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Fluorescent microplate assay method for high-throughput detection of lipase transesterification activity
Jianyong Zheng1, Wei Wei1, Xing Lan1
1Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hang Zhou 310014, Zhejiang, People's Republic of China.
Analytical Biochemistry
|March 18, 2018
Summary
This study presents a sensitive, fluorescent microplate assay for detecting lipase transesterification activity using a model reaction. The method is accurate, efficient, and cost-effective for enzyme analysis.
Area of Science:
- Biochemistry
- Enzymology
Background:
- Lipases are crucial enzymes catalyzing transesterification reactions.
- Accurate detection of lipase activity is essential for various industrial and research applications.
Purpose of the Study:
- To develop a sensitive and efficient fluorescent microplate assay for quantifying lipase transesterification activity.
- To establish a model reaction using butyryl 4-methyl umbelliferone (Bu-4-Mu) and methanol for assay validation.
Main Methods:
- A model transesterification reaction between Bu-4-Mu and methanol in tert-butanol was employed.
- The release of 4-methylumbelliferone (4-Mu) was quantified by measuring fluorescence intensity at specific wavelengths (λex 330 nm, λem 390 nm).
- The assay's accuracy and efficiency were evaluated using various lipases, and kinetic parameters (Km) were determined.
Main Results:
- The developed assay demonstrated high sensitivity in detecting lipase transesterification.
- The method accurately quantified lipase activity across different enzyme samples.
- Kinetic parameters, including apparent Michaelis constant (Km), were successfully calculated for the model reaction.
Conclusions:
- The proposed fluorescent microplate assay is a sensitive, cost-effective, and simple method for detecting lipase transesterification activity.
- This assay provides a reliable tool for enzyme characterization and screening in biochemical research and industrial biotechnology.
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