Cloning, Overexpression, and Characterization of a Thermostable, Organic Solvent-Tolerant Laccase from <i>Bacillus pumilus</i> ARA and Its Application to Dye Decolorization

Abstract

A thermostable and organic solvent-tolerant bacterial laccase from <i>Bacillus pumilus</i> ARA has been expressed heterologously and characterized, which shows potential decolorization capacity to various types of industrial synthetic dyes. The optimal temperature and pH were 85 °C and 3.5, respectively, while the purified recombinant laccase B.P.Lacc was stable under 55-75 °C and pH 5.0-8.0 conditions. The apparent kinetic parameters <i>K</i> _m and <i>V</i> _max of B.P.Lacc for ABTS as the substrate were 0.33 mM and 32.4 U/mg, respectively. Ethanol (1%, v/v) and methanol (2%, v/v) could stimulate the enzyme activity. The recombinant laccase retained over 95% of its initial activity in 10% (v/v) methanol. The optimal expression conditions for the laccase production of B.P.Lacc in LB medium were obtained: induction temperature of 25 °C, 0.4 mM Cu²⁺, and 1.0 mM IPTG added into the culture. After 5 h, the final laccase production was 1283 U/mL. Moreover, the laccase activity increased to 4822 U/mL after follow-up 2 h stationary cultivation, with about a 3.76-fold increase. The purified B.P.Lacc was able to efficiently decolorize synthetic dyes combined with mediators. Adding 1.0 mM ABTS, more than 90% of BRRB was decolorized by the enzyme, whether at pH 4.0 or pH 7.9. The outstanding enzymatic properties suggested that B.P.Lacc may be suitable for a wide application in future biodegradation fields.