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中国精品科技期刊2020
靳燕,李延啸,马俊文,等. 嗜热杜邦菌α-淀粉酶的定向进化及高效表达[J]. 食品工业科技,2022,43(13):139−147. doi: 10.13386/j.issn1002-0306.2021100086.
引用本文: 靳燕,李延啸,马俊文,等. 嗜热杜邦菌α-淀粉酶的定向进化及高效表达[J]. 食品工业科技,2022,43(13):139−147. doi: 10.13386/j.issn1002-0306.2021100086.
JIN Yan, LI Yanxiao, MA Junwen, et al. Directed Evolution and High-level Expression of α-Amylase from Thermomyces dupontii[J]. Science and Technology of Food Industry, 2022, 43(13): 139−147. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2021100086.
Citation: JIN Yan, LI Yanxiao, MA Junwen, et al. Directed Evolution and High-level Expression of α-Amylase from Thermomyces dupontii[J]. Science and Technology of Food Industry, 2022, 43(13): 139−147. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2021100086.

嗜热杜邦菌α-淀粉酶的定向进化及高效表达

Directed Evolution and High-level Expression of α-Amylase from Thermomyces dupontii

  • 摘要: 目的:对嗜热杜邦菌来源α-淀粉酶进行分子改造,以提高其耐热性和产酶水平。方法:基于易错PCR技术构建嗜热杜邦菌来源α-淀粉酶(Td-amy)的随机突变文库,高通量筛选耐热性和比酶活提高的突变体,通过定点突变及同源结构模拟对突变体进行分析,并将其在毕赤酵母中表达。结果:筛选得到一个正向突变体(mTd-amy)。该突变体最适温度(60 ℃)较野生型(55 ℃)提高了5 ℃,比酶活(466.3 U/mg)较野生型(227.9 U/mg)提高至2.0倍。经序列对比,mTd-amy有四个氨基酸发生了变化,分别为Ala4Val、Ala122Val、Lys194Arg和Ala468Asp,定点突变结果表明Ala122Val和Ala468Asp位点为影响其比酶活和最适反应温度的关键。进一步将突变体mTd-amy在毕赤酵母中高效表达,经高密度发酵其酶活达64696 U/mL。结论:定向进化获得了嗜热杜邦菌来源α-淀粉酶的正向突变体,该突变体的最适温度和比酶活力均明显提高,为α-淀粉酶的分子改造以及工业化应用等提供了理论参考。

     

    Abstract: Objective: The α-amylase from Thermomyces dupontii was modified by direct evolution to improve its thermostability and expression level. Methods: A mutation library of α-amylase (Td-amy) from Thermomyces dupontii was constructed by error-prone PCR. The mutants with higher thermostability and specific activity were selected through high-throughput screening, analysis of the mutants by site-directed mutagenesis and homologous structure simulation, and expression in Pichia pastoris. Results: A positive mutant (mTd-amy) was selected. The optimal temperature of mTd-amy was 60 ℃, which was higher than that of the wild type enzyme Td-amy (55 ℃) by 5 ℃. The specific activity of mTd-amy (466.3 U/mg) was 2.0 times higher than that of Td-amy (227.9 U/mg). Sequence and mutation analysis revealed that four sites (Ala4Val, Ala122Val, Lys194Arg and Ala468Asp) in mTd-amy were mutated, and Ala122 Val and Ala468Asp played a key role in the specific activity and optimal temperature of the mutant. mTd-amy was further expressed in Pichia pastoris, and its expression level was up to 64696 U/mL through high cell density fermentation. Conclusion: Through directed evolution, a positive mutant of with high optimal temperature and specific activity was obtained. It provides a theoretical basis for the molecular modification and industrial application of α-amylase.

     

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