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文章摘要
基于角质酶共表达策略的大肠杆菌胞外生产耐高温α-淀粉酶及其发酵条件优化
Enhancing Extracellular Production of Recombinant α-Amylase in Escherichia coli through Co-Expression with Cutinase and Optimizing Fermentation Conditions
  
DOI:10.3969/j.issn.1673-1689.2019.11.002
中文关键词: α-淀粉酶  角质酶  共表达  诱导策略
英文关键词: α-amylase,cutinase,co-expression,induction strategy
基金项目:
作者单位
李祝 食品科学与技术国家重点实验室江南大学江苏 无锡 214122 
段绪果 食品科学与技术国家重点实验室江南大学江苏 无锡 214122
江南大学 生物工程学院江苏 无锡 214122 
宿玲恰 食品科学与技术国家重点实验室江南大学江苏 无锡 214122
江南大学 生物工程学院江苏 无锡 214122 
吴敬 食品科学与技术国家重点实验室江南大学江苏 无锡 214122
江南大学 生物工程学院江苏 无锡 214122 
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中文摘要:
      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0.15 μmol/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6.05×104 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%。
英文摘要:
      The thermostability and catalytic efficiency of Bacillus licheniformis α-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 mutant α-amylase,the Bacillus licheniformis α-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 expressed α-amylase individually,in shake flask and fermenter. The results showed that co-expression system has advantages over individual expression system in extracellular production of α-amylase. Then,the extracellular production of recombinant α-amylase in co-expression system was optimized using different induction strategies. Using the optimal induction strategy of 32 ℃ and combined use of 0.15 μmol/L IPTG and 0.5 g/(L·h) lactose,the co-expression system achieved a maximum extracellular α-amylase activity of 6.05×104 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 extracellular α-amylase activity occupied 93.2% of total α-amylase activity.
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