通过优化表达元件及培养基组分提高地衣芽胞杆菌中碱性丝氨酸蛋白酶产量

Enhancement production of subtilisin in Bacillus licheniformis by screening promoters and signal peptides and optimization of fermentation medium

摘要:

【背景】碱性丝氨酸蛋白酶(Subtilisin)是一种具有广泛用途的工业酶制剂。【目的】旨在通过优化启动子、信号肽及培养基组分来提高地衣芽胞杆菌中碱性丝氨酸蛋白酶产量。【方法】以地衣芽胞杆菌BL10为出发菌株，构建了含有4种不同类型启动子(PbacA、P43、PaprE和PsrfA)及4种不同类型信号肽(SPVpr、SPSacB、SPSacC和SPAprE)的碱性丝氨酸蛋白酶表达菌株，并在获得高产菌株的基础上进行培养基优化。【结果】4种启动子的表达水平为PbacA>PaprE>P43>PsrfA，4种信号肽的分泌效率为SPAprE>SPSacC>SPSacB>SPVpr。其中，菌株BL10/pPbacA-aprE产生最高的碱性丝氨酸蛋白酶酶活(275.21 U/mL)，相比于出发菌株BL10/pHY-aprE (167.98 U/mL)提高了64%。随后，通过对发酵培养基成分进行优化并结合正交优化，获得了一种高产碱性丝氨酸蛋白酶的培养基(g/L)：玉米淀粉40.0，豆粕50.0，(NH4)2SO4 4.0，K2HPO4 3.0，CaCO3 1.0。最后，碱性丝氨酸蛋白酶酶活提高到747.37 U/mL，是初始酶活的4.45倍。【结论】为工业化高产碱性丝氨酸蛋白酶提供了一种有效策略。

Abstract:

[Background] Subtilisin is an important industrial protease with many applications. [Objective] The aim of this study is to improve the subtilisin production by screening promoters and signal peptides, and optimization of fermentation medium. [Methods] Bacillus licheniformis BL10, constructed in our previous research, was served as the host strain for subtilisin production, and four promoters (PbacA, P43, PaprE and PsrfA) and four signal peptides (SPVpr, SPSacB, SPSacC and SPAprE) were screened to improve subtilisin production, and the medium for subtilisin production was further optimized. [Results] Based on our results, the expression ratios among these four promoters were PbacA>PaprE>P43>PsrfA, and the secretion efficiencies were SPAprE>SPSacC>SPSacB>SPVpr, respectively. Among these strains, BL10/pPbacA-aprE displayed with the highest subtilisin activity (275.21 U/mL), which was increased by 64% compared with that of BL10/pHY-aprE (167.98 U/mL). Furthermore, the fermentation medium was optimized, and the orthogonal test was further applied to enhance subtilisin production. The maximum subtilisin activity (747.37 U/mL) was obtained in the optimized medium with 40.0 g/L corn starch, 50.0 g/L soybean meal, 4.0 g/L (NH4)2SO4, 3.0 g/L K2HPO4, 1.0 g/L CaCO3, increased by 4.45-fold compared with that of the initial condition. [Conclusion] Collectively, this study provided an effective strategy for enhanced production of subtilisin.