Amaranthaceae α-amylase inhibitors (AAIs) are knottin-type proteins with selective inhibitory potential against coleopteran α-amylases. Their small size and remarkable stability make them exciting molecules for protein engineering to achieve superior selectivity and efficacy. In this report, we have designed a set of AAI pro- and mature peptides chimeras. Based on in silico analysis, stable AAI chimeras having a stronger affinity with target amylases were selected for characterization. In vitro studies validated that chimera of the propeptide from Chenopodium quinoa α-AI and mature peptide from Beta vulgaris α-AI possess 3, 7.6, and 4.26 fold higher inhibition potential than parental counterparts. Importantly, recombinant AAI chimera retained specificity towards target coleopteran α-amylases. In addition, to improve the inhibitory potential of AAI, we performed in silico site-saturation mutagenesis. Computational analysis followed by experimental data showed that substituting Asparagine at the 6th position with Methionine had a remarkable increase in the specific inhibition potential of Amaranthus hypochondriacus α-AI. These results provide structural–functional insights into the vitality of AAI propeptide and a potential hotspot for mutagenesis to enhance the AAI activity. Our investigation will be a toolkit for AAI’s optimization and functional differentiation for future biotechnological applications.

苋科 α-淀粉酶抑制剂 (AAI) 是结蛋白型蛋白，对鞘翅目 α-淀粉酶具有选择性抑制潜力。它们的小尺寸和卓越的稳定性使它们成为蛋白质工程中令人兴奋的分子，以实现卓越的选择性和功效。在本报告中，我们设计了一组 AAI 前肽和成熟肽嵌合体。基于计算机分析，选择与目标淀粉酶具有更强亲和力的稳定AAI嵌合体进行表征。体外研究证实藜麦前肽α-AI 和甜菜α-AI 成熟肽的嵌合体具有比亲本对应物高 3、7.6 和 4.26 倍的抑制潜力。重要的是，重组 AAI 嵌合体保留了针对鞘翅目 α-淀粉酶靶标的特异性。此外，为了提高 AAI 的抑制潜力，我们进行了计算机位点饱和诱变。计算分析和实验数据表明，将第6位天冬酰胺替换为蛋氨酸后，胁苋α-AI的特异性抑制潜力显着增加。这些结果提供了对 AAI 前肽活力的结构-功能见解，以及增强 AAI 活性的诱变潜在热点。我们的研究将成为 AAI 优化和功能分化的工具包，以用于未来的生物技术应用。