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Dioxygenase Snpd with chiral selectivity to intermediate of aromatic oxyphenoxypropionic acid herbicide, and encoding gene and application thereof

A dioxygenase, stereoselective technology, applied in the field of bioengineering

Active Publication Date: 2019-03-12
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no chiral-specific biological enzymes that can be used to resolve or transform racemic aryloxyphenoxypropionic acid herbicide intermediates have been reported.

Method used

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  • Dioxygenase Snpd with chiral selectivity to intermediate of aromatic oxyphenoxypropionic acid herbicide, and encoding gene and application thereof
  • Dioxygenase Snpd with chiral selectivity to intermediate of aromatic oxyphenoxypropionic acid herbicide, and encoding gene and application thereof
  • Dioxygenase Snpd with chiral selectivity to intermediate of aromatic oxyphenoxypropionic acid herbicide, and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Determination of Degradation S-2-(4-hydroxyphenoxy)propionic acid metabolites by strain B2 of Example 1

[0025] Determination of degradation products: Inoculate strain B2 into 100mL LB liquid medium, culture at 30°C, 180rpm / min until exponential phase, collect bacteria by centrifugation at 6000rpm / min for 10min, wash the bacteria twice with sterile basic salt medium , resuspended in 10ml basal salt medium, inoculated into 100mL inorganic salt liquid medium, and adjusted OD 600 About 1.0, add 0.2mM S-2-(4-hydroxyphenoxy) propionic acid, shake culture at 30°C, 180rpm / min on a shaker. Samples were taken regularly every 3 hours, and an equal volume of methanol was added to the regularly sampled culture solution. After shaking and mixing, the samples were centrifuged at 12000 rpm / min for 5 minutes. The samples were filtered with a 0.22 μm nylon filter membrane and detected by high performance liquid chromatography (HPLC). The HPLC chromatographic conditions are: the chroma...

Embodiment 2

[0030] Example 2 Cloning and functional verification of S-2-(4-hydroxyphenoxy) propionic acid degradation gene

[0031] 2.1 Alignment and search of dioxygenase genes

[0032] Find out the dioxygenase gene tfdA that has been reported to catalyze similar reactions by consulting relevant literature. The TfdA protein sequence was compared with the genome of strain B2, and the result was that 3 similar protein sequences were compared in the genome of strain B2.

[0033] 2.2 Heterologous expression and functional verification of putative dioxygenase genes

[0034] Using the genomic DNA of strain B2 as a template, primers were designed to amplify the putative dioxygenase gene fragment. The primers used are as follows:

[0035] Table 1 Primers used in functional verification experiments

[0036]

[0037]

[0038] Amplification system:

[0039]

[0040] Amplification procedure

[0041] a. Pre-denaturation at 95°C for 3 minutes;

[0042] b. Denaturation at 95°C for 15 se...

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Abstract

The invention discloses a dioxygenase Snpd with chiral selectivity to an intermediate of an aromatic oxyphenoxypropionic acid herbicide, and an encoding gene and application thereof. The dioxygenase gene Snpd with stereoselectivity has the nucleotide sequence of SEQ ID NO.1; and the dioxygenase Snpd encoded by the dioxygenase gene Snp has the amino acid sequence of SEQ ID NO.2. An alpha-ketoglutarate dependent chiral selective dioxygenase gene snpd is successfully cloned through whole-genome sequencing and genetic comparison methods; and the gene is the first disclosed dioxygenase which can degrade S-2-(1-naphthoxy)propionic acid and S-2-(2,4-dichlorophenoxy)propionic acid, S-2-(2,4-hydroxyphenoxy)propionic acid, and herbicide 2,4-dichlorphenoxyacetic acid intermediate, and has very important theoretical value and application prospect in producing an R-type herbicide with high optical purity.

Description

technical field [0001] The invention belongs to bioengineering technology and is applied to the field of enzymatic resolution of low-activity isomers of aryloxyphenoxypropionic acid herbicide intermediates and synthesis of optically pure and effective isomers, and relates to an α-ketopentadiene Acid-dependent, low activity S-isomer with chiral selectivity dioxygenase Snpd and its coding gene and application. Background technique [0002] Chirality is a basic property of three-dimensional molecules. If a compound cannot coincide with its mirror image, the compound is called a chiral compound. Chiral pesticides are an important part of chiral compounds. At present, chiral pesticides account for about 28% of the global pesticide market, while the use of chiral pesticides in my country is even higher, accounting for about 40%. Each enantiomer of the same chiral pesticide not only has enantioselectivity for the insecticidal and herbicidal activity of target organisms, but also p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/02C12N15/70C12P7/40
CPCC12N9/0069C12N15/70C12P7/40
Inventor 蒋建东黄俊伟陈典
Owner NANJING AGRICULTURAL UNIVERSITY
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