AbstractThis is the first report on chemical mutagenesis of Trametes versicolor IBL-04 to develop a hyper-producing mutant for overproduction of manganese peroxidase (MnP) using sugarcane bagasse as a substrate. A freshly prepared inoculum of indigenously isolated T. versicolor IBL-04 was treated with 100 µg mL-1 (v/v) ethyl methane sulfonate (EMS) and ethidium bromide (EB) separately for different time periods. The selected mutants and parent strain were cultured in solid-state fermentation (SSF) conditions to select the hyper-producing mutants. After selection of hyper-producing EMS- and EB-treated mutants, the fermentation parameters, including substrate type, incubation time, initial pH of the medium, temperature, moisture level, and carbon-to-nitrogen ratio (C:N), were optimized by adopting the Classical Optimization Strategy. T. versicolor IBL-04 treated for 90 min with EMS (EMS-90 mutant) gave maximum MnP production (935 U mL-1) after 8 days of fermentation. Supplementation with carbon and nitrogen sources significantly enhanced mutant growth, and under optimum conditions, the maximum MnP production by the mutant strain increased to 3045 U mL-1. The results indicated that the random chemical mutagenesis significantly enhanced the MnP production. The increased production of MnP by the EMS-90 mutant strain suggest its potential for commercial-scale enzyme production and biotechnological applications.