Structure-Function Relationships of Food-Related Enzymes: Aspartic Proteases

Rickey Yada博士由NSERC资助的研究项目的长期目标是从根本上了解酶的功能是如何由其结构决定的。在食品系统中，酶具有广泛的功能，包括防止微生物腐败，提高蛋白质来源的营养价值，工业消毒，以及在各种产品的制造中充当食品加工助剂(例如，奶酪制作)。这项发现拨款提案的重点是天冬氨酸蛋白酶，这是一组结构高度相似的酶，最好的代表是胃酶胃蛋白酶。尽管有这种一致性，但微小的结构差异导致了广泛的不同功能，使它们成为研究酶结构如何决定功能的理想模型。具体地说，将解决两个主要领域的知识差距：首先，了解天冬氨酸蛋白酶对环境条件(热、酸和压力)耐受性的结构基础将涉及能够在高温(来自温泉中的生物体的热蛋白酶)和非酸性条件(来自人肾的肾素)下发挥作用的非典型天冬氨酸蛋白酶的研究。通过蛋白质工程，将在胃蛋白酶(在高温和非酸性条件下都不稳定)中测试存在于温度和肾素中的可疑关键结构特征的稳定效果。此外，还将解决一种名为纤溶酶V的天冬氨酸蛋白酶的结构，以便从其形成其活性结构的独特方式中吸取与稳定性相关的经验教训，这可能导致抑制剂的设计。最后，将描述高压加工(食品工业中的一种新兴加工技术)对几种制作干酪的天冬氨酸蛋白酶结构的影响。第二个研究领域将检测来自植物的天冬氨酸蛋白酶的一部分，称为swaposin‘，它可以抑制食物腐败生物体的生长。为了更好地了解swaposin的作用模式，将最详细地研究它们的结构和与膜的特定接触点。拟议研究计划的影响将是获得必要的知识，以开发解决涉及天冬氨酸蛋白酶的食品相关挑战，如食品腐败、微生物生长和在不同环境条件下涉及酶的食品加工应用。

The long-term goal of Dr. Rickey Yada's NSERC-funded research program has been to fundamentally understand how an enzyme's functionality is determined by its structure. In food systems, enzymes have wide-ranging functions including prevention of microbial spoilage, enhancing nutritional value of protein sources, industrial sanitisation, and acting as food processing aids in the manufacture of a variety of products (e.g., cheese making). This Discovery Grant proposal is focused on aspartic proteases, a group of enzymes which share highly similar structures and are best represented by the stomach enzyme pepsin. Despite this uniformity, minor structural differences result in a wide spectrum of diverse functions, making them an ideal model to study how enzyme structure determines function. Specifically, knowledge gaps in two overarching areas will be addressed: First, understanding the structural underpinnings of aspartic protease tolerance to environmental conditions (heat, acidity, and pressure) will involve the investigation of atypical aspartic proteases that are capable of functioning at high temperature (thermopsin from an organism in hot springs) and non-acidic conditions (renin from human kidney). Through protein engineering, the stabilising effects of suspected key structural features present in thermopsin and renin will be tested in pepsin (which is neither stable at high temperature nor non-acidic conditions). Additionally, the structure of an aspartic protease called plasmepsin V will be resolved so that lessons relevant to stability can be learned from its unique way of forming its active structure, potentially leading to the design of inhibitors. Lastly, the effects of High Pressure Processing (an emerging processing technology in the food industry) on the structures of several cheese making aspartic proteases will be characterised. A second research area will examine a portion of aspartic proteases from plants, termed swaposin', that inhibits the growth of food spoilage organisms. To better understand the swaposin mode of action, their structures and specific points of contact with membranes will be studied at the highest level of detail. The impact of the proposed research program will be to gain knowledge necessary for developing solutions to food-related challenges involving aspartic proteases such as food spoilage, microbial growth and food processing applications involving enzymes in varying environmental conditions.