Molecular cloning, expression, and bioinformatics analysis of the CueO laccase gene from Escherichia coli SDB2.

Abstract

Background: Laccase CueO, a multicopper oxidase, possesses the capability to degrade phenolic compounds. In prior research, a strain of Escherichia coli named SDB2, isolated from chicken cecum, was found to degrade sinapine (a phenolic constituent of rapeseed meal) through the secretion of laccase CueO. Herein, the cloning, expression, and bioinformatics analysis of the CueO gene derived from E. coli SDB2 are reported.

Methods and results: Sequence analysis indicated that SDB2 CueO comprised 1551 bp, 516 amino acids, a putative molecular weight of 56.65 kDa, and an isoelectric point (pI) of 6.21. BLAST comparisons showed that the CueO protein sequence from E. coli SDB2 exhibited 65-90% identity with CueO from other bacteria. Multiple alignment analysis further confirmed the similarity and identity of SDB2 CueO with CueO from other species, and the amino acids surrounding the Cu-binding sites were highly conserved. A phylogenetic tree demonstrated a close evolutionary relationship between CueO from E. coli and CueO from Citrobacter amalonaticus. The three-dimensional (3D) structural model revealed four copper (Cu)-binding regions. Recombinant CueO was successfully obtained by expressing the CueO gene in E. coli BL21 after isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Bioinformatics analysis confirmed the similarity of recombinant CueO with native CueO.

Conclusions: These findings established a basis for understanding the characteristics and functions of laccase CueO from E. coli SDB2, paving the way for future research to explore the properties of recombinant CueO and its potential practical applications in optimizing feed resources, such as rapeseed meal, in the feed production industry.