Bioinformatic analysis and characterization of myxobacteria laccase-like multicopper oxidases

Abstract:

Laccase is a widely-used environment-friendly copper-containing oxidase found in many plants, insects and fungi. Recently, more and more laccases are also found in bacteria. Myxobacteria are an important bacteria resource. However, myxobacteria are much more difficult to isolate and purify than other bacteria. We used bioinformatic approach to screen myxobacteria proteomes available in NCBI. Based on conserved sequences of 4 copper binding sites in multicopper oxidase, 30 potential laccase sequences were obtained. Among them, 9 genes were synthesized and expressed in Escherichia coli BL21 (DE3). Seven proteins showed laccase activity when tested with traditional laccase substrates. One protein, named rSC-2, was chosen for further research because it exhibited the highest activity towards 2,6-dimethyl phenol (DMP). The molecular weight of rSC-2 was 57 kDa. Its specific activity to DMP was 0.27 U/mg. The optimal temperature and the optimal pH were 60 ℃ and 7.0, respectively. About 50% of the original activity was retained after incubation at 60 ℃ and pH 7.0?8.0 for 1 h. Metals showed different effects on rSC-2. rSC-2 activity was enhanced by several metalsat concentration of 1 mmol/L, such as Ca2+ and Mn2+. With a higher concentration of 5 mmol/L, the activity of rSC-2 was apparently inhibited. This is the first report of bioin for matics screening myxo bacteria laccasesin combination with expression in E. coli.

Key words:Bioinformatics, myxobacteria laccase, recombinant expression, separation and purification, enzymology characterization