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.
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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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