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WO2016015668A1 - Enzyme cfh de dégradation de pesticides à base de carbamate, gène cfd codant correspondant et applications des deux - Google Patents

Enzyme cfh de dégradation de pesticides à base de carbamate, gène cfd codant correspondant et applications des deux Download PDF

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Publication number
WO2016015668A1
WO2016015668A1 PCT/CN2015/085603 CN2015085603W WO2016015668A1 WO 2016015668 A1 WO2016015668 A1 WO 2016015668A1 CN 2015085603 W CN2015085603 W CN 2015085603W WO 2016015668 A1 WO2016015668 A1 WO 2016015668A1
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WIPO (PCT)
Prior art keywords
cfd
gene
cfh
carbamate
carbamate pesticide
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Ceased
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PCT/CN2015/085603
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English (en)
Chinese (zh)
Inventor
闫新
洪青
李顺鹏
谷涛
何健
蒋建东
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Nanjing Agricultural University
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Nanjing Agricultural University
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    • A—HUMAN NECESSITIES
    • A62—LIFE-SAVING; FIRE-FIGHTING
    • A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
    • A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
    • A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the field of application environment microorganisms and agriculture, and relates to the application of the enzyme CFH capable of degrading carbamate pesticides such as carbofuran and its coding gene cfd and both.
  • Carbamate pesticides such as carbofuran are a class of insecticides that are widely used worldwide.
  • the toxicity mechanism of this kind of pesticide is similar to that of organophosphorus pesticides, mainly inhibiting cholinesterase activity.
  • Carbamate pesticides can invade the body through the respiratory tract and digestive tract, and can also be slowly absorbed through the skin and mucous membranes, mainly in liver, kidney, fat and muscle tissues. In recent years, due to its high toxicity and frequent pesticide poisoning incidents, some varieties have been restricted or even banned in countries around the world.
  • carbofuran In the case of carbofuran, the EU and Canada have been banned, the United States has been banned from any crop (human consumption); in the 2014 edition of the list of Chinese national banned and restricted pesticides, carbofuran is a restricted pesticide, vegetables, fruit trees, tea trees, Chinese herbal medicines. It is the scope of its ban. This means that carbamate pesticides such as carbofuran are still being used in large quantities, and their residues in soil and agricultural products are still problems we have to face and solve.
  • the object of the present invention is to provide a carbamate-based pesticide degrading enzyme CFH such as carbofuran and its coding gene cfd and the use thereof, in view of the above-mentioned deficiencies of the prior art.
  • the gene cfd can be used to construct transgenic microorganisms or plants that degrade carbamate pesticides, and can also be used to produce enzyme preparations for degrading carbamate pesticides for eliminating carbamate pesticide residues in soils, water bodies and agricultural products.
  • the starting strain of the gene cfd, Sphingobium sp. YBL3 was deposited in the China Center for Type Culture Collection under the accession number CCTCC NO: M208076.
  • the strategy for cloning the carbamate pesticide degrading enzyme gene is the shotgun method.
  • the total DNA of the strain YBL3 was first extracted, and the total DNA was digested with Sau3AI, and then ligated with BamHI-digested plasmid pUC118, and the enzyme-linked product was transformed into E. coli DH5 ⁇ competent cells to obtain a total DNA library.
  • the clones capable of degrading carbofuran were screened by high performance liquid chromatography (HPLC), and then the clones of the degrading ability were sequenced and analyzed to determine the open reading frame (ORF) of the target gene.
  • ORF open reading frame
  • the open reading frame size of the target gene was 2139 bp, named cfd
  • the encoded enzyme was named CFH. This is the first report of a broad-spectrum, highly efficient degrading enzyme gene for carbamate pesticides.
  • the enzyme CFH encoded by the cfd gene has the amino acid sequence of SEQ ID NO.
  • a recombinant expression vector containing the cfd gene A recombinant expression vector containing the cfd gene.
  • the recombinant expression vector inserts the cfd gene between the NdeI and EcoRI sites of pET-29a(+).
  • the starting strain of the genetically engineered bacteria is Escherichia coli BL21 (DE3).
  • the present invention successfully cloned the gene cfd from the strain YBL3 (CCTCC NO: M208076) by the shotgun method.
  • GenBank indicated that the gene is a new gene with a full length (from start codon to stop codon) of 2139 bp, encoding 712 amino acids.
  • the degradation enzyme CFH or the engineered bacteria carrying the cfd gene in the present invention can efficiently degrade carbamate pesticides.
  • the gene cfd can be used to construct transgenic microorganisms or plants that degrade carbamate pesticides, and can also be used to produce enzyme preparations for degrading carbamate pesticides for eliminating carbamate pesticide residues in soils, water bodies and agricultural products.
  • Fig. 2 is a mass spectrometric detection diagram of the gene cfd in BL21 (pET-29a(+)) expressing the product degrading carbofuran to furanol.
  • Figure 2a is a first-order mass spectrum of the degradation product;
  • Figure 2b is a secondary mass spectrum of the substance S1 (carbofuran);
  • Figure 2c is a secondary mass spectrum of the substance S2 (furanol).
  • Sphingomobacter YBL3, classified as Sphingobium sp. YBL3, is deposited in the China Center for Type Culture Collection (CCTCC) at Wuhan University, Wuhan, China, with the accession number CCTCC NO: M208076, and the date of deposit is May 22, 2008. .
  • CTCC China Center for Type Culture Collection
  • YBL3 (CCTCC NO: M208076) was cultured in liquid LB medium (37 ° C, 200 rpm, 18 h), and the total DNA of the high-purity, large-segment strain YBL3 genome was extracted by CTAB method, and dissolved in TE buffer (pH 8.0).
  • the specific method refers to the "Guide to the Experimental Guide to Molecular Biology" edited by F. Osb.
  • the total DNA after digestion was purified by electrophoresis (TAE buffer) and recovered by axygen biosciences (China) recovery kit.
  • TAE buffer electrophoresis
  • the recovered DNA was dissolved in 10 mmol/L Tris ⁇ HCl (pH 8.0) and placed in - Deposited at 20 ° C.
  • the liquid phase conditions are: chromatography column, Kromasil 100-5C18 (4.6mm ⁇ 250mm ⁇ 5 ⁇ m); mobile phase, methanol / water (volume ratio 70/30); flow rate, 1ml ⁇ Min -1 ; UV detector, wavelength 280 nm and 230 nm; injection volume, 20 ⁇ l.
  • the positive clone capable of degrading carbofuran obtained in (6) was entrusted to Shanghai Yingjun Biotechnology Co., Ltd. for sequencing, and the nucleotide sequence of the carbofuran degrading enzyme gene cfd was SEQ ID NO. 1, according to the gene cfd nucleoside.
  • the 712 amino acid sequence to which the acid sequence is derived is SEQ ID NO.
  • Forward primer 5'-TCTGGA CATATG TCTAGTGACAAACTCCAT-3' (SEQ ID NO. 3) and reverse primer: 5'-TCTGGA GAATTC ATTGTCTTTTTGCATCA-3' (SEQ ID NO. 4) as primers, using PCR from strain YBL3
  • the gene cfd sequence was amplified from the total DNA.
  • the reaction was carried out for 3 hours or more in a 37 ° C water bath.
  • the digested product was subjected to 2% agarose gel electrophoresis.
  • the recombinant plasmid pET-29a(+)-cfd was transformed into the expression host strain BL21 (DE3) to obtain recombinant microorganism BL21 (CFH) containing cfd.
  • BL21 (CFH) was cultured in LB medium to an OD 600 nm of 0.6 to 0.8, and IPTG was added to a concentration of 1 mM, and cultured at 30 ° C for 4 hours. Centrifuge 100 ml of the bacterial solution, resuspend the cells in 10 ml (50 mM, pH 7.0) PBS buffer, ultrasonically disrupt (Auto Science, UH-650B ultrasonic processor, 30% intensity) for 5 minutes, centrifuge, collect the supernatant, and use nickel ions to pro The supernatant was purified by chromatography and the carbofuran hydrolase CFH was obtained.
  • Reaction system for hydrolyzing carbamate pesticides by CFH 50 mM phosphate buffer (pH 7.0), 50 mg ⁇ L -1 carbaryl, isoprocarb or carbaryl, CFH 50 ⁇ l, and reacted at 30 ° C for 30 min.
  • the reaction was quenched by the addition of 3 ml of dichloromethane, and the product was extracted with vigorously shaking.
  • the organic phase was dried over anhydrous sodium sulfate, dried with nitrogen, and then dissolved in 100 liters of methanol.
  • the test results show that CFH can also efficiently degrade carbaryl, isoprocarb and carbaryl.
  • the sources of microorganisms used in the present invention are as follows:

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  • Engineering & Computer Science (AREA)
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  • Organic Chemistry (AREA)
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  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Business, Economics & Management (AREA)
  • Emergency Management (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)

Abstract

L'invention concerne une enzyme CFH de dégradation de pesticides à base de carbamate, un gène cfd codant correspondant et des applications des deux. Le cadre de lecture ouvert du gène cfd codant de l'enzyme de dégradation de pesticides à base de carbamate possède une longueur totale de 2139 bp et la séquence représentée par SEQ ID No 1, et le produit CFH codé contient 712 acides aminés et comprend la séquence représentée par SEQ ID No 2. CFH peut dégrader des pesticides à base de carbamate comme le carbofuran.
PCT/CN2015/085603 2014-08-01 2015-07-30 Enzyme cfh de dégradation de pesticides à base de carbamate, gène cfd codant correspondant et applications des deux Ceased WO2016015668A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201410378234.XA CN104178504B (zh) 2014-08-01 2014-08-01 氨基甲酸酯类农药降解酶CFH与其编码基因cfd以及二者的应用
CN201410378234.X 2014-08-01

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CN104178504B (zh) * 2014-08-01 2017-02-01 南京农业大学 氨基甲酸酯类农药降解酶CFH与其编码基因cfd以及二者的应用
CN109232724B (zh) * 2018-09-28 2021-07-27 东北农业大学 降低农药霜霉威残留的基因CsMAPEG及其应用

Citations (5)

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Publication number Priority date Publication date Assignee Title
CN101293131A (zh) * 2007-04-29 2008-10-29 上海普天欣生物技术有限公司 一种消除残留农药的复合酶制剂及应用
CN101338286A (zh) * 2008-08-15 2009-01-07 南京农业大学 一种绿麦隆农药残留降解菌及其生产的菌剂
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CN102164951A (zh) * 2008-07-09 2011-08-24 联邦科学技术研究组织 用于水解苯脲、氨基甲酸酯和有机磷酸酯的酶和方法
CN104178504A (zh) * 2014-08-01 2014-12-03 南京农业大学 氨基甲酸酯类农药降解酶CFH与其编码基因cfd以及二者的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
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CN101293131A (zh) * 2007-04-29 2008-10-29 上海普天欣生物技术有限公司 一种消除残留农药的复合酶制剂及应用
CN102164951A (zh) * 2008-07-09 2011-08-24 联邦科学技术研究组织 用于水解苯脲、氨基甲酸酯和有机磷酸酯的酶和方法
CN101338286A (zh) * 2008-08-15 2009-01-07 南京农业大学 一种绿麦隆农药残留降解菌及其生产的菌剂
CN101684459A (zh) * 2008-09-22 2010-03-31 上海普天欣生物技术有限公司 CehA突变体蛋白、基因、重组载体及其用途和制备方法
CN104178504A (zh) * 2014-08-01 2014-12-03 南京农业大学 氨基甲酸酯类农药降解酶CFH与其编码基因cfd以及二者的应用

Non-Patent Citations (1)

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Title
SUN JIQUAN ET AL.: "Isolation and characterization of three Sphingobium sp. strains capable of degrading isoproturon and cloning of the catechol 1,2-dioxygenase gene from these strains", WORLD J MICROBIOL BIOTECHNOL, vol. 25, 5 November 2008 (2008-11-05), pages 259 - 268, XP019690897, ISSN: 1573-0972 *

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