Understanding enzyme surface kinetics via high content screening methodology

酶是用于衣物洗涤剂和餐具洗涤剂的最重要的分子工具之一，驱动去污、污垢清洁、织物外观/柔软度、白度保持、除臭和去污益处。工业酶在使生物技术更有效和更可靠方面发挥着越来越重要的作用。当用于衣物洗涤剂或餐具洗涤产品时，酶可以提供更好的性能，降低温度，降低水消耗并减轻环境影响。该项目是宝洁公司和学术合作伙伴通过在分子水平上研究酶性能的几个方面来开发新一代高效工业酶的更广泛努力的一部分。具体来说，该项目将研究决定酶如何以及如何与它们作用的底物接触的因素。这很重要，因为这些“吸附/解吸动力学”在确定酶在“真实的生活”消费品中的有效性方面起着关键作用。目前有几种方法用于研究酶吸附/解吸动力学，并将结果与观察到的洗涤性能相关联。例如，表面等离子体共振（SPR）、双偏振干涉（DPI）和具有耗散的石英晶体微天平（QCM-D）。然而，这些方法不能测量与“真实的生活”洗涤剂应用相关的表面上的酶吸附/解吸动力学，因为它们都使用具有模型表面的芯片，所述模型表面与纺织品、陶瓷、钢和与洗涤剂酶相关的其它表面具有可疑的相关性。因此，本项目将开发和应用几种新的方法和技术来研究吸附/解吸动力学。虽然许多不同的酶参与有效的洗衣洗涤剂和餐具洗涤产品，碳水化合物活性酶（CAZymes）将集中在最初。接下来将研究脂肪酶、淀粉酶、蛋白酶和磷酸二酯酶。项目目标：开发一种高含量筛选方法，用于研究织物和硬表面上的酶吸附/脱附动力学。开发高含量筛选方法首先涉及开发合适的荧光酶标记方法。先前的概念验证工作确定脂肪酶可以用DyLight 594荧光标记，其中还发现在有和没有荧光标记的情况下酶活性没有显著差异。将开发一个流动池阵列，同时比较酶的吸附/解吸性能的清洁和污染的织物生物成像后，使用时间推移共聚焦显微镜。应用该方法构建与吸附/解吸动力学、内在活性/底物特异性和洗涤稳定性相关的性能模型。高含量筛选与高含量成像和数据建模将提供一种最先进的方法，该方法利用絮凝细胞成像来测量在洗涤剂存在的真实的时间内酶表面吸附/解吸到相关表面上。这将使P&G能够以自动化和可重复的方式筛选多种条件，复制洗衣和餐具洗涤洗涤循环，使得能够i）通过具有所需表面动力学的配方修饰来增强当前酶的性能，以及ii）与我们的酶供应商一起设计具有所需表面动力学的新酶，以实现用当前酶无法实现的冷洗和快洗中的益处。

Enzymes are one of the most important molecular tools for laundry and dishwashing detergents, driving soil removal, dingy cleaning, fabric appearance/softness, whiteness maintenance, anti-malodour, and soil release benefits. Industry enzymes play increasingly important roles in making biotechnology more effective and substantiable. When used in laundry detergents or dishwashing products, enzymes can provide improved performance with reduced temperatures, lower water consumption and mitigate environmental impacts. This project is part of a wider effort by P&G and academic partners to develop a new generation of highly effective industry enzymes by investigating several aspects of enzyme performance at the molecular level. Specifically, this project, will be investigating the factors that determine how, and how strongly, enzymes become in contact with the substrates they act upon. This is important because these 'adsorption/desorption kinetics' play a critical role in determining how effective enzymes are in 'real life' consumer products. Several methods are currently used to study enzyme adsorption/desorption kinetics and correlate the results to observed wash performance. For example, Surface Plasmon Resonance (SPR), Dual Polarization Interferometry (DPI) and Quartz Crystal Microbalance with Dissipation (QCM-D). However, these methods are unable to measure enzyme adsorption/desorption kinetics on the surfaces relevant to 'real life' detergent applications since they all use chips with model surfaces that have questionable relevance to textiles, ceramics, steel and other surfaces relevant to detergent enzymes. Hence, this project will develop and apply several new approaches and techniques for studying adsorption/desorption kinetics. Whilst many different enzymes are involved in effective laundry detergents and dishwashing products, carbohydrate active enzymes (CAZymes) will be focussed on initially. Following which lipase, amylase, protease and phosphodiesterase will be looked at. Project objectives:Develop a high content screening approach to study enzyme adsorption/desorption kinetics on fabrics and hard surfaces.Developing a high content screening methodology will first involve a suitable fluorescent enzyme tagging approach to be developed. Previous proof of concept work identified that lipase can be fluorescently tagged with DyLight 594 where it was also found that there is no significant difference in enzyme activity with and without fluorescent tag. A flow cell array will be developed to simultaneously compare the adsorption/desorption properties of enzymes on clean and soiled fabrics following bioimaging using time lapse confocal microscopy. Application of the methodology to construct a performance model linked to adsorption/desorption kinetics, intrinsic activity/substrate specificity and in-wash stability.High content screening with high content imaging and data modelling will provide a state-of-the-art approach that utilises imaging of floe cells to measure enzyme surface adsorption/desorption onto relevant surfaces in real time presence of detergent. This will enable P&G to screen multiple conditions in an automated and repeatable manner, replicating the laundry and dishwashing wash cycles enabling to i) enhance the performance of current enzymes via formula modification with desire surface kinetics and ii) engineer new enzymes with desired surface kinetics with our enzyme supplier to achieve the benefits in Cold & Quick wash that can't be achieved with current enzymes.