SOLID PHASE ENZYME SCREENING

固相酶筛选

• 批准号: 2024031
• 负责人: WILLIAM G. COLEMAN
• 金额: $ 10万
• 依托单位: KAIROS SCIENTIFIC, INC.
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 1997-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2024031
• 关键词: bacteria; bacteria characteristic; beta galactosidase; beta glucosidases; biomedical equipment development; catalyst; chemical kinetics; colorimetry; computer program /software; computer system design /evaluation; digital imaging; enzyme activity; indoles; microorganism culture; mutant; spectrometry

Using time-resolved digital imaging spectroscopy, we propose to implement high throughput solid phase filter assays for enzyme activity. This will allow us to screen large numbers (> 10(6)) of enzyme variants for improved activity directly in bacterial colonies. KAIROS' Digital Imaging Spectrophotometer (Colonylmager) will be adapted to the problem of imaging bacterial colony lifts on solid supports (i.e., filters) with colorimetric substrates for specific enzymes. A series of such images versus time will provide information on the substrate turnover rate by each colony. Phase I research and development will focus on improving the filter assay technique and on refining the imaging software used to obtain kinetic data. New software will analyze intra-feature pixel statistics, so as to minimize the distribution of rates obtained from a homogeneous population of colonies. This will enable the identification of colonies exhibiting substrate turnover rates marginally different from wild-type in subsequent mutagenesis experiments. Filter assays and imaging techniques will be made broadly applicable to common colorimetric indicators, beginning with hydrolytic substrates such as X-glu, X-gal, and BCIP. In Phase II, we plan to screen large numbers of enzyme variants for improved catalytic activity and adaptation to harsh conditions, such as high temperature, high/low pH, and organic solvents. We also plan to implement larger format scanners in place of the Colonylmager's CCD camera in order to achieve still higher throughput at lower cost. PROPOSED COMMERCIAL APPLICATION: KAIROS plans to include both commercial services and instrument sales: 1) Provide a service for rapid throughput solid phase analyses of mutagenized libraries for the purpose of identifying clones expressing enzymes with novel (new substrates, high temperature, organic solvents) or improved activity. 2) Market a low cost turn-key instrument to quantitate the results of solid phase enzymatic assays, especially in the emerging field of directed evolution.

使用时间分辨数字成像光谱学，我们建议实施 高通量固相过滤测定酶活性。 这将 允许我们筛选大量 (> 10(6)) 的酶变体以改进 直接在细菌菌落中发挥活性。 KAIROS 的数字成像 分光光度计（Colonylmager）将适应成像问题 使用比色法将细菌菌落提升到固体支持物（即过滤器）上 特定酶的底物。 一系列这样的图像与时间的关系将 提供每个菌落底物周转率的信息。 阶段 我的研发将集中于改进过滤分析 技术和改进用于获得动力学的成像软件 数据。 新软件将分析特征内像素统计数据，以便 最小化从同质总体中获得的比率分布 殖民地。 这将能够识别表现出的菌落 随后的底物周转率与野生型略有不同 诱变实验。 过滤分析和成像技术将 广泛适用于常见的比色指示剂，从 水解底物，例如 X-glu、X-gal 和 BCIP。 在第二阶段，我们 计划筛选大量酶变体以改善催化作用 活动和对恶劣条件的适应，例如高温， 高/低 pH 值和有机溶剂。 我们还计划实施更大规模的 格式化扫描仪来代替 Colonylmager 的 CCD 相机，以便 以更低的成本实现更高的吞吐量。 拟议的商业应用：KAIROS 计划包括商业应用 服务和仪器销售：1）提供快速吞吐量的服务 对诱变文库进行固相分析，目的是 鉴定表达具有新颖酶的克隆（新底物、高 温度、有机溶剂）或提高活性。 2）营销成本低 定量固相酶结果的交钥匙仪器 分析，特别是在新兴的定向进化领域。