Isolation And Characteristics Of Antifungal Proteins From Bacillius Firmu ZP-1

The Bacillius firmu ZP-1 which showed inhibiting efect to lots of pathogens was choosed as material.With SDS-PAGE,antagonistica ctivity tracing,Sephadex G-100 chromatography and Q Sepharose Fast Flow,two kinds of anti-fungal protein P₁ and P₂ which both can inhibited the growth of Pestalotiopsis funereal efectively was purified and characterized.N-terminal aa residues analysis showed proteins P₂ is subtilisin protease and P₁ was identified a kind of Chitinase with enzymatic activity test.Ammonium sulfate was added from 20%saturation to 90%in supernatant after centrifugalization to discard thallus.With 50%(NH₄)₂SO₄ ammonium sulfate solution,we could get a large quantity of antifungal protein.The concentrated material was the applied to Sephadex G-100 and goe two absorption peaks which were seperated obviously;The active fraction was put on a Sepharose Fast Flow after being concentrated was eluted three absorption peaks of which Peak 2 and Peak 3 have antagonistica ctivity but Peak 1 not;Keep the active fraction Peak 2 and Peak 3.SDS-PAGE showed that the proteins in Peak 2 and Peak 3 were both electrophoresis pure protein,the protein in Peak 2 was named P₁ and the protein in Peak 3 was named P₂.The test of antifungal protein P₁ and P₂ isolated from Bacillius firmu showed:P₂ protein of which molecular weight is 67 KDa had strong tolerant to heat,ultraviolet radiation and trypsin,was steady from pH6.0～9.0;P₂ protein of which molecular weight is 27 KDa was steady in ultraviolet radiation,chloroform and trypsin,but not totally steady in high temperature,acid,and proteinase K.N-terminal aa residues analysis showed a seven aa residues sequence: H₂N-Ala-Gln-Ser-Val-Pro-Tyr-Gly.Using this aa sequence as a target,the similarity of P₂ protein was searched on NCBI.It was showed that a high homology between the P₂ protein and the precursor of subtilisin protease from Bacillus.And molecular mass of P₂ is much closed to subtilisin protease.So,protein P₂ is deduced to be a kind of subtilisin protease.N-terminal aa residues analysis of P1 was failed,P₁ was identified a kind of Chitinase with enzymatic activity test.