Expression and characterization of a thermostable organic solvent-tolerant laccase from Bacillus licheniformis ATCC 9945a

Abstract

Bacterial laccases have proven advantages over fungal and plant counterparts in terms of wider pH optimum, higher stability and broader biocatalytic scope. In this work, Bacillus licheniformis ATCC 9945a laccase is produced heterologously in Escherichia coli. Produced laccase exhibits remarkably high temperature optimum at 90 degrees C and possess significant thermostability and resistance to inactivation by organic solvents. Laccase has an apparent melting temperature of 79 degrees C at pH 7.0 and above 70 degrees C in range of pH 5.0-8.0, while having half-life of 50 min at 70 degrees C. Presence of 10% organic solvents such as acetonitrile, dimethylformamide, dimethylsulfoxide or methanol reduces melting temperature to 45-52 degrees C but activity remains practically unimpaired. With 50% of acetonitrile and methanol laccase retained similar to 40% of initial activity. EDTA and 300 mM sodium -chloride have positive effect on activity. Enzyme is active on syringaldazine, ABTS, phenols, amines, naphthol, lignin and lignin model compounds and mediates C-C bond formation via oxidative coupling after one electron oxidation of phenolic group. Successful polymerization of 2 -naphthol was achieved with 77% conversion of 250 mg/L 2-naphtol in only 15 min which may further expand substrate scope of this enzyme towards polymer production and/or xenobiotics removal for environmental applications.