Catechol 2,3-dioxygenase from Pseudomonas sp. strain ND6: gene sequence and enzyme characterization

Area of Science:

• Microbiology
• Biochemistry
• Molecular Biology

Background:

• Naphthalene degradation is crucial for environmental cleanup.
• Catechol 2,3-dioxygenase (C23O) is a key enzyme in naphthalene catabolism.
• Understanding C23O diversity can improve bioremediation strategies.

Purpose of the Study:

• To clone and sequence the C23O gene from Pseudomonas sp. ND6.
• To express and purify the C23O enzyme.
• To compare the biochemical properties of Pseudomonas sp. ND6 C23O with other known C23Os.

Main Methods:

• Gene cloning and sequencing of catechol 2,3-dioxygenase (C23O) from Pseudomonas sp. ND6.
• Gene overexpression in Escherichia coli using the pUC18 vector and lac promoter.
• Enzyme purification using DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography.
• Enzymology experiments to assess specific activity and thermostability.

Main Results:

• The C23O gene from Pseudomonas sp. ND6 consists of 924 nucleotides, encoding a 36 kDa polypeptide.
• High amino acid sequence identity was observed between Pseudomonas sp. ND6 C23O and those from Pseudomonas putida G7 (93%) and Pseudomonas stutzeri AN10 (89%).
• The purified C23O from Pseudomonas sp. ND6 exhibited superior specific activity and thermostability compared to the C23O from Pseudomonas putida G7.

Conclusions:

• The catechol 2,3-dioxygenase (C23O) gene from Pseudomonas sp. ND6 has been successfully cloned, sequenced, and expressed.
• The C23O enzyme from Pseudomonas sp. ND6 demonstrates enhanced biochemical properties, including higher specific activity and thermostability.
• These findings suggest potential applications for Pseudomonas sp. ND6 C23O in bioremediation and industrial processes.