Effect of culture conditions on the production of ligninolytic enzymes by white rot fungi Phanerochaete chrysosporium (ATCC 20696) and separation of its lignin peroxidase

The present work was carried out to determine the optimum culture conditions of Phanerochaete chrysosporium (ATCC 20696) for maximizing ligninolytic enzyme production. Additionally, separation of its lignin peroxidase was conducted. After experiments, an optimized culture medium/condition was constructed (per liter of Kirk’s medium): dextrose 10 g, ammonium tartrate 0.11 g, Tween-80 0.5 g, MnSO₄ 7 mg, and veratryl alcohol 0.3 g in 10 mM acetic acid buffer pH 4.5. Under the optimized experimental condition, both lignin peroxidase (LiP) and manganese peroxidase (MnP) were detected and reach the highest yield at 30°C on the 8th day culture. Salt precipitation methods was used in the extraction and purification processes. Results show that salt precipitation with 60% (NH₄)₂SO₄ yielded the best result, especially toward LiP. Enzyme separation was conducted and two fractions with LiP activity. LiP1 and LiP2 were produced using three columns sequentially: desalting column, Q FF ion exchange column and Sepharyl S-300 HR gel filtration. LiP1 and LiP2 had been purified by 9.6- and 7.6-fold with a yield of 22.9% and 18.6%, respectively. According to the data of sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE), the molecular weights of the enzymes are 38 kDa and 40 kDa, respectively.