Protease Surface Mutations for Stability Under Harsh Conditions
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current enzyme systems, such as serine proteases, face challenges in maintaining activity and functionality under adverse conditions like oxidative agents, chelating agents, extreme temperatures, and pH variations, which limits their effectiveness in applications like cleaning and feed industries.
Innovation Solution
Engineered protease variants with combinable mutations at selected surface positions that affect charge and hydrophobicity are developed, enhancing properties like proteolytic activity, protein expression, and stain removal performance, specifically targeting positions like 24, 45, 101, 109, 118, 213, and 217 in subtilisin enzymes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type subtilisin enzymes are used, then the enzyme system is simple and cost-effective, but the enzyme activity and stability are diminished under adverse conditions such as oxidative agents, chelating agents, extreme temperatures, and pH variations
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations at defined positions in the subtilisin enzyme sequence. These mutations alter the enzyme's physical and chemical parameters (such as charge distribution and hydrophobicity) to enhance its stability and activity under adverse conditions. The mutations are made at specific positions (e.g., positions 24, 45, 101, 109, 118, 213, 217) to optimize the enzyme's performance while maintaining a relatively simple overall structure.
2Reliability
If enzyme variants with improved stability are developed through multiple mutations, then the enzyme performance under adverse conditions is enhanced, but the complexity of enzyme engineering and characterization increases
Solution Approach 1:
The patent applies segmentation by dividing the enzyme engineering process into discrete, manageable steps. Each mutation is introduced and characterized separately at specific positions in the enzyme sequence. This modular approach allows the complex task of creating stable enzyme variants to be broken down into smaller, more manageable experiments, making the overall engineering process more systematic and reproducible.
Solution Approach 2:
The patent systematically varies specific parameters (amino acid sequences at defined positions) to optimize enzyme performance. By changing parameters at specific locations rather than randomly mutating the entire sequence, the complexity of engineering is reduced while still achieving the desired stability improvements.
3Productivity
If wild-type proteases are used in cleaning applications, then the formulation is simple, but the stain removal activity and protein expression level performance are insufficient
Solution Approach 1:
The patent introduces specific amino acid changes at defined positions to enhance the protease's catalytic activity and substrate binding characteristics. These parameter changes result in improved stain removal performance and protein expression levels in cleaning applications, while the overall enzyme structure remains relatively simple compared to fully engineered multi-functional enzymes.
Data Source
AI summary
The present invention provides engineered protease variants. In particular, the protease variants comprise combinable mutations at selected surface positions that affect the charge and/or hydrophobicity of the enzyme to enhance at least one desired property of the resulting variant enzyme in a chosen application. Compositions comprising the protease variants, and methods for using the same are also provided.