乙醛脱氢酶是潜在的酶制剂,可用于从药物中解毒乙醛和其他外源性醛,食物,和生物燃料生产。在这项研究中,我们使用组合策略优化信号肽,增强了来自枯草芽孢杆菌的乙醛脱氢酶(istALDH)的表达,promotors,和生长条件。首先,构建各种信号肽的文库以鉴定有效的istALDH分泌的最佳信号肽。信号肽yqzG达到最高的细胞外istALDH活性(204.85±3.31U/mL)。第二,aprE启动子被组成型启动子取代(即,P43)和诱导型启动子(即,Pglv),导致12.40%和19.97%的istALDH增强,分别。此外,串联启动子P43-Pglv提供了更好的性能,导致istALDH活性增强30.96%。第三,通过一次测试一个因素来优化istALDH的生产。优化了物理参数,包括诱导物(例如,麦芽糖)浓度,孵化温度,和接种量,结果为2.0%,35○C,和2.0%,分别。优化的培养基结果为2.0%葡萄糖,1.5%蛋白胨,2.5%酵母提取物,1%NaCl,和0.5%(NH4)2SO4。细胞外istALDH活性为331.19±4.19U/mL,产量是迄今为止文献报道的最高产量。
Acetaldehyde dehydrogenases are potential enzyme preparations that can be used to detoxify acetaldehyde and other exogenous aldehydes from pharmaceuticals, food, and biofuel production. In this study, we enhanced the expression of acetaldehyde dehydrogenase sourced from Issatchenkia terricola (istALDH) in Bacillus subtilis using a combinatorial strategy for the optimization of signal peptides, promoters, and growth conditions. First, a library of various signal peptides was constructed to identify the optimal signal peptides for efficient istALDH secretion. The signal peptide yqzG achieved the highest extracellular istALDH activity (204.85 ± 3.31 U/mL). Second, the aprE promoter was replaced by a constitutive promoter (i.e., P43) and an inducible promoter (i.e., Pglv), resulting in 12.40% and 19.97% enhanced istALDH, respectively. Furthermore, the tandem promoter P43-Pglv provided a better performance, resulting in 30.96% enhanced istALDH activity. Third, the production of istALDH was optimized by testing one factor at a time. Physical parameters were optimized including the inducer (e.g., maltose) concentrations, incubation temperatures, and inoculation amounts, and the results were 2.0%, 35 ∘C, and 2.0%, respectively. The optimized medium results were 2.0% glucose, 1.5% peptone, 2.5% yeast extract, 1% NaCl, and 0.5% (NH4)2SO4. The extracellular istALDH activity was 331.19 ± 4.19 U/mL, yielding the highest production reported in the literature to date.