Cloning and Heterologous Expression of Laccase from Beauveria pseudobassiana PHF4 in E. coli and its Application in the Degradation of Catechol.

The rapid growth of the human population, coupled with accelerated industrialization, has led to a substantial increase in the release of toxic pollutants into the environment. Catechol is a toxic and carcinogenic phenolic compound extensively utilized in industrial processes and is recognized as a hazardous pollutant due to its persistence and adverse environmental effects. Laccase plays a crucial role in the biodegradation of catechol by catalyzing its oxidation into less toxic products, thereby contributing to the detoxification of phenolic pollutants. In the present study, the laccase gene from Beauveria pseudobassiana PHF4 was successfully amplified using PCR, inserted into the pCR 2.1 cloning vector and initially transformed into E. coli DH5α for plasmid propagation. Subsequently, the recombinant construct was introduced into E. coli BL21 (DE3) cells for heterologous expression of the gene. Induction of E. coli BL21 (DE3) cells with 0.5 mM IPTG and 0.4 mM CuSO₄ at 25 °C for 7 h led to the successful expression of recombinant laccase, achieving an enzymatic activity of 27.15 ± 0.32 U/ml. The purified recombinant laccase exhibited an approximate molecular weight of 65 kDa, as determined by SDS-PAGE analysis. The purified enzyme was further utilized for catechol degradation. To evaluate the catalytic efficiency of laccase, the enzymatic degradation of catechol was systematically monitored using HPLC. The purified laccase exhibited high catalytic activity, degrading 81.7% of catechol within 12 h and 99.47% within 24 h.