Purification and Characterization of a New Thermostable Laccase from Bacillus licheniformis O12 Using One-Step Affinity Chromatography and Its Potential for Decolorization

Abstract

Laccases are copper-containing enzymes that can oxidize a wide variety of substrates. Thanks to this feature of laccase, some dyes that cause environmental pollution can be decolorized. Some bacteria, such as Bacillus licheniformis, naturally produce the enzyme laccase. A new affinity column was tested in this study. For this purpose, the extracellular laccase sepharose 4B-L-tyrosine-ρ-aminobenzoic acid produced by bacteria grown in suitable media was isolated by affinity chromatography method. Its purity was checked by SDS-PAGE method. The decolorization effect of some dyestable in textile wastewater of laccase isolated from B. lichenisformis O12 by affinity column was investigated. No mediator was used in this procedure. .As a result, laccase was purified 4.82-fold purification with a yield of 38.3% respectively, The molecular weight of the purified enzyme was determined as ~70 kDa by the SDS-PAGE method. The enzyme showed optimum activity at pH 4.0 and temperature 92°C. The enzyme was found to retain 100% activity even after 12 hours of incubation at 60°C and 92°C. The kinetic parameters were determined with laccase substrates such as ABTS, 2,6-DMP, and guaiacol. The purified laccase was decolorized with varied efficiencies such as 35% of Reactive black, 31% of Acid black 1, 28% of Methylene blue, and 15% of Acid red 27 without the use of any redox mediators. These properties of B. licheniformis O12 laccase enzyme make it a potential candidate enzyme for use in various biotechnological and industrial applications.