Reducing enzyme of nitryl, phenylnitryl, encoding gene and application

A technology of nitrobenzene nitrate and coding gene, applied in the directions of oxidoreductase, application, genetic engineering, etc., can solve the problems of environmental pollution and high cost

Inactive Publication Date: 2006-07-05
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the production of aromatic compounds such as p-aminophenol and o-aminophenol uses a chemical process of adding pure hydrogen, which is not only costly (needs pure hydrogen), but also easily pollutes the environment

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1, the acquisition of the nitrophenyl nitroreductase gene of the present invention

[0022] Pick the single colony of Comamonas testosteroni CNB1 CGMCC No.1028 strain on the LB plate into the LB culture medium, shake culture at 30°C, collect the bacteria, extract the genomic DNA, and partially digest it with MboI Fragments around 30kb. SuperCos 1 was used as a vector (purchased from Stratagene Company, USA). The vector was digested with XbaI, dephosphorylated, and then cut into two fragments of 6.8 kb and 1.1 kb with BamHI enzyme. Partially digested genome fragments and processed vectors were incubated at 16°C with T 4 DNA ligase ligation. The ligated fragments were packaged with Gigapack III XL packaging protein (purchased from Stratagene Company, USA). Transfected into Escherichia coli XL 1-blue MR (purchased from Stratagene Company, USA) to establish a Cosmid gene library. Use 2-aminophenol as a substrate to screen the library, culture in 2-aminopheno...

Embodiment 2

[0023] Embodiment 2, expression and functional identification of nitrophenyl nitroreductase of the present invention

[0024] 1, the expression of nitrophenyl nitroreductase of the present invention

[0025] Genomic DNA of Comamonas testosteroni (Comamonas testosteroni) CNB1 CGMCC No.1028 strain was extracted and used as a template in primer 1: 5'-GACGTTT CATATG CCGACCAGCCCGTTC-3' (underlined bases are NdeI recognition sites, bold start codons) and primer 2: 5'-TG GGATCC Under the guidance of TAATTCGTGGACGAAGGTGG-3' (the underlined base is the BamHI recognition point, and the bold is the stop codon), PCR amplifies the nitrophenyl nitroreductase gene of the present invention. Among them, the 50μl PCR reaction system is: template (60ng / μl) 0.5μl; dNTP (each 10mM) 1μl; primer (each 25μM) 1μl; 10× buffer 5μl; ddH 2 O 42.1 μl; Taq (5U / μl) 0.4 μl. Wherein, the 10× buffer comes from TaKaRa Taq kit (TaKaRa Company, Code No.: DR100A). The PCR temperature conditions were first 94°C...

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PUM

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Abstract

This invention describes the coding gene and applications of a nitrobenzene nitro-reducing enzyme, comprising a protein from the following amino acid residue sequences: 1) SEQ ID No. 1 in the sequence table; 2) the amino acid residue sequence in SEQ ID No. 1 is a protein substituted, deleted or added with 1-10 amino acid residues and having the nitrobenzene nitro-reducing enzyme function. The nitrobenzene nitro-reducing enzyme and its coding gene in this invention play an important role in the production of ortho-aminophenols and their derivatives.

Description

technical field [0001] The invention relates to a nitrophenyl nitroreductase and its coding gene and application in the technical fields of microbial biotechnology and genetic engineering. Background technique [0002] my country has become the world's largest producer of antipyretic and analgesic drugs. The output of aspirin, paracetamol, and analgin exceeds 10,000 tons, and the output of phenacetin, aminopyrine, and antipyrine exceeds 1,000 tons. Ton. At present, the output of antipyretic and analgesic drugs in my country is growing rapidly, and it is expected to increase at a rate of about 8% per year in the future. With the growth of antipyretic and analgesic drugs, their intermediates have also achieved considerable development. In 2003, domestic paracetamol consumption increased rapidly, and exports also showed a rapid growth momentum. The export volume was 28,163 tons, and the annual export volume increased by about 1 times compared with the previous year. In 2004, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/63C12P7/22
Inventor 刘双江吴建峰刘志培
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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