In Vitro Enzyme Glycosylation: A New Platform for Enzyme Stabilization

项目摘要 第一阶段SBIR的目标是开发一个新的平台，增加稳定性和活动寿命， 用于安培生物传感器制造的氧化酶。该平台基于Design-Zyme的新， 正在申请专利的蛋白质和酶体外糖基化方法。第一阶段提案包括 两个具体目标旨在（1）证明Design-Zyme的新平台可以用来增强 并改善许多氧化酶（如果不是全部）的稳定性和活性寿命，和（2）立即 将三种改进的酶商业化，供大学、政府和工业实体使用。我们 方法将在第二阶段扩展到其他蛋白质家族，可能用于传感应用，例如。 脱氢酶 生物传感器领域已经发展成为一种实用的、具有成本效益的和便携式的筛查模式， 在健康监测、疾病诊断、药物开发、食品安全、 和即时诊断。我们增强的酶将使生物传感器的发展， 迄今为止尚未获得，预计将对开发和制造产生积极影响， 用于新传感应用的器件。这些新的设备，基于酶系统（S）在这里开发，将 还为葡萄糖耐量的临床监测、血乳酸盐监测和其他监测提供了新的范例。 与综合医学相关的疾病状态，最终是人类健康。这些增强的酶， 是开发连续体内分析物监测的必要的第一步 持续数月或数年，几乎没有外界干预。 这一建议是基于我们的重要的初步结果，表明在体外糖基化不 这会损害酶的活性，但相反会增强酶的初始活性和寿命。这是相对 大多数现有的糖基化方法。我们已经发现，当透明质酸共价连接到一个 氧化酶，可以提高其在溶液中的活性寿命，并在高温下提供延长的保护。 作为该阶段I的产物的稳定化的酶可以在任何监测装置中找到实用性， 使用氧化酶。这包括电流生物传感器，可穿戴贴片和其他测量 方式。在第二阶段SBIR结束时，我们将拥有一个完全优化的平台，可以优化任何氧化酶 用于体内（人和动物）生物传感器的酶。

Project Summary The goal of this Phase I SBIR is to develop a new platform that increases the stability and activity lifetime of oxidase enzymes used in amperometric biosensor fabrication. This platform is based upon Design-Zyme’s new, patent-pending approach for in vitro glycosylation of proteins and enzymes. This Phase I proposal consists of two specific aims designed to (1) demonstrate that Design-Zyme’s novel platform can be leveraged to enhance and improve the stability and activity lifetime of many, if not all, oxidase enzymes, and (2) to immediately commercialize three improved enzymes for use by university, governmental, and industrial entities. Our approach will be extended in Phase II to other protein families potentially useful in sensing applications, e.g. dehydrogenase enzymes. The field of biosensors has developed into a practical, cost-effective, and portable screening modality with proven animal and human applications in health monitoring, disease diagnosis, drug development, food safety, and point-of-care diagnostics. Our enhanced enzymes will enable the development of biosensors that have been heretofore unavailable, which is expected to positively impact the development and manufacturing of devices for new sensing applications. These new devices, based on the enzyme system(s) developed here, will also provide a new paradigm for the clinical monitoring of glucose tolerance, blood-lactate monitoring, and other disease states relevant to integrative medicine and ultimately, human health. These enhanced enzymes, and the underlying platform, are a necessary first step to the development of continuous in vivo analyte monitoring lasting months or years with little outside intervention. This proposal is based on our significant preliminary results showing that in vitro glycosylation does not compromise enzyme activity, but instead can enhance an enzyme’s initial activity and lifetime. This is in contrast to most existing methods of glycosylation. We have found that hyaluronic acid, when covalently attached to an oxidase, can improve its activity lifetime in solution and provide extended protection at elevated temperatures. The stabilized enzymes that are a product of this Phase I can find utility in any monitoring device that presently uses oxidase enzymes. This includes amperometric biosensors, wearable patches and other measurement modalities. At the end of the Phase II SBIR, we will have a fully optimized platform that can optimize any oxidase enzyme for use in in vivo (human and animal) biosensors.