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以质粒pHY300PLK为骨架构建Bacillus stearothermophilus来源的α/β-环糊精葡萄糖基转移酶(α/β-CGTase)在枯草芽孢杆菌(Bacillus subtilis)表达系统的表达质粒。为了提高α/β-CGTase的表达量,考察了9个单启动子(PamyQ, PamyQ', Papr E, Pgsi B, Phpa Ⅱ, Pnpr E, Psrf, Pxyl, Pxyl')对α/β-CGTase表达量的影响,摇瓶发酵表明最优单启动子为PamyQ',α/β-CGTase表达量为6.52 U/mL,其次为Phpa Ⅱ和Pnpr E,分别对应酶活5.80 U/mL和5.73 U/mL。用PamyQ'与PamyQ'、Phpa Ⅱ、Pnpr E两两组合,构建双启动子PamyQ'-PamyQ'、Phpa Ⅱ-PamyQ'、Pnpr E-PamyQ',摇瓶发酵表明最优双启动子为Phpa Ⅱ-PamyQ',α/β-CGTase表达量为11.15 U/mL,是用单启动子PamyQ'表达时的1.7倍。测定Phpa Ⅱ-PamyQ'、PamyQ'-PamyQ'、PamyQ'-PamyQ'、PamyQ'、Pgsi B、Phpa Ⅱ为启动子时,α/β-CGTase m RNA转录水平,结果表明α/β-CGTase表达量随m RNA转录水平的提高而提高。使用含有启动子Phpa Ⅱ-PamyQ'的表达质粒进行3 L罐发酵,培养96 h时,α/β-CGTase酶活达到最高110.4 U/mL。
Abstract：The expression plasmid was constructed with plasmid pHY300 PLK to express α/β-Cyclodextrin glucosyltransferases(α/β-CGTase) from Bacillus stearothermophilus in Bacillus subtilis expression system. The effects of 9 promoters(PamyQ, (PamyQ', Papr E, Pgsi B, Phpa Ⅱ, Pnpr E, Psrf, Pxyl, Pxyl') on the CGTase expression were investigated in order to increase the α/β-CGTase expression. Shaking flask fermentation showed that the optimal promoter was (PamyQ'and the expression of α/β-CGTase was 6.52 U/mL. Followed by Phpa Ⅱand Pnpr E, corresponding to enzyme activity of 5.80 U/mL and 5.73 U/mL, respectively. The double promoter (PamyQ'-(PamyQ', Phpa Ⅱ-(PamyQ', Pnpr E-(PamyQ'were constructed by combining (PamyQ'with (PamyQ', Phpa Ⅱand Pnpr E, and the optimal double promoter was Phpa Ⅱ-(PamyQ'and the expression of α/β-CGTase was 11.15 U/mL, which is 1.7 times that of the single promoter (PamyQ'. The transcription levels of α/β-CGTase mRNA in Phpa Ⅱ-(PamyQ', (PamyQ'-(PamyQ', (PamyQ'-(PamyQ', (PamyQ', Pgsi Band Phpa Ⅱwere determined. The results showed that the expression of mRNA increased with the increase of mRNA transcript level. Fermentation in3 L cans was performed using the expression plasmid containing the promoter Phpa Ⅱ-(PamyQ'and α/β-CGTase reached a maximum of 110.4 U/mL at 96 h.
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基本信息:
DOI：10.13417/j.gab.038.003540
中图分类号:TQ920.1;TS201.3
引用信息:
[1]李云菲,宿玲恰,吴敬.启动子对枯草芽孢杆菌表达环糊精葡萄糖基转移酶的影响[J].基因组学与应用生物学,2019,38(08):3540-3547.DOI:10.13417/j.gab.038.003540.
基金信息:
国家自然科学基金(31501419);; 国家杰出青年基金(34125020)共同资助
2019-08-25
2019-08-25