Cloning, expression in Escherichia coli, and enzymatic properties of laccasefrom Aeromonas hydrophila WL-11
Jing Wu , Kyoung-Sook Kim , Jai-Heon Lee , Young-Choon Lee
DOI:
Received June 25, 2009,Revised November 10, 2009, Accepted , Available online
Volume 22,2010,Pages 635-640
- Summary
- References
- Related Articles
A strain WL-11 with high laccase activity was isolated from activated sludge collected from the e uent treatment plant of a textile
and dyeing industry. It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis. A gene encoding
of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized. Nucleotide sequence analysis showed an
open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids. The primary structure of the enzyme predicted
the structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. The
predicted amino acid sequence showed a high homology (more than 60%) with bacterial laccases in the genome and protein databases
and the highest degree of similarity (61% identity) was observed with the multicopper oxidase of Klebsiella sp. 601. When expressed
in Escherichia coli, the recombinant enzyme was overproduced in the cytoplasm as soluble and active form. The purified enzyme
had an optimum pH of 2.6 and 8.0 for ABTS (2,2’-azino-bis(3-ethylbenzthiazolinesulfonic acid) and DMP (2,6-dimethoxyphenol),
respectively. The kinetic study on ABTS revealed a higher a nity of this enzyme to this substrate than DMP.
