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CN-121991983-A - 4-Chlorocatechol 1,2 dioxygenase CatA and encoding gene cat A and application thereof

CN121991983ACN 121991983 ACN121991983 ACN 121991983ACN-121991983-A

Abstract

The invention discloses 4-chlorocatechol 1,2 dioxygenase CatA and a coding gene cat A and application thereof. The cat A has a nucleotide sequence of SEQ ID NO.1 and an amino acid sequence of SEQ ID NO.2, can simultaneously degrade 4-chlorocatechol and catechol enzymes, has good stability in a higher temperature range and a wider pH range, has obvious differences between the gene sequences and known functional genes, has important significance in constructing genetic engineering bacteria for efficiently degrading chlorinated aromatic pollutants, developing novel bio-enhancement treatment processes and preparations, and provides a brand-new technical approach for solving the environmental problem of intractable chlorinated organic pollution, and has remarkable social and economic benefits.

Inventors

  • LI NA
  • WANG BOHAN
  • HU SHIYU
  • GAO AO
  • ZHANG HAO

Assignees

  • 南阳师范学院

Dates

Publication Date
20260508
Application Date
20260324

Claims (6)

  1. 1. A4-chlorocatechol 1,2 dioxygenase gene catA is characterized in that the nucleotide sequence is SEQ ID NO.1.
  2. 2. The use of the 4-chlorocatechol 1,2 dioxygenase gene catA of claim 1 in the construction of transgenic engineering bacteria for the degradation of catechol and 4-chlorocatechol.
  3. 3. Use of the 4-chlorocatechol 1,2 dioxygenase gene catA of claim 1 in the preparation of a formulation for degrading or removing catechol, 4-chlorocatechol, and related chlorinated aromatic hydrocarbon contaminants in soil, water.
  4. 4. A4-chlorocatechol 1,2 dioxygenase CatA, characterized in that the nucleotide sequence of the 4-chlorocatechol 1,2 dioxygenase gene CatA is encoded by the nucleotide sequence of the 4-chlorocatechol 1,2 dioxygenase gene of claim 1, and the amino acid sequence is SEQ ID NO.2.
  5. 5. Use of 4-chlorocatechol 1,2 dioxygenase CatA according to claim 4 for the preparation of an enzyme preparation for the degradation of 4-chlorocatechol and catechol.
  6. 6. Use of 4-chlorocatechol 1,2 dioxygenase CatA of claim 4 in the preparation of a formulation for removal of catechol, 4-chlorocatechol, and related chlorinated aromatic hydrocarbon contaminants in soil, water.

Description

4-Chlorocatechol 1,2 dioxygenase CatA and encoding gene cat A and application thereof Technical Field The present invention relates to the field of environmental microbiology and agricultural technology. In particular to 4-chlorocatechol 1,2 dioxygenase CatA and a coding gene cat A and application thereof. Background Aromatic compounds are a class of nondegradable pollutants commonly existing in industrial wastewater of paper making, petrochemical industry, pesticides, medicines, dyes and the like. Among them, chlorinated aromatic compounds, such as chlorophenols, have more stable structure, higher biotoxicity, teratogenicity and carcinogenicity due to the introduction of chlorine atoms with strong electronegativity and large steric hindrance on benzene rings, become typical representatives of persistent organic pollutants in the environment, and are a serious challenge for the environmental field in terms of efficient degradation and treatment. In the metabolic pathways of microorganisms for aerobic degradation of chlorinated aromatic compounds, they are usually first converted into chlorocatechol as a key intermediate metabolite. Among these, 4-chlorocatechol is a core intermediate in the degradation pathways of a variety of important contaminants (e.g., chlorinated aromatic hydrocarbon contaminants such as 5-chlorosalicylic acid, 2, 4-dichlorophenoxyacetic acid, 4-chlorophenol, and 2, 4-dichlorophenol). The benzene ring opening reaction of catechol intermediates is the rate limiting step in the whole degradation process, and directly determines the flux and degradation efficiency of the whole metabolic pathway. The key enzyme catalyzing this step is catechol 1, 2-dioxygenase (EC 1.13.11.1), which catalyzes the cleavage (ortho cleavage) of the catechol benzene ring between two adjacent hydroxyl groups in the presence of molecular oxygen, generating mucofuroic acid, thereby opening the benzene ring structure and allowing it to enter the subsequent central metabolism. For many years, a variety of catechol 1, 2-dioxygenases from different microbial strains have been isolated, purified and characterized, the corresponding coding genes (commonly designated catA genes) of which have also been cloned and sequenced. However, further studies revealed that these known enzymes have the following prominent technical drawbacks and limitations when applied to the degradation of chlorinated aromatic contaminants: 1. The known catechol 1, 2-dioxygenases mostly show a high catalytic efficiency and specificity towards catechol, the natural substrate of catechol, with low catalytic activity towards chlorinated substrates or complete absence. However, this high degree of specificity results in a structurally similar chlorinated substrate, such as 4-chlorocatechol, which tends to exhibit very low affinity and catalytic rates. The molecular mechanism is that the spatial structure of the active center of the enzyme cannot effectively contain chlorine atoms, or the strong electron-withdrawing effect of the chlorine atoms reduces the electron cloud density of catechol rings, so that the enzyme is difficult to effectively combine with the catechol rings and catalyze the ring opening of the catechol rings. 2. The enzyme stability is insufficient, and the practical application environment is difficult to adapt, and the enzyme activity and stability of many known catechol 1, 2-dioxygenase can be drastically reduced under the environment deviating from the optimal condition. For example, in an actual industrial wastewater treatment scenario, the pH may fluctuate widely (meta-acid or meta-base), the temperature may be high, or inhibitors of heavy metals, organic solvents, etc. are present in the wastewater. Known enzymes are susceptible to inactivation under these harsh conditions, resulting in their efficacy in practical applications far below laboratory ideal conditions. 3. The known enzyme genes have high homology and function convergence, the homology among the sequences of the currently reported catA genes capable of efficiently degrading catechol is generally higher (more than 80 percent), the coded enzymes are similar in catalytic property, and further research on the catarrh genes is likely not to have unexpected new functions breaking through the bottleneck of the prior art. Therefore, how to obtain the novel catechol 1, 2-dioxygenase can effectively overcome the defects of the prior art, has important significance for constructing genetic engineering bacteria for efficiently degrading chlorinated aromatic pollutants and developing novel bio-enhancement treatment process and preparation, and also provides a brand-new technical means for solving the environmental problem of refractory chlorinated organic pollution. Disclosure of Invention Therefore, the technical problem to be solved by the invention is to provide the 4-chlorocatechol 1,2 dioxygenase CatA, the coding gene cat A and the application thereof, which can effectively