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HIGH THROUGHPUT HYDROLASE SCREENING

高通量水解酶筛选

基本信息

批准号：
6311126
负责人：
FRANCISCO MORIS-VARAS
金额：
$ 9.1万
依托单位：
THERMOGEN,INC.
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-03-01 至 2001-12-01
项目状态：
已结题

项目摘要

Description: (Applicant's abstract) High throughput screening of biocatalysts is of great relevance for the fields of chemical processing, biotransformation, and drug discovery. Continuous progress in molecular biology, especially DNA cloning and mutagenesis, has facilitated access to a number of biocatalysts unpredicted a few years ago. Conventional screening methods practiced by organic chemists are unable to provide answers within the process development timeline for new pharmaceuticals. ThermoGen proposes to develop a comprehensive screening system for hydrolases. This system will use actual substrates instead of fluorogenic/chromogenic substrate analogs and will also perform enantioselectivity measurements. In Phase I we will develop an assay for isolated enzymes based on pH indicators using a solid phase format. We will use fluorescence indicators as an alternative for screening heterogeneous reaction mixtures, like in vivo assay of clones and screening of poorly soluble substrates. In Phase II we will design assays for the synthetic use of hydrolytic enzymes (in anhydrous media) and will develop data analysis software compatible with the mainstream hardware available. Phase III will see commercialization of the solid phase and the data analysis software and the offer of the screening technology to synthetic organic chemists and pharmaceutical manufacturers. PROPOSED COMMERCIAL APPLICATION: ThermoGen proposes to develop a high throughput assay to asses activity and enantioselectivity of hydrolytic enzymes. This assay will shorten and simplify the currently tedious and timeconsuming screening step to discover and implement industrial scale biotransformation processes.

描述：（申请人摘要）生物催化剂的高通量筛选对于化学加工，生物转化，和药物发现。分子生物学的不断进步，尤其是DNA 克隆和诱变，促进了获得一些生物催化剂，几年前没有预料到。传统的筛选方法，有机化学家无法在工艺开发过程中提供答案新药的时间轴。 ThermoGen公司建议开发一种水解酶的综合筛选系统。该系统将使用实际底物，而不是荧光/显色底物底物类似物，并且还将进行对映选择性测量。在第一阶段，我们将开发一种基于pH指示剂的分离酶测定方法使用固相形式。我们将使用荧光指示剂作为用于筛选非均相反应混合物的替代方法，如体内测定克隆和筛选难溶性底物。在第二阶段，我们将设计水解酶合成用途的测定法（在无水介质中）并将开发与主流硬件兼容的数据分析软件 available.第三阶段将看到固相和数据的商业化分析软件和筛选技术的提供，以合成有机化学家和制药商。拟定商业应用： ThermoGen建议开发一种高通量检测方法来评估活性，水解酶的对映体选择性。该分析将缩短和简化目前繁琐和耗时的筛选步骤，以发现和实施工业规模的生物转化方法。