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<p>Glucan 1,4-alpha-maltohydrolase (3.2.1.133, GMH) is an important biocatalyst in the baking industry, which could delay the retrogradation of bread and improve its cold-storage durability. In the present study, a newly cloned <i>Thgmh</i> was characterized and secreted by <i>Pichia pastoris</i> (<i>Komagataella pastoris</i>). After computationally assisted rational design that promotes peptide folding, the maltogenic activity in supernatant was enhanced 1.6-fold in comparison with the base strain. The signal leading sequence screening and the gene dosage increment further improved secretion by approximately 6.4-fold. The purified rationally designed ThGMHs exhibited maximal activity against soluble starch at pH 7.0 and 60&#xa0;℃, and maltose is the main catalytic product. In a 5-L bioreactor, conventional fed-batch fermentation resulted in 6130 U mL<sup>−1</sup> extracellular maltogenic activity. Therefore, a promising strain for GMH production was developed, which provides a useful reference for the secretory production of other industrial enzymes.</p>

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Effect of Fold-Promoting Mutation and Signal Peptide Screening on Recombinant Glucan 1,4-Alpha-maltohydrolase Secretion in Pichia pastoris

  • 期刊论文

  • 发表日期：

    2025年1月8日

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  • Siyi Wang,
  • Kai Zhu,
  • Pulin Liu

摘要

Glucan 1,4-alpha-maltohydrolase (3.2.1.133, GMH) is an important biocatalyst in the baking industry, which could delay the retrogradation of bread and improve its cold-storage durability. In the present study, a newly cloned Thgmh was characterized and secreted by Pichia pastoris (Komagataella pastoris). After computationally assisted rational design that promotes peptide folding, the maltogenic activity in supernatant was enhanced 1.6-fold in comparison with the base strain. The signal leading sequence screening and the gene dosage increment further improved secretion by approximately 6.4-fold. The purified rationally designed ThGMHs exhibited maximal activity against soluble starch at pH 7.0 and 60 ℃, and maltose is the main catalytic product. In a 5-L bioreactor, conventional fed-batch fermentation resulted in 6130 U mL−1 extracellular maltogenic activity. Therefore, a promising strain for GMH production was developed, which provides a useful reference for the secretory production of other industrial enzymes.