Transglutaminase Pro-Peptide Variants for Stability and Activity
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Solution Overview
Problem
Existing transglutaminase enzymes suffer from poor stability in solution, leading to enzymatic activity reduction due to self-crosslinking, and there is a lack of identified residues for beneficial mutations in pro-peptide binding that can modulate enzyme activity effectively.
Innovation Solution
Development of pro-peptide variants through directed evolution to enhance pH responsiveness, binding affinity, stability, and yield of mature transglutaminase, including specific amino acid modifications and truncations to alter binding properties without significantly changing the mature domain.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If transglutaminase is formulated as a powder for commercial application, then stability is improved, but enzymatic activity is reduced due to self-crosslinking when taken into solution
Solution Approach 1:
The pro-peptide acts as an intermediary that binds to the mature transglutaminase domain, preventing self-crosslinking and aggregation in solution. This mediator approach allows the enzyme to maintain both stability and activity by blocking the active site without permanently inactivating it.
Solution Approach 2:
The invention modifies the pro-peptide sequence to optimize its binding properties and pH responsiveness, changing the parameters of the regulatory domain to improve both stability and enzymatic activity simultaneously. Specific amino acid substitutions are made to enhance binding affinity and modulate pH-dependent activity.
2Stability of the object's composition
If the pro-peptide binding affinity is increased, then stability is improved, but enzymatic activity is reduced due to stronger inhibition
Solution Approach 1:
The pro-peptide is engineered to exhibit dynamic binding characteristics with pH responsiveness, allowing it to bind strongly at certain pH levels for stability while releasing or binding weakly at other pH levels to permit enzymatic activity. This dynamic regulation resolves the contradiction between stability and productivity.
3Adaptability or versatility
If site-directed mutagenesis is used to modify pro-peptide binding affinity, then binding properties are altered, but the lack of identified beneficial residues limits effective modulation
Solution Approach 1:
The invention systematically varies pro-peptide sequence parameters through directed evolution and identifies specific beneficial mutations at key residues. By changing amino acid parameters at identified positions, the invention achieves effective binding affinity modulation without excessive complexity.
Solution Approach 2:
The invention employs screening methods that provide feedback on binding affinity and enzymatic activity, allowing iterative optimization of pro-peptide variants. This feedback mechanism identifies beneficial mutations efficiently, reducing the complexity of mutation identification while improving adaptability.
Data Source
AI summary
Pro-peptide variants useful in modulating the activity of transglutaminases are disclosed herein.