Purification and characterization of catechol 1,2-dioxygenase (EC 1.13.11.1; catechol-oxygen 1,2-oxidoreductase; C12O) using the local isolate of phenol-degrading Pseudomonas putida.

Publisher: Springer Country of Publication: United States NLM ID: 0376757 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1874-9356 (Electronic) Linking ISSN: 00155632 NLM ISO Abbreviation: Folia Microbiol (Praha) Subsets: MEDLINE

Publication: New York : Springer
Original Publication: Praha, Academia Scientiarum Bohemoslovenica.

Pseudomonas putida*/enzymology ; Pseudomonas putida*/genetics ; Pseudomonas putida*/metabolism ; Biodegradation, Environmental* ; Catechol 1,2-Dioxygenase*/metabolism ; Catechol 1,2-Dioxygenase*/genetics ; Phenol*/metabolism ; Soil Microbiology*; Phylogeny ; Bacterial Proteins/metabolism ; Bacterial Proteins/genetics ; Bacterial Proteins/chemistry ; Bacterial Proteins/isolation & purification ; Hydrogen-Ion Concentration ; RNA, Ribosomal, 16S/genetics ; Temperature

The purpose of the present study was to purify and characterize the catechol 1,2-dioxygenase (EC 1.13.11.1; catechol-oxygen 1,2-oxidoreductase; C12O) enzyme from the local isolate of Pseudomonas putida. This enzyme catalyzes the initial reaction in the ortho-pathway for phenol degradation in various gram-negative bacteria, including the genus of Pseudomonas. Pseudomonads are commonly used in the biodegradation of xenobiotics due to their versatility in degrading a wide range of chemical compounds. Eighty-nine soil samples were taken from the contaminated soil of the Midland Refineries Company (MRC) of Al-Daura refinery area at Baghdad from April to August 2021. The samples were grown in a mineral salt medium containing 250 mg per L of phenol to test their ability to biodegrade phenol. The pH was adjusted to 8.0 at 30 °C using a shaking incubator for 24-48 h. A number of 62 (69.6%) isolates of the total number were able to degrade phenol efficiently. The findings of the VITEK system and the housekeeping gene 16S rDNA confirmed that out of the positive isolates for phenol degradation, 36 from 62 (58.06%) were identified as Pseudomonas spp. isolates. Those isolates were distributed as P. aeruginosa 30 (83.3%) and P. putida 6 (16.6%). The enzyme production capabilities of the isolates were evaluated, and the highest activity was 2.39 U per mg for the isolate No. 15 which it was identified as P. putida. The previous isolate was selected for enzyme production, purification, and characterization. The enzyme was purified using ion exchange and gel filtration chromatography, with a combined yield of 36.12% and purification fold of 15.42 folds. Using a gel filtration column, the enzyme's molar mass was calculated to be 69 kDa after purification. The purified enzyme was stable at 35 °C and a pH of 6.0.
(© 2023. Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i.)

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Keywords: Pseudomonas putida; Catechol 1,2-dioxygenase (EC 1.13.11.1; Catechol-oxygen 1,2-oxidoreductase; C12O); Characterization; Phenol degradation; Purification

EC 1.13.11.1 (Catechol 1,2-Dioxygenase)
339NCG44TV (Phenol)
0 (Bacterial Proteins)
0 (RNA, Ribosomal, 16S)

Date Created: 20230913 Date Completed: 20240529 Latest Revision: 20240917

20250114

10.1007/s12223-023-01090-8

37702837