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Dismutase of hydroxyamino-benzene, its coded gene, and application

A technology encoding gene and dismutase, which is applied in the field of axanil dismutase and its encoding gene and application, can solve the problems of high cost and environmental pollution

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 o-aminophenol and o-hydroxyaminobenzene aromatic compounds adopts techniques such as iron powder reduction method or liquid-phase catalytic hydrogenation method, which not only has high cost (requires pure hydrogen), but also easily causes pollution to the environment (produces a large amount of iron slime with organic poison)

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1, the acquisition of the axanil dismutase gene of the present invention

[0022] Pick a single colony of Comamonas testosteroni CNB1 CGMCC No.1028 strain on LB plate into LB culture medium, shake culture at 30°C, collect the bacteria, extract genomic DNA, and partially digest with Mbo I Fragments up to about 30kb. SuperCos 1 was used as a vector (purchased from Stratagene Company, USA). The vector was first digested with Xba I, dephosphorylated, and then cut into two fragments of 6.8 kb and 1.1 kb with BamH I 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-aminophenol aqueous sol...

Embodiment 2

[0023] Embodiment 2, the expression and functional identification of axanil dismutase of the present invention

[0024] 1, the expression of azanol dismutase 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'-GTCC GAATTC AAGGAGACCCCCTTCATGCC-3' (underlined base is the EcoR I recognition site, bold is the start codon) and primer 2: 5'-GTCA AAGCTT Under the guidance of TGCGGGAAGTCTGATGGT-3' (the underlined base is the Hind III recognition site, and the bold is the stop codon), PCR amplifies the hydroxylamine dismutase 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 (5 U / 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 for ...

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PUM

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Abstract

This invention describes a hydroxylamine benzene dismutase, its coding gene and applications. The hydroxylamine benzene dismutase in this invention comprises 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 hydroxylamine benzene dismutase 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 hydroxylamine dismutase and its encoding gene and application in the technical fields of microbial biotechnology and genetic engineering. Background technique [0002] O-aminophenol is an intermediate in the production of dyes, medicines, and spices. It is mainly used to manufacture naphthol AS-PH, widely used in the dye industry, and also used in pharmaceutical technology and spice industries, such as: o-vanillin, etc. Therefore, with the development of printing and dyeing industry, spice industry and medical industry, the market demand for intermediates for synthesizing these products will also continue to increase. In addition, aromatic compounds have both amino groups and hydroxyl groups in adjacent positions, which can make such aromatic compounds polymerized into polymer compounds and form some molecular materials with new physical and chemical properties. [0003] At present, the production of o-aminophenol and o-hydr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/52C12N15/63C12P7/22
Inventor 刘双江吴建峰刘志培
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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