Mutant Creatinase Enzyme for Stable Biosensor Operation
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Solution Overview
Problem
Current biosensors for measuring creatinine in physiological fluids have a shortened lifespan due to inactivation by thiol-interactive agents like MIT, which are used to prevent bacterial growth, and lack thermostability, making them unsuitable for elevated temperatures.
Innovation Solution
A mutant creatine amidinohydrolase polypeptide with specific amino acid substitutions, such as C175A and C299A, that retains activity in the presence of thiol-agents and exhibits enhanced thermostability, allowing for the use of preservative agents like Neolone 950 without reducing sensor lifespan.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If thiol-interactive agents like MIT are used to prevent bacterial growth in biosensors, then microbial contamination is controlled, but the enzymatic activity of creatinase is inactivated and sensor lifespan is shortened
Solution Approach 1:
The patent applies this principle by using thiol-interactive agents (MIT) that would normally harm the creatinase enzyme, but by introducing a mutant creatinase with thiol-resistant properties, the harmful agent becomes beneficial for preventing microbial contamination while the mutant enzyme remains protected and functional, thus extending sensor lifespan
Solution Approach 2:
The patent changes the chemical structure parameter of the creatinase enzyme by introducing specific amino acid substitutions (C175A, C299A, and/or C304A mutations) that alter the enzyme's sensitivity to thiol agents, making it resistant to MIT while maintaining catalytic activity, thereby resolving the contradiction between contamination control and enzyme stability
2Object-affected harmful factors
If preservative solutions containing thiol-agents are used to control microbial growth, then bacterial and fungal contamination is prevented, but the thiol groups of creatinase are modified and enzyme activity is lost
Solution Approach 1:
The patent transforms the harmful effect of thiol-agents on creatinase into a beneficial situation by creating a mutant enzyme that is resistant to these agents. The preservative solution continues to effectively control microbial contamination while the mutant creatinase (with C175A, C299A, and/or C304A mutations) remains unaffected and maintains its enzymatic activity
Solution Approach 2:
The mutant creatinase acts as an intermediary that is compatible with thiol-agent preservatives. The specific amino acid substitutions create a protective effect, allowing the enzyme to function as a mediator between the preservative system and the biochemical reactions, enabling both contamination control and enzyme functionality
3Ease of operation
If standard creatinase is used in biosensors, then the sensor can measure creatinine, but the enzyme lacks thermostability and is inactivated at elevated temperatures
Solution Approach 1:
The patent changes the thermal stability parameter of creatinase by introducing specific amino acid substitutions (C175A, C299A, and/or C304A). These mutations alter the enzyme's three-dimensional structure and bonding characteristics, increasing its thermostability while preserving its ability to catalyze creatinine measurement reactions
4Reliability
If creatinase is used in the presence of isothiazolinone-derived agents, then microbial growth is controlled, but the enzyme activity is inhibited and sensor performance deteriorates
Solution Approach 1:
The patent changes the chemical resistance parameters of creatinase through site-directed mutagenesis, specifically substituting cysteine residues at positions 175, 299, and/or 304 with amino acids that are resistant to isothiazolinone-derived agents. This allows the enzyme to maintain full productivity and sensor performance while the preservative agents effectively control microbial growth
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 creatine amidinohydrolase extends the lifespan of biosensors by maintaining enzymatic activity at elevated temperatures and in the presence of thiol-agents, ensuring accurate and prolonged creatinine measurement in physiological fluids.
Implementation Method 1
A mutant polypeptide having creatine amidinohydrolase activity, where the polypeptide both retains activity in the presence of reagents that modify thiol groups and has enhanced thermostability
Data Source
AI summary
The invention relates to a mutant polypeptide having creatine amidinohydrolase activity, where the polypeptide both retains thermostable activity in the presence of reagents that modify thiol groups. The invention further relates to methods for producing the mutant polypeptide; and to a sensor comprising the mutant polypeptide for use in the measurement of creatinine in samples of physiological fluids. Additionally, the invention teaches how to use the mutant polypeptide to enhance the life-time of a creatinine sensor, and a method for producing the sensor.