信号肽筛选优化提高耐热α-环糊精酶在枯草芽胞杆菌中的表达  

Enhanced Thermophilicα-Cyclodextrin Glycosyltransferase Expression by Optimizing Target Signal Peptide in Bacillus subtilis

作　　者：陈龙军[1] 林陈强[1] 贾宪波[1] 方宇[1] 张慧[1] 陈济琛[1] CHEN Longjun;LIN Chenqiang;JIA Xianbo;FANG Yu;ZHANG Hui;CHEN Jichen(Soil and Fertilizer Institute,Fujian Academy of Agricultural Sciences,Fuzhou,Fujian 350003,China)

机构地区：[1]福建省农业科学院土壤肥料研究所,福建福州350003

出　　处：《福建农业学报》2022年第3期414-422,共9页Fujian Journal of Agricultural Sciences

基　　金：福建省农业科学院对外合作项目(DWHZ2021-17);福建省科技计划公益类专项(2022R1022-5、2018R1022-7);福建省农业科学院探索创新项目(AA2018-13);。

摘　　要：【目的】探索嗜热α-环糊精葡萄糖基转移酶(α-CGTase)在枯草芽胞杆菌(Bacillus subtilis RIK1285)中的高效胞外表达条件。【方法】以来源于枯草芽胞杆菌的173种信号肽为基础构建信号肽文库,筛选获得9条表达效率更高的信号肽,其中citH信号肽引导分泌效率最高。在此基础上,为进一步优化α-CGTase的分泌表达,对信号肽citH的Gly2、Asn3及Thr4进行饱和突变,并比较不同突变体的引导分泌效率。【结果】突变体G2R-NK-T4LCGT的引导分泌效率最高,重组枯草芽胞杆菌的胞外α-CGTase活力高达(14.2±0.11)U·mL^(−1),相比未突变信号肽[(9.6±0.29)U·mL^(−1)],分泌效率提高了47.9%;是野生菌株嗜热地芽胞杆菌Geobacillus caldoxylosilyticus.CHB1(0.66 U·mL^(−1))的21.5倍。重组α-CGTase的最适反应pH值为6.0,最适反应温度为60℃,在50℃以内稳定;Mg^(2+)、Ca^(2+)对α-环糊精酶活性具有一定的激活作用。【结论】信号肽对环糊精酶在芽胞杆菌中的高效表达具有重要影响,为外源蛋白在芽胞杆菌中的表达提供一定借鉴。【Objective】Conditions to achieve high-efficiency secretory expression of the thermophilicα-cyclodextrin glucosyltransferase(α-CGTase)in Bacillus subtilis RIK1285 were explored to understand the role played by signal peptide.【Method】A library based on 173 signal peptides from B.subtilis was constructed to identify the highest secretion efficiency candidate.Using the saturation mutagenesis of segments in the selected signal peptide,the secretion expression ofα-CGTase was enhanced.【Result】There were 9 signal peptides with high expression efficiency identified from the library.Among them,the signal peptide citH showed the highest secretion efficiency.Subsequently,the saturation mutagenesis of Gly2,Asn3,and Thr4 of citH produced a mutant,G2R-NK-T4L-CGT,with an extracellular activity ofα-CGTase as high as 14.2 U·mL^(−1) and a secretion efficiency increased by 47.9%over the non-mutated signal peptide(9.6 U·mL^(−1)),which was 21.5 times higher than that of the wild-type Geobacillus caldoxylosilyticus CHB1(0.66 U·mL^(−1)).The purifiedα-CGTase had a maximal activity at pH 6.0 and 60℃,was thermally stable within 50℃,and could be activated by Mg^(2+)and Ca^(2+).【Conclusion】The important effect signal peptide had on the high-efficiency expression of cyclodextrinase in B.subtilis was verified.The result provided a new and valuable reference for studies on the expression of extraneous proteins in the bacteria.

关 键 词：信号肽 Α-环糊精葡萄糖基转移酶 饱和突变 枯草芽胞杆菌 

分 类 号：Q786[生物学—分子生物学]