Expression of Protease Gene sph of Bacillus sphaericus 2297 in Pichia pastoris and Characterization of the Recombinant Enzyme

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.