Mutant Lactate Oxidase Stability Enhancement
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Solution Overview
Problem
The stability of lactate oxidase enzymes used in biosensors and in vitro tests is low, leading to short shelf-life and in-use times, necessitating frequent replacements and increased costs, which is inconvenient and wasteful, and requires maintaining devices at reduced temperatures for blood analysis.
Innovation Solution
A mutant lactate oxidase with increased stability is developed by substituting tyrosine at position 191 with amino acids like phenylalanine or leucine, enhancing its half-life and maintaining enzymatic activity, allowing for operation at higher temperatures and extended usage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type lactate oxidase is used in biosensors and in vitro tests, then the device can perform lactate determination, but the enzyme stability is low leading to short shelf-life and in-use times
Solution Approach 1:
The patent applies parameter changes by substituting amino acid residues at specific positions (191, 95, 103, 160, 198, 212, 232, 277) in the lactate oxidase sequence to modify enzyme stability parameters. The Tyr191Phe substitution specifically increases thermostability and overall enzyme stability, extending both shelf-life and in-use time without compromising catalytic function.
2Ease of operation
If wild-type lactate oxidase is used, then lactate determination can be performed, but frequent replacements are needed increasing costs and inconvenience
Solution Approach 1:
The patent implements preliminary action by pre-modifying the enzyme through amino acid substitution to enhance its stability before use. The mutant lactate oxidase with improved stability is prepared in advance, eliminating the need for frequent replacements during operation and reducing maintenance time and costs.
3Device complexity
If wild-type lactate oxidase is used, then the enzyme can catalyze lactate oxidation, but the device must be maintained at reduced temperatures complicating the system
Solution Approach 1:
The patent changes the temperature parameter requirement through amino acid substitution. The mutant enzyme with Tyr191Phe substitution exhibits increased thermostability, allowing operation at higher temperatures (up to 37°C and potentially higher) without requiring complex refrigeration or temperature control systems, thereby simplifying device design.
4Reliability
If wild-type lactate oxidase is used, then lactate measurement can be performed, but the enzyme becomes inactivated during use requiring frequent sensor replacement
Solution Approach 1:
The patent modifies the enzyme's stability parameters through site-directed mutagenesis, specifically substituting Tyr191 with Phe. This amino acid substitution increases the enzyme's resistance to inactivation during use, extending the functional lifetime of biosensors and reducing replacement frequency.
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 mutant lactate oxidase exhibits a significantly increased half-life, reducing the need for frequent replacements and allowing for stable operation at 37°C, thus simplifying devices and reducing costs, especially in emerging markets.
Implementation Method 1
lactate oxidase which becomes inactivated during use and shelf-life
Implementation Method 2
The hydrogen peroxide produced by this reaction may be then used in an enzymatic reaction to generate a colored dye
Data Source
Figure 1A~1B
Figure 2
AI summary
The present invention relates to a mutant lactate oxidase having increased stability, a nucleic acid encoding the mutant lactate oxidase, an expression vector comprising the nucleic acid, a host cell comprising the nucleic acid or the expression vector, a method of determining lactate in a sample, the use of the mutant lactate oxidase for determining lactate, a device for determining lactate in a sample using the mutant lactate oxidase and a kit for determining lactate comprising the mutant lactate oxidase.