Presentation Information
[3GteX-03-KL]Rational design of hyperactive enzymes and activity-sensing biosensors
○Munehito Arai1 (1. The University of Tokyo (Japan))
Keywords:
enzyme,rational design,biosensor
[Purpose]
Enhancing enzymatic activity is essential for improving the efficiency of biological manufacturing. Two major approaches are commonly used to obtain hyperactive enzymes: directed evolution, which relies on sensitive biosensors, and rational design guided by structural insights. In this study, we attempted to advance both strategies by designing product-responsive biosensors for activity detection and by developing computational methods for improving catalytic activity of enzymes.
[Method]
For directed evolution, we rationally designed biosensors that emit fluorescence upon binding to reaction products, enabling rapid detection of enzyme activity. For computational enzyme design, we used structural information to introduce mutations predicted to increase catalytic turnover while preserving substrate affinity.
[Results]
The biosensors produced fluorescence signals in response to enzymatic products, indicating their potential utility for high-throughput activity screening. In parallel, our rational design approach identified mutations that enhanced the turnover number of a model enzyme without significantly altering its Michaelis constant, demonstrating that catalytic efficiency can be improved while maintaining substrate binding.
[Consideration]
These results show that both product-responsive biosensors and rational design strategies are effective routes for enhancing enzymatic activity. The biosensors enable rapid screening of enzyme libraries, whereas the rational design method reduces the number of enzyme variants that need to be explored experimentally, thereby improving the efficiency of designing hyperactive enzymes. Together, these approaches have the potential to serve as complementary tools for enzyme optimization.
[Conclusion]
In this study, we developed activity-sensing biosensors for directed evolution and rational design strategies for improving catalytic activity, providing two practical approaches for obtaining hyperactive enzymes. These tools are expected to facilitate the efficient development of high-performance biocatalysts and to contribute to advances in biological manufacturing.
Enhancing enzymatic activity is essential for improving the efficiency of biological manufacturing. Two major approaches are commonly used to obtain hyperactive enzymes: directed evolution, which relies on sensitive biosensors, and rational design guided by structural insights. In this study, we attempted to advance both strategies by designing product-responsive biosensors for activity detection and by developing computational methods for improving catalytic activity of enzymes.
[Method]
For directed evolution, we rationally designed biosensors that emit fluorescence upon binding to reaction products, enabling rapid detection of enzyme activity. For computational enzyme design, we used structural information to introduce mutations predicted to increase catalytic turnover while preserving substrate affinity.
[Results]
The biosensors produced fluorescence signals in response to enzymatic products, indicating their potential utility for high-throughput activity screening. In parallel, our rational design approach identified mutations that enhanced the turnover number of a model enzyme without significantly altering its Michaelis constant, demonstrating that catalytic efficiency can be improved while maintaining substrate binding.
[Consideration]
These results show that both product-responsive biosensors and rational design strategies are effective routes for enhancing enzymatic activity. The biosensors enable rapid screening of enzyme libraries, whereas the rational design method reduces the number of enzyme variants that need to be explored experimentally, thereby improving the efficiency of designing hyperactive enzymes. Together, these approaches have the potential to serve as complementary tools for enzyme optimization.
[Conclusion]
In this study, we developed activity-sensing biosensors for directed evolution and rational design strategies for improving catalytic activity, providing two practical approaches for obtaining hyperactive enzymes. These tools are expected to facilitate the efficient development of high-performance biocatalysts and to contribute to advances in biological manufacturing.
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