Study On Antiviral Effect And Enzyme Characterization Of Alkaline Protease From Bacillus Subtilis

Alkaline Protease are serine proteases which can hydrolyze protein at alkaline pH. Alkaline Protease have extensive sources, and the advantages of the method to get alkaline protease from microorganism are low cost, scientific and practical. The aim of the study was to find a new approach on controlling tobacco mosaic by the alkaline protease which can hydrolyze the coat protein of TMV. And the research results were as follows:(1) The alkaline protease producing strain W-QX-1 was screened out of 4 Bacillus subtilis which had been isolated by our laboratory.(2) The fermentation conditions of a high yield strain producing high alkaline protease were confirmed: initial pH 8.0, a 250mL flask containing 20mL medium for 36h.(3) The alkaline protease was purified from the fermentation broth of Bacillus subtilis W-QX-1 by salting-out of ammonium sulfate followed by Native-PAGE. The purified proteinase was demonstrated to be electrophoretic homogeneity by SDS-PAGE, with a relative molecular weight of 42400.(4) The alkaline protease from Bacillus subtilis W-QX-1 had the ability of inactivating TMV in vitro by half leaf method on Nicotiana glutinosa. The inhibition rate against TMV was 45.20%,53.40%,60.42% and 76.73% when the concentration of the alkaline protease was 20mg/L,50mg/L,100mg/L and 200mg/L; The experiment results indicated that the efficiency of inhibiting infectivity of TMV of 48h applied before inoculation was 50.35% when concentration of the alkaline protease was 200mg/L. And it could also inhibit multiplication of TMV in a certain extent in Nicotiana glutinosa, but the effect was not obvious.(5) The optimum temperature for hydrolysis of casein were 50℃. The optimum pH was 7.8 in borate buffer and 8.0 in Tris buffer, respectively. The enzyme was stable up to 45℃, within the pH range of 6¹0; The enzyme activity was enhanced in presence of Ca²⁺,K⁺,Mg²⁺ and enzyme activity was inhibited by Cu²⁺,Ag⁺; Proteolytic activity was completely inhibited by EDTA and was activated significantly by SDS.(6)The alkaline protease from Bacillus subtilis W-QX-1 had a broad action spectrum. It could not only degrade casein, but also hydrolyze soluble silk fibroin, pure fibroin, bovine serum albumin and gelatin.