Abstract: The protease gene sph of Bacillus sphaericus 2297 was optimized by partly replacing the P.pastoris preferred codons(synonymous codons),the gene was synthesized,and the recombinant P. pastoris X33-ppicZalphA-sph was constructed,and the enzymatic properties of recombinant enzyme were studied. The results showed that,the optimal temperature of the recombinant enzyme was 40 ℃,the optimum reaction pH was 8.0,more than 80% enzyme activity was retained when incubated at 20~30 ℃ for 10 h,and enzyme activity was retained more than 60% after treated at pH7.0~9.0 for 24 h. Fe³⁺ and Ba²⁺ significant inhibited enzyme activity,while K⁺ and Sr²⁺ significant activated enzyme activity. The recombinant protease SPH had good stability in organic solvent with polar constant between 0.8~3.1,more than 50% enzyme activity was retained after 6 d incubation in 25% n-butanol,cyclohexane and xylene. This paper laid the foundation for studying the mechanism of organic solvent tolerance proteases from B. phaericus 2297 and B. phaericus DS11.