium containing chlorantraniliprole for 3 days, centrifugated at 5000rpm for 10 minutes, the supernatant and the precipitate are respectively collected, firstly, the supernatant is added with an equal volume of acetone precooled at-20 ℃, stirred for 2 hours in an ice bath, all liquid is collected, centrifugated at 14000rpm for 20 minutes, and the precipitate is redissolved by PBS, thus the extracellular protein is obtained. The collected bacterial cells are crushed based on an ultrasonic crushing method, and the supernatant is centrifugally collected to be intracellular proteins.
In conclusion, the enterococcus buttermidis EMBL-3 and the culture solution thereof can effectively degrade the pesticide chlorantraniliprole and can be applied to pesticide pollution restoration. Therefore, the invention also provides an insecticide pollution repairing agent which contains the strain or the culture solution of the strain, and the insecticide pollution repairing agent is sprayed on a to-be-repaired object when in use. With further research on enterococcus casseliflavus EMBL-3, the enterococcus casseliflavus EMBL-3 is found to be very likely to have degradation effect on other types of pesticides or pesticides, and is worthy of more extensive and intensive research.
The foregoing is only a partial embodiment of the present invention, and many variations and modifications will be apparent to those skilled in the art in light of the present description, drawings and claims, without departing from the spirit and scope of the invention as defined in the appended claims. Any modification, modification or equivalent variation of the above embodiments according to the technical ideas and entities of the present invention falls within the scope of protection defined by the claims of the present invention.
Additional Material 1
16S rRNA gene sequence of EMBL-3 strain
AGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCTTTTTCTTTCACCGGAGCTTGCTCCACCGAAAGAAAAAGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACACTATTTTCCGCATGGAAGAAAGTTGAAAGGCGCTTTTGCGTCACTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAAAATGTTCATCCCTTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTCTAGAGATAGAGCTTCCCCTTCGGGGGCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTTAGTTGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAGTACAACGAGTTGCGAAGTCGCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCTAAGGTGGGATAGATGATTGGGGTGAAGTCGTAACAAGGTAACC
Claims (7)
1. Enterococcus casseliflavus @Enterococcus casseliflavus) Strain characterized by the deposit name: EMBL-3, accession number: china center for type culture Collection, date of preservation: 2023, 17, 07, accession number: cctccc M20231305.
2. Use of an enterococcus buttermidis strain according to claim 1 for degrading the insecticide chlorantraniliprole.
3. A method for degrading chlorantraniliprole, comprising culturing the strain of claim 1 and contacting the chlorantraniliprole to be degraded with a strain culture.
4. The method for degrading chlorantraniliprole according to claim 3, wherein the temperature required for degradation is 20-40 ℃.
5. A method for degrading chlorantraniliprole according to claim 3, wherein the concentration of chlorantraniliprole is reduced by 25% within 3 days of contact.
6. A method for degrading chlorantraniliprole according to claim 3, wherein the potential degradation process of EMBL-3 p-chlorantraniliprole is cleavage of amide bond and dehalogenation reaction, resulting in benzene ring derivative and pyridine ring compound.
7. A pesticidal contamination repair agent comprising the strain of claim 1 or a culture solution or dry powder formulation of said strain.
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| JP2000044949A (en) * | 1998-05-25 | 2000-02-15 | Shinei Ferumentekku:Kk | Soil improver and method for improving soil |
| CN1990503A (en) * | 1993-11-08 | 2007-07-04 | 康诺特实验室有限公司 | Method for producing purified recombinant haemophilus influenzae transferrin binding proteins |
| CN103764814A (en) * | 2011-04-29 | 2014-04-30 | 丹尼斯科美国公司 | Production of mevalonate, isoprene, and isoprenoids using genes encoding polypeptides having thiolase, HMG-COA synthase and HMG-COA reductase enzymatic activities |
| CN113151102A (en) * | 2021-05-13 | 2021-07-23 | 西湖大学 | Klebsiella strain for degrading plastics and application thereof |
| CN114107109A (en) * | 2021-11-29 | 2022-03-01 | 宜宾五粮液股份有限公司 | Enterococcus casseliflavus and application thereof in production of caproic acid through microbial fermentation |
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| US20210221720A1 (en) * | 2020-01-20 | 2021-07-22 | Chio Kang Medical, Inc. | Pathogenic bacteria and method for degrading ethylene oxide |
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| CN1990503A (en) * | 1993-11-08 | 2007-07-04 | 康诺特实验室有限公司 | Method for producing purified recombinant haemophilus influenzae transferrin binding proteins |
| JP2000044949A (en) * | 1998-05-25 | 2000-02-15 | Shinei Ferumentekku:Kk | Soil improver and method for improving soil |
| CN103764814A (en) * | 2011-04-29 | 2014-04-30 | 丹尼斯科美国公司 | Production of mevalonate, isoprene, and isoprenoids using genes encoding polypeptides having thiolase, HMG-COA synthase and HMG-COA reductase enzymatic activities |
| CN113151102A (en) * | 2021-05-13 | 2021-07-23 | 西湖大学 | Klebsiella strain for degrading plastics and application thereof |
| CN114107109A (en) * | 2021-11-29 | 2022-03-01 | 宜宾五粮液股份有限公司 | Enterococcus casseliflavus and application thereof in production of caproic acid through microbial fermentation |
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