Endonuclease V Variants for Thermostable DNA Cleavage
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Solution Overview
Problem
Endonuclease V enzymes from E. coli lack thermostability and stability under severe conditions, limiting their efficiency in high-temperature and high-salt processes without stabilizing agents.
Innovation Solution
Development of variants with specific amino acid substitutions such as C169Q, K133T, K138D, K155G, K194R, and D206H, which enhance thermostability, solubility, and residual activity, allowing for efficient cleavage of deoxyinosine-containing DNA even without reducing agents like BSA or TCEP.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Endonuclease V from E. coli is used under high temperature and high salt conditions, then the enzyme can perform cleavage activity, but the enzyme loses stability and residual activity
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues (C136, C169, C192, K133, K138, K155, K194, D206) in the enzyme sequence to alter its thermal and chemical stability properties, enabling it to maintain activity under harsh conditions without requiring stabilizing agents
Solution Approach 2:
The invention creates a composite enzyme system by combining multiple amino acid substitutions at different positions to achieve synergistic effects that improve both thermostability and residual activity simultaneously
2Reliability
If stabilizing agents such as BSA or reducing agents such as TCEP/DTT are added to maintain enzyme activity, then the enzyme retains residual activity under thermal stress, but the system complexity and cost increase
Solution Approach 1:
The engineered enzyme variants perform self-service by inherently maintaining their own structural stability and catalytic activity under harsh conditions through their modified amino acid sequences, eliminating the need for external stabilizing agents like BSA or reducing agents
3Productivity
If the enzyme is used for prolonged periods under non-reducing conditions, then the cleavage reaction can be sustained, but the enzyme activity decreases over time
Solution Approach 1:
The patent modifies the enzyme's chemical parameters through amino acid substitutions that enhance its resistance to denaturation and inactivation under prolonged non-reducing conditions, thereby extending its operational lifespan and maintaining productivity over time
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 variants exhibit improved stability and cleavage kinetics, maintaining activity at elevated temperatures and reducing the need for stabilizing agents, thus enhancing the enzyme's performance in harsh conditions.
Implementation Method 1
Endonuclease V (herein after referred to as Endo V), also called deoxyinosine 3' endonuclease, is a DNA repair enzyme that recognizes DNA containing deoxyinosine (a deamination product of a deoxyadenosine, also referred to as inosine and hypoxanthine) residues. Endo V primarily cleaves the second and third phosphodiester bond 3' to an inosine residue in the same strand, leaving a nick with a 3'-hydroxyl and a 5'-phosphate.
Data Source
AI summary
The present invention relates to novel polypeptides having an endonuclease V activity, and uses thereof.