Protein Disulfide Bond Cleavage Using Electrode-Regenerated Redox Protein
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Solution Overview
Problem
Conventional methods for cleaving disulfide bonds in proteins, such as those described in PTL 1 and PTL 2, require excessive amounts of enzymes or thioredoxin, leading to inefficient digestion of digestion-resistant proteins and residual enzyme in food.
Innovation Solution
A method and device that utilize a reduced redox protein to cleave disulfide bonds, with electron donation from an electrode connected to an external power supply to repeatedly activate and reduce oxidized redox proteins, and a device comprising an electrode, power supply, and controller to control voltage application.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional alkaline treatment or thioredoxin addition is used to improve protein digestibility, then disulfide bonds are cleaved and digestibility improves, but excessive amounts of enzymes or thioredoxin are required
Solution Approach 1:
The patent replaces the chemical/biological reduction system (thioredoxin or alkaline treatment) with an electrochemical system. An electrode applies electrical potential to directly reduce disulfide bonds in proteins, eliminating the need for excessive thioredoxin or enzyme additions. The electrical energy directly drives the reduction reaction: R-S-S-R + 2e⁻ → 2R-S⁻
Solution Approach 2:
The patent changes the fundamental parameter for disulfide bond reduction from chemical concentration (thioredoxin amount) to electrical potential (voltage applied to electrode). By controlling the electrode potential to be more negative than the reduction potential of the disulfide bond, efficient reduction occurs with minimal material input. The system monitors and adjusts voltage to maintain optimal reduction conditions.
2Reliability
If excessive amounts of thioredoxin are added to cleave all disulfide bonds, then complete cleavage is achieved, but a large amount of unreacted enzyme remains in the food
Solution Approach 1:
The patent replaces the biological enzyme system with an electrochemical reduction system. The electrode directly donates electrons to disulfide bonds without requiring thioredoxin as an intermediary. This eliminates the problem of unreacted enzyme remaining in the food, as the electrical energy is fully consumed in the reduction process and leaves no biological material behind.
Solution Approach 2:
The electrochemical system is self-regulating through the applied voltage. The electrode continuously supplies electrons as long as voltage is applied, ensuring complete reduction without needing excess reagent. The system automatically adjusts to reduce all available disulfide bonds based on the electrical energy supplied, not on the amount of thioredoxin present.
3Reliability
If conventional methods are used to reduce disulfide bonds, then some cleavage occurs, but the process is inefficient and requires large amounts of reducing agents
Solution Approach 1:
The patent substitutes the slow, concentration-dependent enzymatic reduction with rapid, potential-dependent electrochemical reduction. The electrode directly transfers electrons to disulfide bonds at rates determined by electrical current, which can be precisely controlled and scaled. This eliminates the kinetic limitations of enzymatic systems where reaction rate depends on enzyme concentration and substrate availability.
Solution Approach 2:
The electrochemical system provides continuous reduction as long as voltage is applied to the electrode. Unlike batch enzymatic reactions that reach equilibrium based on reagent amounts, the electrical system can continuously drive reduction by maintaining the potential difference, ensuring complete and efficient conversion of disulfide bonds to thiol groups throughout the treatment period.
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
Efficient reduction and cleavage of disulfide bonds in proteins using a small amount of redox protein, improving protein digestibility by breaking disulfide bonds and enhancing enzyme activity.
Implementation Method 1
reducing an oxidized redox protein produced by oxidation of the reduced redox protein in the cleaving to the reduced redox protein by donating an electron from an electrode connected to an external power supply outside the reaction system to the oxidized redox protein
Implementation Method 2
cleaving a disulfide bond in a protein present in a reaction system by reduction via a reduced redox protein
Data Source
AI summary
A method for cleaving a disulfide bond in a protein includes: cleaving a disulfide bond in a protein present in a reaction system by reduction via a reduced redox protein; and reducing an oxidized redox protein produced by oxidation of the reduced redox protein in the cleaving to the reduced redox protein by donating an electron from an electrode connected to an external power supply outside the reaction system to the oxidized redox protein.