研究目的
To optimise a high-throughput fluorescamine assay for the detection of N-acyl-L-homoserine lactone acylase activity.
研究成果
The optimised fluorescamine-based assay can be used as a high-throughput screen to detect enzymes with AHL acylase activity, with optimal conditions being fluorescamine dissolved in acetone, potassium phosphate buffer, and pH 7.
研究不足
The assay requires purified enzymes to avoid high background fluorescence from crude cell lysates. The high-throughput format is semi-quantitative due to variations in enzyme solubility and yield.
1：Experimental Design and Method Selection:
The study focused on optimising the reaction conditions for the fluorescamine-based assay to detect AHL acylase activity, including testing different buffer conditions and pH levels.
2：Sample Selection and Data Sources:
The study used homoserine lactone (HSL) and fluorescamine to test the reaction under various conditions.
3：List of Experimental Equipment and Materials:
Fluorescamine, acetone, acetonitrile, various buffers spanning pH
4：5–0, and a CLARIOstar microplate reader were used. Experimental Procedures and Operational Workflow:
The reaction conditions were tested by adding fluorescamine to HSL in different buffers and measuring fluorescence immediately and after 60 minutes.
5：Data Analysis Methods:
The fluorescence signal intensity was measured and compared across different conditions to determine the optimal assay conditions.
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