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Phenol hydroxylase gene and uses thereof

A phenol hydroxylase and gene technology, applied in organic chemistry, sugar derivatives, bacteria, etc., can solve the problems of no treatment method and poor treatment effect of aromatic compounds

Inactive Publication Date: 2004-06-02
INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the phenol concentration is too high, the activated sludge will produce toxicity inhibition, resulting in poor treatment effect (such as the phenol concentration is 800mg / L, the activated sludge disintegrates)
Therefore, there is no feasible treatment method for aromatic compounds widely present in wastewater from printing and dyeing, pharmaceutical, rubber, petrochemical and other industries.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Screening of PHEA-2 strain

[0033] Use a variety of aromatic compounds as carbon sources for preliminary screening from phenol-containing refining wastewater (specific method: take 1ml of phenol-containing wastewater, add sterile water to 10ml, shake and shake, and apply 100μl of chlorobenzene, phenol, toluene, Catechol 200mg / l each on LB plates), respectively inoculated on MS plates or upper liquid medium containing 3mM phenol, cultures were cultured at 30°C, and single colonies that grew significantly in one day were selected. The 16S rDNA sequence analysis and physiological and biochemical determination of a microbial strain that can grow well with phenol as the sole carbon source shows that the strain is resistant to chloramphenicol and ampicillin. It was inoculated on MS medium with 3mM phenol as the sole carbon source. In, sampling every 2-3 hours to determine the phenol content in the culture broth (the phenol content is determined according to the nation...

Embodiment 2

[0039] Example 2: Construction of PHEA-2 strain gene library

[0040] The conventional method (CTAB method, Wilson K. Preparation of genomic DNA from bacteria. Preparation of genomic from bacteria. 1987. P.2.10-2.12. In FMAusubul, R. Bent, et al. Current protocols in molecular biology.J.Wiley & Sons, New York, NY), extract the total DNA of Acinetobacter calcoaceticus PHEA-2, use appropriate amount of restriction endonuclease Sau3A I at 37 ℃, water bath conditions 50 ~ 100μg The total DNA was partially digested, and the digested fragments were electrophoresed on 0.4% agarose gel. After staining with ethidium bromide, the agarose gel containing 15-23 kb fragments was cut under ultraviolet light and placed in a 1.5ml centrifuge tube. Add 3 to 4 times the volume of Binding Buffer, and react for about 7 minutes at 55-65°C in a water bath until the gel is completely dissolved. Place the DNA / agarose solution in the recovery column and centrifuge at room temperature for 1 minute at 10,000...

Embodiment 3

[0041] Example 3: Cloning of phenol hydroxylase gene library

[0042] By comparing the nucleotide sequence of the phenol hydroxylase gene of different phenol-degrading bacteria, a pair of specific primers Lphl (5′-AGG CAT CAA GAT CAC CGA CTG-3) and Lph2 (5′-CGC) of the gene were designed CAG AAC CATTTA TCG ATC-3′), a PCR product of 684 bp was obtained. Using this product as a probe, 6 positive recombinant phages were screened from the gene library by hybridization, and the foreign DNA of the positive recombinant phage P21 was analyzed. Sequence analysis showed that the sequence contained 4668bp of all phenol hydroxylase genes, and the insert contained 6 complete open reading frames (ORF), named ORF2~ORF7, respectively. The deduced amino acids of the sequence were similar to those of A.CalcoaceticusNCIB8250 phenol hydroxylase. The 6 subunits MopK, L, M, N, O, P have 58.5%, 72.4%, 80.9%, 93.5%, 60.8%, 84.7% homology. ORF2~ORF7 are called mphK, L in Table 1. , M, N, O, P.

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PUM

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Abstract

The present invention relates to phenol hydroxylase gene and its use. The present invention provides metabolic encoding gene capable of converting phenol into catechol; constitutes HF conjugal transfer plasmid; and obtains genetic engineering strain capable of degradation to phenol or enlarging degradation range of substrate. When the phenol hydroxylase gene is transferred to immobile calcium acetate bacillus PHEA-2, the phenol degrading capacity of the strain will be raised. When the hydroxylase gene is transferred to toluene degrading bacillus, the bacillus strain will obtain phenol degrading capacity and enlarged substrate degrading range.

Description

Technical field: [0001] The present invention relates to a phenol hydroxylase gene cloned from Acinetobacter calcoaceticus PHEA-2, and the use of the gene in increasing or enhancing the ability of recipient bacteria to degrade phenol. Background technique: [0002] Phenol is a priority pollutant listed in my country as a pollutant that seriously pollutes the environment and harms human health. The use of environmental biotechnology is currently the main method to eliminate such compounds in the environment. The core of biological treatment is to oxidize and decompose organic matter in wastewater into inorganic matter through the metabolic activities of microorganisms. The effect of the treatment has a great relationship with the microbial community structure and quantity in the treatment system, and the degradation ability of degrading bacteria. However, the microbial community structure in the existing treatment system is unreasonable, the degradation ability of degrading bacter...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C12N1/21
Inventor 徐玉泉陈明张维林敏
Owner INST OF AGRO FOOD SCI & TECH CHINESE ACADEMY OF AGRI SCI
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