Purification, Characterization, Gene Cloning And Application Of Catechol 2,3-Dioxygenase From The Novel Strain Burkholderia Cepacia L68

Modern industry produced a variety of aromatic compounds and resulted in the environmental pollution. Bacterial expressing the enzymes of the pathways for the degradation of aromatic compounds were used to cleaning up contaminated environments.A strain that can grow on phenol as the sole source of carbon and energy, L68, was isolated, and identified as Burkholderia cepacia. The phenol-degradation conditions of the bacteria were analyzed. The optimal growth temperature is 35°C and the optimal growth pH value is 7.0. No phenol was detected after the strain growing on the inorganic salt medium containing 0.5g/L of phenol at 35°C for 24h. Strain L68 was capable of degrading catechol when the phenol concentration reached 0.8g/L. Immobilized strain L68 could degrade phenol when the phenol concentration was as high as 1.5g/L.Strain L68 was capable of degrading catechol via the meta-cleavage pathway. In this thesis, the purification and characteristics of catechol 2,3-dioxygenase from Burkholderia were reported for the first time. Culture conditions improvement for the production of catechol 2,3-dioxygenase by strain L68 was investigated using orthogonal array designs. The experimental results revealed that the volume of medium and the percent of phenol had remarkable effects on the enzyme production, while the percent of inoculation played little role in the production of the enzyme. The improved culture conditions are 250 ml medium consisted of 0.2‰ yeast extract and 19‰ phenol, pH 6.0, and 2.5 % inoculation. Catechol 2,3-dioxygenase was purified to homogeneity from the cell extract of the strain by ammonium sulfate-fraction, Sephadex G-150 and DEAE-Sepharose Fast Flow column chromatography. The specific activity of the purified enzyme was 309.66 folds as high as that of crude extract and the yield was about 55.43%. The molecular mass of enzyme subunit is 34±lkDa and the pI was about 4.2. When catechol and 4-methyl-catechol were used as substrate, the Km values of the enzyme were 4.9μM and 9.9μM, respectively. The UV spectrum of catechol 2,3-dioxygenase from L68 was the same as most of the protein and the fluorescence spectrum showed the λ _emmax was 336nm. At 20°C, the circular dichroism spectrum showed that the contents of α-helix, β-sheet, β-turn and random coil were 45.8%, 15.6%, 23.4% and 15.3%, respectively. The dependence of...