碱性蛋白酶SubC在枯草芽孢杆菌中的高效异源表达

Efficient heterologous expression of alkaline protease SubC in Bacillus subtilis

摘要:

[背景] 碱性蛋白酶是工业用酶中占比最大的酶类，广泛应用于清洁、食品、医疗等行业。近期研究发现碱性蛋白酶在生产生物活性肽方面有巨大潜力，这将进一步拓宽其在保健食品领域中的应用。[目的] 利用枯草芽孢杆菌异源表达地衣芽孢杆菌来源的碱性蛋白酶SubC。[方法] 通过筛选3种枯草芽孢杆菌宿主菌株（Bacillus subtilis 1A751、MA07、MA08）和6种信号肽（AmyE、AprE、NprE、Pel、YddT、YoqM），同时优化诱导剂浓度、发酵培养基和发酵时长，最终得到最优重组菌株MA08-AmyE-subC_opt。[结果] 重组菌株MA08-AmyE-subC_opt的胞外酶活力为3.33×10³ AU/mL，胞外蛋白分泌量为胞内可溶蛋白表达量的4倍，与携带野生型信号肽的对照组菌株WT相比，酶活提高了73.4%。[结论] 异源碱性蛋白酶SubC在枯草芽孢杆菌中成功表达，为碱性蛋白酶SubC的表达和在保健食品领域的工业化应用提供了理论基础。

Abstract:

[Background] Alkaline protease is the enzyme with the largest proportion among industrial enzymes and is widely used in cleaning, food, medical and other industries. Recent studies have found that alkaline protease has great potential in the production of biologically active peptides, which will further broaden its application in the field of health food. [Objective] In this study, Bacillus subtilis was used to heterologously express the alkaline protease SubC derived from Bacillus licheniformis.[Methods] By screening three kinds of Bacillus subtilis host strains (B. subtilis 1A751, MA07 and MA08) and six kinds of signal peptides (AmyE, AprE, NprE, Pel, YddT and YoqM), as well as optimizing the inducer concentration, fermentation medium and fermentation duration, we finally obtained the optimal recombinant strain MA08-AmyE-subC_opt. [Results] The extracellular enzyme activity of the recombinant strain MA08-AmyE-subC_opt was 3.33×10³ AU/mL. Secretory expression level is four times more than that of intracellular soluble protein expression. Compared with the control group WT, the enzyme activity increased by 73.4%.[Conclusion] The heterologous alkaline protease SubC was successfully expressed in Bacillus subtilis, which provided a theoretical basis for the expression of alkaline protease SubC and its industrial application in the field of health food.