Molecular and bioinformatics studies and determination of functional properties of Catechol 2,3-dioxygenase from Aneurinibacillus migulanus Znu12

Catechol 2,3-dioxygenases play a key role in the metabolism of aromatic molecules in the environment by soil bacteria. The aim of this study is to introduce the new catechol 2,3-dioxygenase and also to survey bioinformatics and biochemical characteristics in the presence of various substrates as well as to study its activity in various pH ranges for the widespread use of such enzymes in bioremediation industry.In this study, the DNA sequences of Catechol 2,3-dioxygenase were separated from Aneurinibacillus migulanus Znu12 and recorded with accession number MN197546.1 in NCBI, then it was cloned in plasmid pET28a. The accuracy of cloning and not changing the frame shift was confirmed by sequencing. After transferring of plasmid into E. coli BL21 (DE3) cells, protein expression was assessed by SDS-PAGE. Afterwards, the purification, assay and optimization of enzyme activity were evaluated.The results of PCR reaction on agarose gel indicated a sequence of 912 bp, an enzyme with 304 amino acid residues. Results of sequence alignment by ESPript3 server also confirmed highly conserved residues of His199 and Tyr 255 in the enzymes active site. Biochemical characterization of the enzyme showed that the enzyme had the highest (100%) activity at pH 7.2, while it had only 70% activity at pH 7.4. As compared with pyrogallol and phenol, catechol by optimal concentration of 0.032 was the most preferred substrate for the enzyme. Bioinformatics results confirmed also such substrate preference based on the type and position of amino acid residues in the enzyme active site.