Purification And Characterization Of Extracellular Protease From Ascosphaera Apis

Honeybee chalkbrood disease is a fungal epidemic disease of apian larva which is caused by Ascosphaera apis. Fungi can secrete some proteases, which can degrade the cell wall or tissue. These proteases are closely related with its virulence. Virulence is an important indicator for the degree of infection decisions. Serine protease is suggested to be a key enzyme involved in the fungal penetration to invertebrates. Therefore, researching on the protease systematically is good for us to be acquainted with the way how pathogenetic Fungi infect insects, so as to prevent the disease finally.In this paper, based on other researchers, we built a set of simpler, more effective and lower-cost circuit to purify extracellular protease from crude extract of A. apis..We purified a kind of serine protease from crude extract of A.apis, and studied its physical and chemical properties.We inoculated 10 strains of A.apis from different areas on the plate with casein, ND-2 was chosen to be the material for further experiment. The result of the orthogonal experiment which was used to choose the best factor on crude extract was:20 mM Tris-HCl, pH7.8, and 80% of (NH₄)₂SO₄.Crude extract of 6 days cxtracellular protease from A.apis was purified by 80% (NH₄)₂SO₄ precipitation, DEAE-Sapharose Fast Flow (eluted with linear gradient of 0⁰.5 mol/LNaCl)and Sephadex G-75 gel filtration, a class of serine protease was obtained. Specific activity was improved to 345.7 U/mg from 1.9 U/mg, it was 181.9-fold purified with yield of 5.7%.The purified protease was performed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE), its molecular weight was about 29.5 kDa, called pro-AAND-2. The characterization to the pro-AAND-2 showed that Cu²⁺and Fe³⁺can strongly inhibited its enzyme activity, the enzyme activity would lost completely when the Concentration of Ag⁺ and Cu²⁺ reached 20 mM and 50 mM, enzyme activity could not be detected when it was in l0 mmol/L Phenvlmethanesulfonyl fluoride, indicating that it might be a serine protease. The optimum reaction temperature and pH were 55℃and 7.0, it was comparative steady in a wide range of pH(3.0¹1.0), but with the temperature increasing, the stability became weaker and weaker.