Studies On Purification And Characteristics Of Protease From Aschersonia Placenta

Aschersonia is an important entomogenous fungus.The protease produced during the infection process of this fungus is of great importance.Isolate WYSD-16 was identified as Aschersonia placenta based on its morphological characteristics.The medium optimization for protease production by WYSD-16 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,mental ions and vitamins on protease production. Among various candidates employed,maltose,peptone,magnesium and V_B6 were the most suitable for protease production,respectively.Subsequently,optimal medium for the combination of carbon sources,nitrogen sources,mineral sources and vitamins,as well as submerged culture conditions were investigated using an orthogonal layout.Media and cultural conditions including Maltose 40g/L(w/v),Peptone 20g/L,Magnesium 4×10^-4mol/L,V_B6 400mg/L,pH 6.0,inoculum volume 8 ml,medium capacity 150 ml/250 ml flask,mycelial age 5 days,culture time 10 days were employed,respectively.Under optimal culture conditions, maximum protease activity reached 73.18 U/mL after 10d of fermentation.The strong protease 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 66 kDa measured by SDS-PAGE.The optimal reaction temperature and pH for protease were 50℃and 8.0,respectively.Enzyme activity was significantly enhanced by Na⁺ and alcohol.