Modified KZ144 Endolysin Sequence Stability
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Solution Overview
Problem
Existing endolysin enzymes exhibit suboptimal characteristics, particularly in terms of stability and processing, which limits their effectiveness in degrading the peptidoglycan of Gram-negative bacteria such as Pseudomonas and Campylobacter.
Innovation Solution
Development of polypeptides with amino acid sequences exhibiting at least 90% sequence identity to a specific endolysin sequence, incorporating modifications such as cysteine residue substitutions and additional amino acid sequence stretches, to enhance stability and activity, including fusion proteins with tags for improved expression and purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the native KZ144 endolysin sequence is used, then the enzyme exhibits lytic activity against Gram-negative bacteria, but the stability and processing characteristics are suboptimal
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the KZ144 endolysin through site-directed mutagenesis. Specific residues were changed to improve structural stability and processing characteristics while preserving the enzyme's lytic activity against Gram-negative bacteria. This direct modification of molecular parameters resolves the contradiction between maintaining native activity and improving stability/processing.
Solution Approach 2:
The patent implements local quality by introducing specific point mutations at targeted positions within the endolysin sequence rather than globally altering the protein. The modifications were made at specific local regions to enhance stability and processing without compromising the overall functional integrity of the enzyme, thus resolving the contradiction between native activity and improved characteristics.
2Temperature
If amino acid sequence modifications are introduced to improve stability, then temperature stability increases, but sequence identity to the native endolysin decreases
Solution Approach 1:
The patent deliberately changes the amino acid sequence parameters to enhance temperature stability. Through controlled site-directed mutagenesis, specific residues were modified to increase thermal stability while maintaining sufficient sequence identity to the native endolysin to preserve its fundamental structure and function. This resolves the contradiction between temperature stability and sequence identity.
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 polypeptides demonstrate increased stability and maintained or improved activity against Pseudomonas and Campylobacter bacteria, with enhanced temperature stability and effective peptidoglycan degradation, as evidenced by increased melting temperatures and minimal inhibitory concentrations.
Implementation Method 1
the endolysin KZ144, a highly lytic peptidoglycan hydrolase
Implementation Method 2
degrade the peptidoglycan of Gram-negative bacteria, in particular of Pseudomonas and/or Campylobacter bacteria
Data Source
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AI summary
The present invention relates to polypeptides comprising an amino acid sequence exhibiting at least about 90% sequence identity with the sequence of SEQ ID NO: 1. Said polypeptides preferably degrade the peptidoglycan of Gram-negative bacteria, in particular of Pseudomonas and/or Campylobacter bacteria. In addition, the present invention relates to nucleic acids encoding such polypeptides, vectors comprising such nucleic acids, and corresponding host cells. Finally, the present invention relates to compositions comprising such polypeptides, nucleic acids, vectors, and/or host cells according to the present invention.