The New Zealand Institute for Plant and Food Research Limited, Batchelar Road, Palmerston North 4410, New Zealand.
Kiwifruit Breeding Centre (previously The New Zealand Institute for Plant and Food Research Limited), Te Puke, New Zealand.
Anal Methods. 2022 Sep 22;14(36):3552-3561. doi: 10.1039/d2ay01007k.
The actinidin proteinase family has a striking sequence diversity; isoelectric points range from 3.9 to 9.3. The biological drive for this variation is thought to be actinidin's role as a defense-related protein. In this study we map mutations in the primary sequence onto the 3D structure of the protein and show that the region with the highest diversity is close to the substrate binding groove. Non-conservative substitutions in the active site determine substrate preference and therefore create problems for quantification of actinidin activity. Here we use a peptide substrate library to compare two actinidin isoforms, one from the kiwiberry cultivar 'Hortgem Tahi' (), and the other from the familiar kiwifruit cultivar 'Hayward' ( var. ). Among 360 octamer substrates we find one substrate (RVAAGSPI) with the useful property of being readily cleaved by all the functionally active actinidins in a set of and var. isoforms. In addition, we find that two substrates (LPPKSQPP & ILRDKDNT) have the ability to differentiate different isoforms from a single fruit. We compare actinidins from 'Hayward' and for their ability to digest the allergenic gluten peptide (PFPQPQLPY) but find the peptide to be indigestible by all sources of actinidin. The ability to inactivate salivary amylase is shown to be a common trait in cultivars due to proteolysis by actinidin and is particularly strong in 'Hortgem Tahi'. A mixture of 10% 'Hortgem Tahi' extract with 90% saliva inactivates 100% of amylase activity within 5 minutes. Conceivably, 'Hortgem Tahi' might lower the glycaemic response in a meal rich in cooked starch.
植物凝乳蛋白酶家族具有显著的序列多样性；等电点范围为 3.9 至 9.3。这种变异的生物学驱动力被认为是植物凝乳蛋白酶作为防御相关蛋白的作用。在这项研究中，我们将一级序列中的突变映射到蛋白质的 3D 结构上，并表明具有最高多样性的区域靠近底物结合槽。活性位点的非保守取代决定了底物的偏好，因此给植物凝乳蛋白酶活性的定量带来了问题。在这里，我们使用肽底物文库比较了两种植物凝乳蛋白酶同工型，一种来自猕猴桃品种“Hortgem Tahi”（），另一种来自常见的猕猴桃品种“Hayward”（var. ）。在 360 个八肽底物中，我们发现有一种底物（RVAAGSPI）具有有用的特性，即容易被一组 和 var. 同工型中的所有功能活性的植物凝乳蛋白酶切割。此外，我们发现两种底物（LPPKSQPP 和 ILRDKDNT）具有区分来自单个果实的不同同工型的能力。我们比较了“Hayward”和 中的植物凝乳蛋白酶消化过敏原谷蛋白肽（PFPQPQLPY）的能力，但发现所有来源的植物凝乳蛋白酶都不能消化该肽。由于植物凝乳蛋白酶的蛋白水解作用，显示出使唾液淀粉酶失活的能力是 品种的共同特征，在“Hortgem Tahi”中尤为强烈。将 10%的“Hortgem Tahi”提取物与 90%的唾液混合，可在 5 分钟内使 100%的淀粉酶活性失活。可以想象，“Hortgem Tahi”可能会降低富含煮熟淀粉的膳食中的血糖反应。
