灵杆菌胞外核酸酶在短短芽孢杆菌中的高效表达

High-Efficiency Expression of Serratia marcescens Non-Specific Nuclease in Brevibacillus choshinensis

摘要:

利用短短芽孢杆菌（Brevibacillus choshinensis）原核表达系统对灵杆菌胞外核酸酶（Serratia marcescens non-specific nuclease，SMNE）进行重组表达，以期获得高产量重组SMNE。利用大肠杆菌-芽孢杆菌穿梭载体构建SMNE重组质粒，实现其在B. choshinensis HD31-SP3菌株中的表达。通过粗酶活检测验证其活性后，首先对温度、甘油和发酵时长等发酵条件进行优化，随后将获得的重组SMNE经亲和层析、凝胶阻滞层析分离纯化后进行酶活性质表征检测。经B. choshinensis HD31-SP3菌株重组表达的SMNE，初测胞外酶活为4.07×10⁶ U/L。经发酵条件的优化测试，最终在培养基中加入终体积分数5%甘油，于30 ℃下摇瓶培养56 h，获得的发酵上清胞外酶活可达2.6×10⁷ U/L，是未优化条件的6倍；经蛋白质纯化条件筛选，最终经一步亲和纯化后SMNE回收产量即达30~40 mg/dL，比活力为1.3×107 U/mg，为商业化对照的2~3倍。通过对该酶的酶学性质鉴定后发现：该酶最适反应条件为37~45 ℃，pH 8~9；在不存在Mg²⁺/Mn²⁺的情况下仍保持47%的活性，且在300 mmol/L Na+/K+条件下可保持35%~45%的活性。

Abstract:

High-efficiency recombinant expression of Serratia marcescens non-specific nuclease (SMNE) was obtained using the prokaryotic Brevibacillus choshinensis expression system. The SMNE recombinant expression plasmid was constructed using the Escherichia coli-Bacillus spp. shuttle vector. After confirmation of the enzyme activity of SMNE in crude supernatant, the recombinant expression conditions were optimized towards temperature, glycerol content in the culture, and incubation time. The enzyme activity of recombinant SMNE wascharacterizedafter purificationby affinity and gel filtration chromatography. Recombinant SMNE was expressed in B. choshinensis HD31-SP3 strain. The initial enzyme activity in crude cell lysate was 4.07×10⁶ U/L. The expression condition was determined to be 5% glycerol at 30℃ for 56 h after optimization, which increased the activity to 2.6×107 U/L and obtained 6 folds of the initial activity yield. The purification procedure was also optimized and simplified to be a one-step affinity chromatography. The pure enzyme yield reached 30~40 mg/dL and the specific activity reached 1.3×10⁷ U/mg, which was 2~3 folds of that of the commercialcontrol. Characterization of the recombinant SMNE indicated that the optimal reaction condition was 37~45 ℃, pH 8~9. 47% of the activity remained without Mg²⁺/Mn²⁺, while 35%~45% of the activity remained with 300 mmol/L of Na⁺/K⁺.