In Vitro Enzyme Glycosylation: A New Platform for Enzyme Stabilization

项目摘要 此第一阶段SBIR的目标是开发一种新平台，以提高稳定性和活动寿命 用于电流型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的体外糖基化方法。此第一阶段提案包括 两个具体目标旨在(1)展示Design-Zyme的新颖平台可用于增强 并提高许多(如果不是全部)氧化酶的稳定性和活性寿命，以及(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。我们的 该方法将在第二阶段扩展到其他可能在传感应用中有用的蛋白质家族，例如 脱氢酶。 生物传感器领域已经发展成为一种实用、成本效益高和便携的筛查方式 动物和人类在健康监测、疾病诊断、药物开发、食品安全、 和医疗点诊断。我们增强的酶将使生物传感器的开发成为可能 到目前为止还无法获得，预计将对 用于新的传感应用的设备。这些基于这里开发的酶系统(S)的新设备将 也为临床糖耐量监测、血乳酸监测等提供了新的范例 疾病状态与综合医学相关，最终与人类健康有关。这些增强的酶，以及 基础平台是发展连续体内分析物监测的必要的第一步 在几乎没有外部干预的情况下持续数月或数年。 这一建议是基于我们重要的初步结果，即体外糖基化不会 降低了酶的活性，但反而可以提高酶的初始活性和寿命。这与之形成鲜明对比 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 氧化物酶，可以延长其在溶液中的活性寿命，并在高温下提供延长的保护。 作为这一阶段I的产品的稳定酶可以在任何监测设备中找到用途，目前 使用氧化物酶。这包括安培生物传感器、可穿戴贴片和其他测量 医疗模式。在第二阶段SBIR结束时，我们将拥有一个完全优化的平台，可以优化任何氧化剂 用于活体(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.