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 inEscherichia colithrough 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

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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，theBacillus licheniformisα-amylase was co-expressed withThermobifida fuscacutinase inEscherichia coliand 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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