在淀粉类食品面团发酵的过程中,低pH环境对麦芽糖淀粉酶的稳定性提出了严峻挑战。目前大多数麦芽糖淀粉酶对酸性条件敏感,在低pH环境下快速失活,限制了其在面包制作中的应用。该文针对来源于类食品乳杆菌的麦芽糖淀粉酶,从催化中心pKa值和表面电荷两方面设计突变位点进行耐酸性改造。通过单点突变与组合突变,成功筛选出最佳正向耐酸突变体K322D/K361D,其最适pH从5.0降至4.5,pH 4.5时水解活力高达433.23 U/mg,较野生型酶提升2.27倍,且该突变体在pH 4.0~5.0稳定性大于68%。结构与静电势能分析表明,突变体表面净电荷的变化有助于改善酶的构象状态,增强柔性结构调整能力,从而提高pH稳定性。该研究为改善麦芽糖淀粉酶在面制品抗老化应用效果提供了理论基础,展现出良好的应用前景。
During the fermentation process of starchy foods, the low pH environment poses a severe challenge to the stability of maltogenic amylase.At present, most maltogenic amylase is sensitive to acidic conditions and rapidly inactivates in low pH environments, which limits its application in bread making.This research studied the maltogenic amylase derived from Lactobacillus paralimentarius and designed mutation sites for acid-resistant modification from both the pKa value of the catalytic center and surface charge.Through single-point mutation and combined mutation, the best forward acid-resistant mutant K322D/K361D was successfully screened.Its optimal pH dropped from 5.0 to 4.5, and the hydrolysis activity at pH 4.5 increased to 433.23 U/mg, which was 2.27 times higher than that of the wild type.Its stability was greater than 68% in the pH range of 4.0-5.0.Structural and electrostatic potential energy analysis showed that the change in the net charge on the surface of the mutant helped to improve the conformational state of the enzyme, enhanced the ability to adjust the flexible structure, and thereby improving pH stability.This study provides a theoretical basis for improving the anti-aging effect of maltogenic amylase in flour products and shows good application prospects.
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