Abstract

Enzymes are important tools for various applications. We have studied structural transitions and functional stability of a Kunitz trypsin inhibitor from Chickpea (CaTI2), a potent insect gut-protease inhibitor, under different stress conditions like non-neutral pH, elevated temperature and co-solvent concentrations. CaTI2 was cloned and expressed in an eukaryotic system P. pastoris and was investigated for conformational transitions using circular dichroism spectroscopy, differential scanning fluorimetry and activity assay. Native CaTI2 has a sheet dominant structure with 40% β sheets and possess a single tryptophan residue situated in the hydrophobic core of the enzyme. The recombinant inhibitor maintained its maximum activity under alkaline pH with its secondary structure intact between pH 6–10. CaTI2 was observed to be thermally stable up to 55 °C with a T_m of 61.3 °C above which the protein unfolds. On treating with chemical denaturant (urea), the CaTI2 lost its inhibitory potential and native conformation beyond 2 M urea concentration. Moreover, the protein unfolded at lower temperatures as the concentration of denaturant increased, suggesting more complex structural changes. Further, the stability of the inhibitor was found to be directly proportional to the solvent polarity. The data, herein offers significant information of inhibitor stability and activity which could be exploited for its further development into an effective pesticide.

Graphic Abstract

中文翻译：

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鹰嘴豆的Kunitz胰蛋白酶抑制剂（CaTI2）的功能稳定性和结构转变。

摘要

酶是用于各种应用的重要工具。我们研究了鹰嘴豆的Kunitz胰蛋白酶抑制剂（CaTI2）（一种有效的昆虫肠道蛋白酶抑制剂）在不同的胁迫条件下（如非中性pH，升高的温度和助溶剂浓度）的结构转变和功能稳定性。CaTI2被克隆并在真核系统巴斯德毕赤酵母中表达并使用圆二色谱，差示扫描荧光法和活性测定法研究其构象转变。天然CaTI2具有40％β片层的片层优势结构，并具有位于酶疏水核心中的单个色氨酸残基。重组抑制剂在碱性pH下保持其最大活性，其二级结构在pH 6-10之间保持完整。CaTI2观察到是热稳定的高达55℃用Ť_米在61.3°C时蛋白质会展开。用化学变性剂（尿素）处理后，CaTI2失去了超过2 M尿素浓度的抑制能力和天然构象。此外，随着变性剂浓度的增加，蛋白质在较低的温度下展开，表明结构更加复杂。此外，发现抑制剂的稳定性与溶剂极性成正比。本文中的数据提供了抑制剂稳定性和活性的重要信息，可将其用于进一步开发成有效农药。

图形摘要