Optimized Laccase production from the white rot fungi Pleurotus ostreatus and Trametes versicolor

Laccases are predominantly found in bacteria, fungi, plants, and insects, and they have numerous industrial and biotechnological applications. Laccases are utilized in the pharmaceutical, food, pulp, and paper industries, and their cost-effective production in submerged culture conditions is of significant commercial value. Attempts were made to overexpress three laccase isoforms from Trametes versicolor (TV) in heterologous systems. Recombinant TV laccases were either insoluble in E.coli or poorly expressed in Pichia pastoris. Hence a submerged fermentation process was developed to produce these commercially important laccases from two non-recombinant white rot fungi: Pleurotus ostreatus (PO) and Trametes versicolor (TV). Molasses and corn steep liquor (CSL) were used as carbon and nitrogen sources, respectively, while 2,5-xylidine, sodium lignosulfonate, and copper sulfate were used as inducers, making the entire process economical. Laccase activity reached a maximum of 374,000 U/L (or 374 kU/L) in 20 days. When evaluated at 45 °C, TV laccase outperformed PO laccase in terms of stability, a crucial factor in the delignification of biomass. TV laccase demonstrated superior stability over PO laccase at 45 °C, making it the preferred choice for biomass pre-treatment applications. We demonstrate the use of this laccase in two industrial applications.

1. Lignocellulose Depolymerization: Treatment of rice straw with Pleurotus ostreatus laccase resulted in visible structural distortion, as observed under a scanning electron microscope.

2. Industrial Wastewater Treatment: Significant decolorization of indigo carmine and Remazol Brilliant Blue dyes was achieved overnight, with reductions of up to 70 % and 74 %, respectively, when incubated with Trametes versicolor laccase.