Amadoriase Mutants for HbA1c Measurement Specificity
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Solution Overview
Problem
Current amadoriases used for measuring glycated hemoglobin (HbA1c) have insufficient substrate specificity, leading to measurement errors due to reactivity with ε-fructosyl lysine (ε-FK), which is a contaminant generated during the hemoglobin treatment process.
Innovation Solution
Specific amino acid substitutions in the amadoriase derived from the genus Coniochaeta, such as glutamic acid at position 98, and other targeted positions, reduce the enzyme's reactivity to ε-FK relative to α-fructosyl valyl histidine (α-FVH) and α-fructosyl valine (α-FV), enhancing substrate specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If amadoriases with broad substrate reactivity are used for HbA1c measurement, then the enzyme can detect multiple glycated proteins, but measurement precision deteriorates due to cross-reactivity with ε-FK contaminants
Solution Approach 1:
The patent applies local quality by introducing specific amino acid substitutions at particular positions (e.g., position 98, 110, 154, 259) in the amadoriase molecule. These localized changes modify the enzyme's active site properties to selectively reduce reactivity with ε-FK while preserving activity toward α-FVH and α-FV, thereby achieving high substrate specificity without compromising overall catalytic function
Solution Approach 2:
The patent employs parameter changes by systematically varying amino acid residues at specific positions in the amadoriase sequence. Through site-directed mutagenesis, parameters such as hydrophobicity, charge, and steric properties at key positions are altered to optimize the enzyme's substrate recognition profile, reducing cross-reactivity with ε-FK while maintaining high activity for HbA1c measurement
2Reliability
If amino acid substitutions are introduced to alter substrate specificity, then reactivity to ε-FK is reduced, but enzyme stability may deteriorate
Solution Approach 1:
The patent applies parameter changes by systematically varying amino acid residues at specific positions in the amadoriase sequence. Through site-directed mutagenesis, parameters such as hydrophobicity, charge, and steric properties at key positions are altered to optimize the enzyme's substrate recognition profile, reducing cross-reactivity with ε-FK while maintaining high activity for HbA1c measurement
Solution Approach 2:
The patent employs self-service by selecting amino acid substitutions that maintain the enzyme's overall structural integrity and catalytic mechanism. The modified amadoriases retain their ability to catalyze the oxidation of fructosyl amino acids and peptides, demonstrating that the core functional properties are preserved despite local sequence changes
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 modified amadoriases exhibit significantly lower ratios of ε-FK/α-FVH and ε-FK/α-FV, reducing measurement errors and enabling accurate quantification of HbA1c, thus providing a more reliable diabetes mellitus marker.
Implementation Method 1
An amadoriase oxidizes iminodiacetic acid or a derivative thereof (also referred to as an 'Amadori compound') in the presence of oxygen to catalyze a reaction to generate glyoxylic acid or α-ketoaldehyde, amino acid or peptide, and hydrogen peroxide
Implementation Method 2
An amadoriase oxidizes iminodiacetic acid or a derivative thereof (also referred to as an 'Amadori compound') in the presence of oxygen to catalyze a reaction
Data Source
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AI summary
This invention provides an amadoriase having high substrate specificity to fructosyl valyl histidine. Such amadoriase comprises substitution of one or more amino acid residues at positions corresponding to amino acids selected from the group consisting of position 98, position 259, position 154, position 125, position 261, position 263, position 106, position 103, position 355, position 96, position 66, position 67, position 70, position 100, position 110, position 113, position 114, and position 156 in the amadoriase derived from the genus Coniochaeta. This invention enables accurate measurement of α-fructosyl valyl histidine derived from the β-chain amino terminus in glycated hemoglobin in the presence of ε-fructosyl lysine.