Advancing pyrene biodegradation via RSM-based optimization and characterization of catechol 1, 2-dioxygenase and 2, 3-dioxygenase in Acinetobacter baumannii BJ5 strain

Catechol 1, 2-dioxygenase (C12O) and catechol 2, 3-dioxygenase (C23O) are key enzymes involved in the biodegradation pathways, catalyzing the breakdown of aromatic pollutants including polycyclic aromatic hydrocarbons (PAHs) in environmental systems. The current study employed Response Surface Methodology (RSM) with a Box-Behnken Design (BBD) to optimize key biochemical parameters, namely pH, temperature, and substrate concentration, affecting the activity of these dioxygenases from Acinetobacter baumannii BJ5, in the biodegradation of the high molecular weight polycyclic aromatic hydrocarbon (HMW PAH), pyrene. Optimization was performed within pH range of 6.4–8.4 for C12O and 6.0 to 8.0 for C23O, temperature range of 30 °C–40 °C for C12O and 25 °C–35 °C for C23O, and substrate concentration from 2.0 to 6.0 mM for C12O and 1.5–5.5 mM for C23O. The optimal conditions determined were 7.5 and 7.0 (pH), 35 °C and 30 °C (temperature), and 6.0 mM and 4.0 mM (Substrate concentration) for C12O and C23O, respectively. Partial purification of C12O increased purity 1.10-fold with 57.83 % yield and raised specific activity from 75.50 to 87.33 μmol/min/mg. The findings underline the significant potential of dioxygenases in degradation of recalcitrant PAHs, thereby supporting practical deployment in contaminated soil and water systems. This investigation constitutes one of the first efforts to employ RSM for optimizing PAH degrading dioxygenases in a gram-negative strain, A. baumannii BJ5 with respect to degradation of HMW PAH, pyrene and provide valuable insights toward developing enzyme based strategies for the sustainable management of persistent industrial pollutants.