Mutated Cellobiose Dehydrogenase for Selective Lactose Detection
Solution Overview
Problem
Cellobiose dehydrogenases (CDHs) lack specificity in detecting lactose in the presence of glucose and galactose, making them less effective in sensors for analyzing mixtures of substrates.
Innovation Solution
Modified CDHs with reduced glucose and galactose oxidation activity are developed by substituting specific amino acids in the flavodehydrogenase domain, maintaining high lactose oxidation activity, allowing for selective lactose detection using direct or mediated electron transfer-based electrodes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If natural CDH is used for lactose detection, then it can oxidize lactose, but it also oxidizes glucose and galactose causing interference
Solution Approach 1:
The patent applies parameter changes by substituting specific amino acid residues in the CDH enzyme structure (e.g., N721Q, N721I, N721T, N721L mutations) to alter the enzyme's substrate specificity. These parameter changes at the molecular level modify the enzyme's catalytic properties, reducing its ability to oxidize glucose and galactose while maintaining lactose oxidation capability, thus resolving the interference issue in lactose detection
Solution Approach 2:
The patent applies local quality by making targeted modifications at specific locations within the CDH enzyme structure, particularly in the flavin domain around residue 721. These localized amino acid substitutions change the properties of specific regions of the enzyme without affecting the overall structure, enabling selective reduction of glucose and galactose oxidation while preserving lactose oxidation activity
2Measurement precision
If amino acid substitution is made to reduce glucose and galactose activity, then specificity improves, but lactose oxidation activity may decrease
Solution Approach 1:
The patent optimizes parameter changes by testing multiple amino acid substitutions (Q, I, T, L) at position 721 and selecting those that provide the best balance between reducing glucose/galactose activity and maintaining lactose oxidation activity. The selected mutations achieve at least 50% reduction in glucose/galactose oxidation while preserving sufficient lactose oxidation capability for effective detection
Solution Approach 2:
The patent applies partial action by achieving moderate reduction in glucose and galactose oxidation activity (at least 50% reduction) rather than complete elimination, which is sufficient to resolve interference issues while minimizing impact on lactose oxidation activity. This partial modification approach maintains adequate productivity for sensor 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 modified CDHs effectively reduce interference from glucose and galactose, enhancing the specificity and accuracy of lactose detection, with minimal reduction in lactose oxidation activity, thus improving sensor performance.
Implementation Method 1
Cellobiose dehydrogenase (EC 1.1.99.18, CDH) was first discovered in 1974 in the extracellular enzyme system of Phanerochaete chrysosporium
Implementation Method 2
By its catalytic activity the natural substrate cellobiose is oxidised in a reaction which reduces the FAD of the flavin domain
Implementation Method 3
the combination of a catalytically active flavodehydrogenase domain (also called 'flavin domain'), hosting a non-covalently bound FAD
Data Source
Figure 1
Figure 1
Figure 2~3
AI summary
The present invention relates to a modified cellobiose dehydrogenase (CDH) or its functional flavodehydrogenase domain having a substitution at the amino acid corresponding to N721 of SEQ ID NO: 5 (CDH from M. thermophilum) by glutamine, isoleucine, threonine or leucine or a substitution of the asparagine in the active canter motif having the amino acid sequence NHW by glutamine, isoleucine, threonine or leucine.