Modified Nuclease Salt Tolerance Temperature Stability
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Solution Overview
Problem
Conventional nucleases, such as the Serratia marcescens nuclease, exhibit limited activity at high temperatures, low temperatures, and high salt concentrations, making them unsuitable for various industrial applications in biotechnology.
Innovation Solution
Development of modified nucleases with amino acid substitutions at specific positions, such as P51, T77, and others, to enhance their activity and stability across a broader range of temperatures and salt concentrations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional nucleases are used, then they exhibit high activity at optimal temperature and salt concentration, but their activity is limited at high temperatures, low temperatures, and high salt concentrations
Solution Approach 1:
The patent applies parameter changes by modifying amino acid residues at specific positions (e.g., P51, T77, N58, K84, D191) in the nuclease sequence to alter the enzyme's physical-chemical properties. These substitutions change the enzyme's stability and activity parameters, enabling it to maintain reliable enzymatic activity across a broader temperature range (4°C to 60°C) and at higher salt concentrations (0.05M to 1.00M) without losing its core hydrolytic function
Solution Approach 2:
The patent creates composite enzyme variants by combining multiple amino acid substitutions within a single nuclease molecule. These composite modifications (e.g., P51A/T77R/N58K/K84G/D191P) work synergistically to simultaneously improve temperature stability and salt tolerance while preserving enzymatic activity, achieving adaptability across diverse industrial conditions
2Adaptability or versatility
If amino acid substitutions are made to improve temperature and salt tolerance, then the nuclease becomes more versatile, but the sequence complexity increases
Solution Approach 1:
The patent applies local quality by making targeted amino acid substitutions at specific positions (P51, T77, N58, K84, D191) rather than throughout the entire sequence. Each substitution is strategically placed to address specific stability issues (temperature or salt tolerance) while leaving the rest of the enzyme structure unchanged, thus improving versatility with minimal increase in overall sequence complexity
Solution Approach 2:
The patent segments the complexity management by treating different functional improvements as separate modular substitutions. Each amino acid change can be independently designed, tested, and combined, allowing the complex task of improving temperature and salt tolerance to be broken down into manageable, interchangeable genetic modules
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 nucleases demonstrate high activity at salt concentrations ranging from 0.05M to 1.00M and at temperatures between 4°C and 60°C, thereby expanding their industrial applicability.
Implementation Method 1
Nucleases are hydrolytic enzymes that cleave nucleic acids (DNA or RNA)
Data Source
AI summary
The invention relates to nucleases with improved properties such as enzymatic activity at high temperature, at low temperature, and/or high salt concentration. The invention also relates to methods for hydrolyzing polynucleotide substrates using the nucleases at high temperatures, at low temperature, or at high salt concentration. The invention further relates to uses of the nucleases for hydrolyzing polynucleotides in the manufacture of biopharmaceuticals, pharmaceutical compositions, vaccines, or viral vectors. Furthermore, the invention relates to kits comprising the nucleases.