| Lecanicillium lecanii, with a wide geographical distribution and broad host range, is of considerable importance in agriculture as an entomopathogenic fungus of insect and nematodes. L. lecanii was evaluated as protease producer and fungal protease played an important role in biological control of pests. Lecanicillium FJVL-12 showed some insecticidal activity on the tobaco whitefly, and it was identificated as L. lecanii based on nr-LSU sequences. The medium optimization for protease production by FJVL-12 based on one-factor-at-a-time and orthogonal array design methods was described in this dissertation. The one-factor-at-a-time method was used to study the effects of carbon sources, nitrogen sources, metal ions and vitamins on protease production. Among various candidates employed, maltose, peptone, magnesium and VB6 were the most suitable for protease production, respectively. Subsequently, optimal medium for the combination of carbon sources, nitrogen sources, vitamins and mineral sources, as well as submerged culture conditions were investigated using an orthogonal layout. Media and cultural conditions including Maltose1% (w/v), Peptone0.5%, Magnesium 4×10-4mol/L, VB6 0.02%, pH 6.0, inoculum volume 5 mL, medium capacity 100 mL/250 mL flask, mycelial age 7 days, culture time 5 days were employed, respectively. Under optimal culture conditions, maximum protease activity reached 85.46 U mL-1 after 5 d of fermentation. The enzyme was purified to electrophoretic homogeneity using the combination of various chromatographic steps: DEAE-cellulose, Sephadex G-100. The purified enzyme was a monomer with an apparent molecular weight of 45 kDa measured by SDS-PAGE. The optimal reaction temperature and pH for protease were 40℃and 8.0, respectively. Enzyme activity was significantly enhanced by Na+,Fe3+ and Ethanol, but completely inhibited by Ba2+ and Methanol. In phosphate buffer and at 40℃, the apparent Km and Vmax of protease were calculated to be 1.093 mg/mL and 2.523μmol/min, respectively.
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