Directed Evolution and High-level Expression of <i>α</i>-Amylase from <i>Thermomyces dupontii</i>

JIN Yan; LI Yanxiao; MA Junwen; WANG Yuchuan; YAN Qiaojuan; JIANG Zhengqiang

doi:10.13386/j.issn1002-0306.2021100086

Volume 43 Issue 13

Jun. 2022

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Science and Technology of Food Industry > 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.

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

1.
Key Laboratory of Food Bioengineering (China National Light Industry), College of Engineering, China Agricultural University, Beijing 100083, China
2.
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China
3.
College of Food Science and Engineering, Nanjing University of Finance and Economics/Collaborative Innovation Center for Modern Grain Circulation and Safety, Nanjing 210023, China

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Received Date: October 12, 2021
Available Online: April 30, 2022

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Abstract

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.
- α-amylase,
- directed evolution,
- specific activity,
- site-directed mutation,
- high cell density fermentation,
- Thermomycesdupontii

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References (34)

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