摘要
在前期工作中通过分子改造提高了来源于地衣芽孢杆菌(Bacillus licheniformis)α-淀粉酶在高温酸性条件下的稳定性,同时改良了催化效率。为了实现α-淀粉酶在大肠杆菌BL21(DE3)中的胞外高效表达,作者尝试采用共表达嗜热放线菌(Thermobifida fusca)角质酶来增强大肠杆菌膜透性,同时优化诱导策略以提高重组α-淀粉酶的胞外表达。首先构建了能同时表达角质酶和α-淀粉酶的工程菌BL21(DE3)/pETDuet-amy-cutinase,并将此菌株与能单独表达α-淀粉酶的工程菌BL21(DE3)/pET-20b-amy进行摇瓶和3 L发酵罐的产酶发酵对比。结果显示,共表达菌株在胞外产酶方面具有明显的优势。进一步对共表达菌株的诱导条件进行优化,在32℃、诱导剂为0.15μmol/L IPTG与0.5 g/(L·h)乳糖条件下,发酵32 h后胞外α-淀粉酶最高酶活力可达6.05×10^4 U/mL,是单表达菌株摇瓶发酵水平的28.3倍。此时胞外重组α-淀粉酶质量浓度为8.92 g/L,重组蛋白质的分泌效率为93.2%。
The thermostability and catalytic efficiency of Bacillus licheniformisα-amylase have been improved at high temperature and acidic conditions using site-directed mutations in our previous study.To achieve highly efficient extracellular production of this mutantα-amylase,the Bacillus licheniformisα-amylase was co-expressed with Thermobifida fusca cutinase in Escherichia coli and the induction strategy was also optimized.First,the co-expression system was compared with individual expression system,which expressedα-amylase individually,in shake flask and fermenter.The results showed that co-expression system has advantages over individual expression system in extracellular production ofα-amylase.Then,the extracellular production of recombinantα-amylase in co-expression system was optimized using different induction strategies.Using the optimal induction strategy of 32℃and combined use of 0.15μmol/L IPTG and 0.5 g/(L·h)lactose,the co-expression system achieved a maximum extracellularα-amylase activity of 6.05×10^4 U/mL(8.92 g/L)at 32 h,which was 28.3 fold to that of achieved by individual expression system in shake flask.In addition,the extracellularα-amylase activity occupied 93.2%of totalα-amylase activity.
作者
李祝
段绪果
宿玲恰
吴敬
LI Zhu;DUAN Xuguo;SU Lingqia;WU Jing(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2019年第11期9-17,共9页
Journal of Food Science and Biotechnology
基金
国家自然科学基金杰出青年基金项目（31425020）
江苏省自然科学基金项目（BK20140132）
江苏省产学研前瞻性联合研究项目（BY2015019-32）
