Mutant Glucose Dehydrogenase Substrate Specificity
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Solution Overview
Problem
Wild-type cytochrome-c-containing glucose dehydrogenase (CyGDH) and PQQGDH struggle with accurate blood glucose measurement due to reactivity with xylose and galactose, in addition to glucose, limiting their substrate specificity.
Innovation Solution
A mutant-type glucose dehydrogenase with specific amino acid substitutions in the α-subunit, such as replacing glycine at position 10, histidine at position 4, and asparagine at position 4 with other amino acids, reduces reactivity with xylose and galactose while maintaining glucose specificity, utilizing peptide sequences (i) to (v) and optionally additional substitutions at specific positions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If wild-type CyGDH or PQQGDH is used for glucose measurement, then glucose dehydrogenase activity is sufficient, but substrate specificity is poor due to reactivity with xylose and galactose
Solution Approach 1:
The patent applies local quality by making specific amino acid substitutions at particular positions (326, 365, 472) in the α-subunit of glucose dehydrogenase. These localized changes modify the enzyme's substrate binding pocket to selectively recognize glucose while excluding xylose and galactose, thereby improving substrate specificity without compromising overall enzyme function.
Solution Approach 2:
The patent employs parameter changes by altering the amino acid sequence parameters of the GDH α-subunit through site-directed mutagenesis. Specific residues are substituted to change the chemical and physical properties of the active site, optimizing substrate recognition and reducing cross-reactivity with interfering sugars.
2Object-generated harmful factors
If mutant GDH with mutations at positions 326, 365, and 472 is used, then reactivity with maltose is reduced, but reactivity with xylose and galactose remains high
Solution Approach 1:
The patent applies partial or excessive action by combining the mutations at positions 326, 365, and 472 with additional mutations at positions 363 and/or 475. This cumulative approach ensures that while maltose reactivity is reduced, the additional mutations specifically address the insufficient reduction of xylose and galactose reactivity, achieving comprehensive substrate specificity improvement.
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 GDH exhibits improved substrate specificity for glucose, suitable for use in glucose sensors and assays, with decreased reactivity with xylose and galactose, enhancing measurement accuracy.
Implementation Method 1
a mutant-type glucose dehydrogenase having glucose dehydrogenase activity
Implementation Method 2
glucose dehydrogenase activity
Data Source
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AI summary
The present invention provides a mutant-type glucose dehydrogenase having glucose dehydrogenase activity and having decreased reactivity with xylose, wherein said mutant-type glucose dehydrogenase comprises a mutant-type α-subunit comprising an amino acid sequence of 520 to 550 amino acids containing the following peptide sequences (i) to (v) in this order, except that one or more amino acid residue(s) selected from the group consisting of the glycine at position 10 in the peptide sequence (iii), the histidine at position 4 in the peptide sequence (iv), and the asparagine at position 4 in the peptide sequence (v) is/are substituted with another/other amino acid(s): (i) Val/Ile Val/Ile Val/Ile Gly Ser Gly Val Ala Gly (SEQ ID NO:21); (ii) Cys Cys Gly Asn Asn Asn Cys Met Pro Ile Cys (SEQ ID NO:22); (iii) Val Gly Arg Asn Leu Met Asp His Pro Gly Thr Gly (SEQ ID NO:23); (iv) Lys Lys Ile/Leu His Leu Ser Asn (SEQ ID NO:24); (v) Phe Ala Pro/Asn Asn Asn His Ile (SEQ ID NO:25).