Osorio Claudia E, Wen Nuan, Mejías Jaime H, Mitchell Shannon, von Wettstein Diter, Rustgi Sachin
Department of Crop and Soil Sciences, Washington State University, Pullman, WA, United States.
Agriaquaculture Nutritional Genomic Center, Temuco, Chile.
Front Nutr. 2020 Feb 18;7:11. doi: 10.3389/fnut.2020.00011. eCollection 2020.
Wheat gluten proteins are the known cause of celiac disease. The repetitive tracts of proline and glutamine residues in these proteins make them exceptionally resilient to digestion in the gastrointestinal tract. These indigested peptides trigger immune reactions in susceptible individuals, which could be either an allergic reaction or celiac disease. Gluten exclusion diet is the only approved remedy for such disorders. Recently, a combination of a glutamine specific endoprotease from barley (EP-B2), and a prolyl endopeptidase from (Fm-PEP), when expressed in the wheat endosperm, were shown to reasonably detoxify immunogenic gluten peptides under simulated gastrointestinal conditions. However useful, these "glutenases" are limited in application due to their denaturation at high temperatures, which most of the food processes require. Variants of these enzymes from thermophilic organisms exist, but cannot be applied directly due to their optimum activity at temperatures higher than 37°C. Though, these enzymes can serve as a reference to guide the evolution of peptidases of mesophilic origin toward thermostability. Therefore, a sequence guided site-saturation mutagenesis approach was used here to introduce mutations in the genes encoding Fm-PEP and EP-B2. A thermostable variant of Fm-PEP capable of surviving temperatures up to 90°C and EP-B2 variant with a thermostability of up 60°C were identified using this approach. However, the level of thermostability achieved is not sufficient; the present study has provided evidence that the thermostability of glutenases can be improved. And this pilot study has paved the way for more detailed structural studies in the future to obtain variants of Fm-PEP and EP-B2 that can survive temperatures ~100°C to allow their packing in grains and use of such grains in the food industry.
小麦麸质蛋白是乳糜泻的已知病因。这些蛋白质中脯氨酸和谷氨酰胺残基的重复序列使它们对胃肠道消化具有极强的抵抗力。这些未消化的肽会在易感个体中引发免疫反应，可能是过敏反应或乳糜泻。麸质排除饮食是治疗此类疾病的唯一批准疗法。最近，当在小麦胚乳中表达时，来自大麦的谷氨酰胺特异性内切蛋白酶（EP-B2）和来自[未提及具体来源]的脯氨酰内切蛋白酶（Fm-PEP）的组合在模拟胃肠道条件下显示出能合理地解毒免疫原性麸质肽。然而，这些“麸质酶”由于在高温下变性而应用受限，而大多数食品加工过程都需要高温。存在来自嗜热生物的这些酶的变体，但由于它们在高于37°C的温度下具有最佳活性，不能直接应用。不过，这些酶可作为参考，以指导中温来源的肽酶向热稳定性进化。因此，这里采用了序列引导的位点饱和诱变方法在编码Fm-PEP和EP-B2的基因中引入突变。使用这种方法鉴定出了一种能够在高达90°C的温度下存活的Fm-PEP热稳定变体和一种热稳定性高达60°C的EP-B2变体。然而，所达到的热稳定性水平还不够；本研究提供了证据表明麸质酶的热稳定性可以提高。并且这项初步研究为未来更详细的结构研究铺平了道路，以获得能够在约100°C的温度下存活的Fm-PEP和EP-B2变体，以便将它们包装在谷物中并在食品工业中使用此类谷物。
