2-amino phenol 1, 6-dioxygenase, its gene and use thereof

A dioxygenase and aminophenol technology, applied in genetic engineering, oxidoreductase, plant gene improvement, etc., can solve the problems of biotransformation of chloronitrobenzene compounds that have not been reported.

Inactive Publication Date: 2005-06-01
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In addition, bioprocessing and transformation using enzymatic reactions is an emerging bioengineering technology in recent years. It can be transformed and processed in a very economical and efficient way under normal temperature and pressure conditions to obtain high-purity and high-quality products. It has already been used in biopharmaceuticals It has been applied in the field of biochemical industry, such as the production of chiral drugs and the production of acrylamide, but no report has been seen in the biotransformation of chloronitrobenzene compounds

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Cloning of 2-aminophenol 1,6-dioxygenase gene

[0019]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 the total 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 digested with XXba I, dephosphorylated, and then cut into two fragments of 6.8 kb and 1.1 kb with BamH I enzyme. The partially digested genome fragment and the treated vector were ligated with T4 DNA ligase at 16°C. The ligated fragments were packaged with Gigapack IIIXL packaging protein (purchased from Stratagene Company, USA). Finally, it was transfected into Escherichia coli XL 1-blue MR (purchased from Stratagene Company, USA) to establish a Cosmid gene library. Use 2-aminophenol as the substrate to screen the library, incubate at 30°C for two days, the clones whose solution is ...

Embodiment 2

[0020] Embodiment 2: Contain the transformant construction and the expression of enzyme of the gene provided by the invention

[0021] After the gene provided by the invention is connected to the carrier SuperCos 1, it is packaged with Gigapack III XL packaging protein, or the gene can also be connected to other expression vectors, and then the packaged vector or connected vector is transformed into competent Escherichia coli or other Bacteria are cultured in LB medium or other medium, and the colony containing the linked gene fragment is the transformant. The transformant using SuperCos 1 as the vector can express the enzyme activity without induction, while the transformation using other expression vectors as the vector Substitutes need the corresponding inducer of the vector to induce the expression of enzyme activity. These transformants can be used for the production of enzymes or these transformants can be directly used for the treatment of waste water containing p-nitro...

Embodiment 3

[0022] Example 3: Determination of 2-aminophenol 1,6-dioxygenase activity

[0023] The reaction product of 2-aminophenol, 2-aminomuconic acid semialdehyde, has a maximum absorption peak at 380nm. The increase in the light absorption value at 380nm can be measured by an ultraviolet spectrophotometer, and the enzyme specific activity of dioxygenase can be measured. The reaction system includes 0.3 μmol of 2-aminophenol, 1 mL of 50 mmol / L phosphate buffer, cell extract containing 0.3-0.9 mg of protein, and the total volume of the enzymatic reaction is 3 mL. The molar extinction coefficient of 2-aminomuconic acid semialdehyde ε=15.1L mmol -1 cm -1 . One unit of enzyme activity is defined as 1 mg of protein produces 1 nmol of 2-aminomuconic acid semialdehyde per minute.

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Abstract

The present invention relates to one p-nitrobenzene chloride ring opening enzyme gene and its expression product. The gene is from Comamonas testosteroni CNB1 CGMCC No. 1028 strain, has size of 1770 bp, and contains two ORF opening read frames in the same direction of 942 bp and 813 bp separately, separated by 15 bp, coding two different protein products beta-subunit and alpha-subunit. Two beta-subunits and two alpha-subunits constitute one active enzyme molecule. The enzyme can catalyze the ring opening cracking in the 1 and the 6 place of 2-amino phenol and the direct ring opening cracking of p-nitrobenzene chloride. Therefore, the gene may be used in constituting gene engineering bacterian and produce active enzyme for treating sewage containing p-nitrobenzene chloride and similar compound and in biological conversion engineering.

Description

technical field [0001] The invention belongs to the technical field of microbial biotechnology and genetic engineering, in particular, it relates to a gene derived from Comamonas testosteroni CNB1 CGMCCNo.1028 bacterial strain, the gene size is 1770 bp base pairs, its An active enzyme is expressed, which can catalyze the ring-opening and cleavage of 1 and 6 positions of 2-aminophenol, and can also catalyze the direct ring-opening and cleavage of p-nitrochlorobenzene. Therefore, the gene can be used to construct genetically engineered bacteria, produce active enzymes, be used for sewage treatment containing p-nitrochlorobenzene compounds, and be used for biotransformation processing engineering. technical background [0002] Chloronitrobenzenes (CNBs), as important chemical intermediates, are widely used in the production and manufacture of rubber, dyes, pesticides, pharmaceuticals, etc. Taking p-chloronitrobenzene as an example, the output in my country in 2000 was 12 tons. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C02F3/34C12N9/02C12N15/53C12S99/00
Inventor 刘双江吴建峰刘志培
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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