Phenanthracene is a highly toxic organic compound capable of contaminating water and soils, and biodegradation is an important tool for remediating polluted environments. This study aimed to evaluate the effects of trace elements, cations, and environmental conditions on the activity of the protocatechol 3,4-dioxygenase (P3,4O) enzyme produced by the isolate Leifsonia sp. in cell-free and immobilized extracts. The isolate was grown in Luria Bertani broth medium (LB) amended with 250 mg L−1 of phenanthrene. Various levels of pH (4.0-9.0), temperature (5-80 °C), time (0-90 min), trace elements (Cu2+, Hg2+ and Fe3+), and cations (Mg2+, Mn2+, K+ and NH4+) were tested to determine which conditions optimized enzyme activity. In general, the immobilized extract exhibited higher enzyme activity than the cell-free extract in the presence of trace elements and cations. Adding iron yielded the highest relative activity for both cell-free and immobilized extracts, with values of 16 and 99 %, respectively. Copper also increased enzyme activity for both cell-free and immobilized extracts, with values of 8 and 44 %, respectively. Enzyme activity in the phosphate buffer was high across a wide range of pH, reaching 80 % in the pH range between 6.5 and 8.0. The optimum temperatures for enzyme activity differed for cell-free and immobilized extracts, with maximum enzyme activity observed at 35 ºC for the cell-free extract and at 55 ºC for the immobilized extract. The cell-free extract of the P3,4O enzyme exhibited high activity only during the first 3 min of incubation, when it showed 50 % relative activity, and dropped to 0 % after 60 min of incubation. By contrast, activity in the immobilized extract was maintained during 90 min of incubation. This isolate has important characteristics for phenanthrene biodegradation, producing high quantities of the P3,4O enzyme that forms part of the most important pathway for PAH biodegradation.