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Halogenase for catalyzing formation of C-F and C-Cl bonds

A halogenase, -FDA technology, applied in the field of halogenase, can solve the problems of unsatisfactory selectivity, poor solubility of fluorine-containing inorganic substances, high toxicity, etc.

Inactive Publication Date: 2014-04-02
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reason is that in nature, CaF 2 The solubility of fluorine-containing inorganic substances is very poor, and most of them exist in the form of precipitation. Therefore, fluorine cannot be well utilized by living organisms
At present, only relying on the very small amount of natural fluorine-containing organic compounds found is far from meeting the needs of production and life.
Although some methods for the chemical synthesis of organic fluorine-containing compounds have been developed in the past few decades, due to the special chemical properties of fluorine atoms, these methods often require harsh and highly toxic chemical environments to complete the fluorination of organic compounds. , and the selectivity is not satisfactory

Method used

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  • Halogenase for catalyzing formation of C-F and C-Cl bonds
  • Halogenase for catalyzing formation of C-F and C-Cl bonds
  • Halogenase for catalyzing formation of C-F and C-Cl bonds

Examples

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Effect test

Embodiment 1

[0045] Example 1. Determination of the Halogenase Gene Sequence

[0046] 1.1 Obtaining the original gene of halogenase

[0047] Using FramePlot 4.0beta (http: / / nocardia.nih.go.jp / fp4 / ) and NCBI database BLAST method (http: / / www.ncbi.nlm.nih.gov / blast / ) to obtain Nocardia from Brazil The original gene sequence information of the halogenase (SEQ ID No 1).

[0048] 1.2 Acquisition of Halogenase Escherichia coli Optimized Expression Sequence

[0049] The halogenase gene derived from Nocardia brasiliensis in GenScript Biotechnology Co., Ltd., using Optimum Gene TM Algorithm was used to synthesize the optimized expression gene of Escherichia coli, and the halogenase gene sequence (SEQ ID No 2) with 82% identity to the original sequence was obtained. And constructed on the pUC57 simple plasmid.

[0050]

Embodiment 2

[0051] Example 2. Construction of Halogenase Expression Plasmid

[0052] restriction endonuclease Nde I and Hin dⅢ Plasmid with Halogenase Gene Linked nob A / pUC57 simple and plasmid pET28a(+) were digested with double enzymes, and the 900bp halogenase DNA fragment and the pET28a(+) vector large fragment were recovered by electrophoresis, and then the vector and the fragment were ligated with T4 polymerase solution I from TaKaRa Company, and reacted overnight at 16°C . Then transform Escherichia coli DH5 α Competent cells were spread on LB selective plates added with kanamycin antibiotic, and incubated upside down at 37°C for 15 h. The transformant that can grow on the LB plate with kanamycin may be the transformant transformed with the recombinant plasmid, select a single colony and inoculate it in the liquid LB medium containing kanamycin, and culture it on a shaker at 37 °C and 220 rpm for 15 h , to amplify the pET28a(+) plasmid that may contain the halogenase gene. ...

Embodiment 3

[0054] Embodiment 3. The acquisition of engineering bacteria

[0055] Transform the correct recombinant plasmid into Escherichia coli BL21 (DE 3 ) Competent cells, the genetically engineered bacteria pWHU2401 / BL21 (DE 3 ), and finally spread the obtained engineered bacteria on the LB selective plate added with kanamycin antibiotic, culture it upside down at 37 ℃ for about 20 h, and grow on the LB plate added with kanamycin antibiotic It is the transformant transformed with the recombinant plasmid.

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Abstract

The invention belongs to the technical field of gene engineering, and relates to halogenase capable of catalyzing formation of C-F bonds and C-Cl bonds, which is obtained through a gene recombination technology. A preparation method of the halogenase comprises the following steps: by taking a halogenase gene derived from Nocardia brasiliensis as an original gene, optimizing according to codon preference expressed by the halogenase gene in Escherichia coli, thus synthesizing an optimized halogenase gene sequence taking the Escherichia coli as an expression host; recombining the optimized halogenase gene sequence into a cloning vector pUC57 simple, performing NdeI and HindIII double digestion, and connecting to an expression vector pET28a(+) segment having the same endoenzyme site, thus obtaining an expression vector pWHU2401; and converting the expression vector pWHU2401 into an Escherichia coli competent cell to obtain an engineering bacterium capable of heterologously expressing halogenase, performing amplified fermentation, and purifying to obtain the halogenase. According to the invention, the halogenase can specifically synthesize 5'-FDA, and the catalytic activity thereof is equivalent to that of known halogenase FlA. When L-amino acid oxidase is added, the halogenase also can catalyze formation of 5'-ClDA.

Description

[0001] technical field [0002] The invention belongs to the technical field of genetic engineering, and in particular relates to obtaining a halogenase capable of catalyzing the formation of C-F bonds and C-Cl bonds through gene recombination technology. Background technique [0003] Halogenases are a large class of enzymes that catalyze halide ions (including F - , Cl - , Br - , I - ) Enzymes that form stable carbon-halogen bonds can be divided into heme-dependent, flavin-dependent, ketoglutarate-dependent, vanadium-dependent and SAM-dependent halogenases from the catalytic mechanism (C. T. Walsh, Chemistry & Biology, 2008, 15, 99-109), among which the halogenase that can catalyze the formation of C-F bonds is particularly important. [0004] Organic fluorine compounds are widely used in medicines, medical testing, pesticides and materials. The introduction of fluorine atoms into medicinal compounds can often improve the activity and stability of drugs. According to s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P19/40
CPCC12N9/1085C12P19/40C12Y205/01063
Inventor 瞿旭东王娅娅邓子新
Owner WUHAN UNIV
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