Bacillus sphaericus 2297蛋白酶基因sph在毕赤酵母中的表达及重组酶酶学性质

将Bacillus sphaericus 2297蛋白酶基因sph进行毕赤酵母密码子优化后,进行全基因合成,并构建了重组酵母菌P. pastoris X33-ppicZalphA-sph,对重组酶进行酶学性质研究。结果表明,重组酶的最适反应温度和最适反应pH为40 ℃和8.0,且其为20~30 ℃中保温10 h仍有80%以上的酶活力、在pH7.0~9.0条件下孵育24 h,仍能保持60%以上的酶活性。K+、Sr2+对酶活有明显激活作用,而Fe3+、Ba2+对酶活有明显抑制作用;重组蛋白酶SPH在极性常数为0.8~3.1的25%的正丁醇、环己烷、二甲苯中孵育6 d后,仍能保留50%以上的酶活性。本研究为B. phaericus 2297和B. phaericus DS11蛋白酶有机溶剂耐受性机制的研究奠定基础。

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.