Abstract: Protease is a kind of enzyme widely used in the food,detergent and medicine industries due to its advantages of catalytic efficiency and specificity.A protease-producing strain designated as CF-02 was isolated from soil by casein nutrient medium,subsequently its identification and enzymatic properties were investigated in this paper.The results indicated that strain CF-02 was identified as Bacillus subtilis by physiological-chemical test and 16S rDNA sequencing with ability to produce large amount of protease.The strain began to produce protease at the late stationary phase of growth period in fermentation medium and the enzyme reached the maximum amount at 36 h.The optimum conditions of pH and temperature catalyzed by protease in our research were pH 8.0 and 55℃ respectively,indicating that this enzyme was a mesophilic and neutral protease.Enzyme activity decreased sharply when the reaction pH value was higher than 9.0 or lower than 5.0,or the reaction temperature higher than 70℃,and these results indicated that the protease was susceptible to high temperature,acidity and alkalinity.2.5 mmol/L of Ca²⁺,Mn²⁺and Cu²⁺could increase 10% of the protease activity,whereas Fe²⁺and Zn²⁺decreased approximate 7% of the protease activity;Mg²⁺,K⁺and Na⁺had little significant effects on its activity.The enzyme was purified using ammonium sulfate precipitation and anion-exchange chromatography prior to MS identification.MS analysis showed that the protease was an amino peptidase and similar to Bacillus subtilis YtoP protein sequence,which could degrade protein and polypeptides at N-terminal.Additionally,SDS-PAGE revealed that the molecular of the protease was about 34 kDa and this isolated protease producing strain might have potential application in industry.