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.

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

  • 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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