Isolation of Mitochondria

M. circinelloides were grown for 72 h, and mycelium was filtered on preweighed Whatman no. 1 filter paper. The resulted mycelium was washed twice with buffer (50 mM Tric/HCl and 1.2 M sorbitol, pH 6.5); 1.5 g of lysing enzymes (containing β-glucanase, cellulase, protease, and chitinase activities; Sigma) was added to 250 ml stabilizing buffer (1.2 M MgSO₄ and 10 mM KH₂PO₄ at pH 6.0). The filtered mycelium in buffer was incubated for 3 h at 30°C with speed of 100 rpm for protoplast preparation (Yusuf et al., 2018). The resulting mixture was subjected to gradient centrifugation 500, 1,000, and 2,000 g for 5 min individually to remove cell impurity substances and 11,000 g for 20 min for mitochondria isolation (BestBio kit BB-36017). Isolated mitochondria were washed and weighted and were resuspended in the buffer; the final concentration of mitochondria was adjusted to 1 mg/ml and stored at 0°C for further study.