Firefly Luciferase Mutations Enhance Thermostability
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Firefly luciferase enzymes are unstable under heat and have reduced luminescence persistence, making them unsuitable for high-sensitivity assays and long-term measurements due to their low thermostability and storage stability.
Innovation Solution
Introducing specific mutations at positions 287, 392, 344, 326, and 467 in the firefly luciferase gene, such as mutating the amino acid at position 287 to alanine and 392 to isoleucine, and combining these with other mutations like 326S and 467I, significantly enhances the stability and recovery of reduced stability caused by other mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If firefly luciferase is used for high-sensitivity measurement, then measurement sensitivity is improved, but the enzyme is easily inactivated by heat and loses luminescence rapidly
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations at positions 287, 392, 344, 326, and 467 in the firefly luciferase sequence. These mutations alter the enzyme's structural parameters to enhance thermostability and storage stability while preserving its luminescence function for high-sensitivity measurements.
Solution Approach 2:
The patent applies local quality by making targeted amino acid substitutions at specific positions (287, 392, 344, 326, and 467) rather than global modifications. Each mutation is strategically placed to stabilize specific regions of the enzyme structure, thereby improving overall stability without compromising the active site functionality.
2Duration of action of moving object
If mutation is introduced to improve persistent luminescence, then luminescence duration is improved, but enzyme stability deteriorates
Solution Approach 1:
The patent applies merging by combining multiple mutations (287A, 392I, 344A, 326S, and 467I) into a single luciferase variant. This combination allows the enzyme to simultaneously achieve persistent luminescence through the 344A mutation and enhanced stability through the other mutations, resolving the trade-off between luminescence duration and enzyme stability.
3Reliability
If salt or reagent composition is modified to improve stability, then storage stability is improved, but reagent versatility is reduced and interference increases
Solution Approach 1:
The patent applies taking out by extracting the stability enhancement from the reagent composition and transferring it to the enzyme itself through genetic mutation. Instead of modifying reagents with salts or additives that limit versatility, the stability is built into the luciferase protein structure, allowing the enzyme to maintain stability across diverse reagent formulations and applications.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The mutations result in a firefly luciferase with improved thermostability and storage stability, allowing for increased activity persistence after heat treatment and prolonged storage, effectively addressing the limitations of existing luciferase enzymes.
Implementation Method 1
Firefly luciferase is an enzyme that, in the presence of magnesium ion and oxygen, converts adenosine triphosphate (ATP), D-luciferin and oxygen to adenosine monophosphate (AMP), oxyluciferin and carbon dioxide, generating light.
Data Source
Figure 1~2
Figure 3~4
AI summary
There are provided, firefly luciferase with excellent thermostability and/or storage stability and a process for its production. Firefly luciferase having the amino acid sequence of firefly luciferase, wherein the amino acid corresponding to position 287 of Heike firefly luciferase has been replaced with alanine, or wherein the amino acid corresponding to position 392 has been replaced with isoleucine, and a gene for the firefly luciferase. By utilizing this gene, it is possible to efficiently produce firefly luciferase with increased stability. It is also possible to obtain firefly luciferase with further increased stability by combination with a mutation in which the amino acid at position 326 has been replaced with serine, and/or a mutation in which the amino acid at position 467 has been replaced with isoleucine.